FIELD OF THE INVENTION This invention relates to methods of identifying agents that inhibit early stages of delayed type hypersensitivity reactions that typically occur within the first few hours after antigen exposure. The invention also discloses methods of preventing or mitigating delayed type hypersensitivity reactions, by preventing or decreasing activation of the classical complement cascade, through the modulation of IgM antibodies or antibody fragments synthesized by a B-1 type subpopulation of B cells. The invention also discloses methods of identifying agents that inhibit the hypersensitivity response by inhibiting the B-1 cell or its IgM antibody or antibody fragment production, or by inhibiting IgM antibody activation of the complement cascade.

FEDERAL FUNDING This invention arose from research funded by the following federal grant monies: R01 AI-12211, R01 AI-26889 and AI-07174.

BACKGROUND Delayed type hypersensitivity (DTH) reactions are a form of inflammatory response linked to many clinical indications such as tissue destruction in response to pathogen infection, autoimmune diseases, diabetes, multiple sclerosis and allergic asthma (for a review of DTH see IMMUNOLOGY, IMMUNOPATHOLOGY & IMMUNITY, Stewart Sell editor., 1996, Appleton & Lange, Stamford, CT, as well as Askenase (1993) IN ALLERGY:

PRINCIPLES AND PRACTICE, 4th edition, E. Middleton et al. editors, C. V. Mosby CO., St.

Louis, MO. 362-389). Loss of the ability to mount a DTH response has been linked to disease progression during human immunodeficiency (HIV) infection, as well as the progression of certain cancers. DTH reactions are typically unleashed when antigens react with CD4+ TDTH-sensitized cells. These specifically sensitized CD4+ Thl cells are attracted to gradients of antigen released from tissues and interact with activated endothelial cells through reaction of lymphocyte receptors such as L-selectin, a4 (3, integrin (VLA-4), aLP2 integrin (LFA-1) and H-CAM (CD44) with E-selectin, VCAM-1, ICAM-1 and carbohydrates on endothelial cells. Contact sensitivity (CS) is another manifestation of a T cell-mediated cutaneous inflammatory response similar to DTH (P. W. Askenase. (1993) "Effector and regulatory mechanisms in delayed-type hypersensitivity (DTH),"IN ALLERGY: PRINCIPLES AND PRACTICE 362-89 (4th edition., E. Middleton, Jr. et al., eds., C. V. Mosby Co., St. Louis, MO)).

Upon reaction with soluble antigens in tissues, sensitized TDTH cells release lymphokines, such as tumor necrosis factor (TNF), interleukin-2 (IL-2), interferon-gamma (INF-y) and other inflammatory initiating factors which attract and activate macrophages.

In turn, activated macrophages produce TNF-a, IL-1, IL-6 and PGE2 which contribute to the late phase of the DTH reaction, as well as phagocytosis and the digesting of tissue and cellular debris.

Little is known about the early stages of DTH initiation. IgE and IgG, antibodies may initiate DTH through their binding to mast cells (W. Ptak et al., (1991) J. Immunol.

146: 3929-36). Other evidence seems to suggest that the involvement of innate immunity via complement is an important component of DTH initiation. For instance, it has been recently shown that C5a receptor antagonists exhibit immunosuppressant effects on delayed-type hypersensitivity (R. F. Tsuji et al., (1992) Biosci. Biotechnol. Biochem. 56: 1686-9; R. F. Tsuji et al., (1992) Biosci. Biotechnol. Biochem. 56: 2034-6) and contact sensitivity (R. F. Tsuji et al., (1995) Immunopharmacologv 29 : 79-87). While the ability to suppress DTH initiation with C5a receptor antagonists may suggest a role for complement in DTH initiation, their potential cross reactivity and toxic side effects, such as humoral

immune suppression and weight reduction, complicate the elucidation of a potential role for innate immunity in the initiation of DTH reactions.

SUMMARY OF THE INVENTION The present inventors have discovered an unexpected cascade of events believed to be at least partially responsible for initiation of DTH and CS. Studies pertaining to the instant invention indicate that B-1 cells, their secreted IgM antibodies or fragments of the secreted IgM antibodies, as well as a subsequent generation of the complement C5a fragment are involved in the early initiation of DTH and CS reactions.

The present invention provides methods to modulate a B-1 cell mediated delayed type hypersensitivity reaction in a subject comprising the step of administering a therapeutically effective amount of an agent that modulates an early phase step in DTH initiation. Such agents include agents which modulate B-1 cell production or secretion of IgM pentamer, IgM halfrner, or antigen binding IgM fragments, such as anti-C5 antibodies, anti-C5a antibodies, anti-C5a receptor antibodies, anti-IgM antibodies, as well as antagonists of the C5a receptor.

The invention further provides methods to identify agents that inhibit B-1 cell IgM initiated delayed type hypersensitivity comprising the step of determining whether an agent complexes with IgM pentamer, palmier, or heavy chain fragments, which initiate B-1 cell delayed-type hypersensitivity.

BRIEF DESCRIPTION OF THE FIGURES Figure 1. Reconstitution of B-1 cell Deficient CBA/N-xid mice with anti-TNP monoclonal IgM antibody. 13.4 IgM monoclonal antibody (mAb) was purified and injected i. v. into CBA/N-xid B-1 cell deficient mice that were PC1 sensitized and PC1 challenged.

The 13.4 IgM mAb restored (hatched bars, Group G), the defective control 24 hr CS responses (Group D), but had no effect on unsensitized CBA/N-xid mice (Group E). In contrast, positive CBA/J controls that were PCl-sensitized and PCl-challenged had brisk CS responses (Group B), compared to negative responses in vehicle sensitized CBA/J that were PC1 ear challenged (Group A). Positive control B-1 cells also restored CS (Group F).

Figure 2. The role of B-1 cells in Contact Sensitivitv (CS !. Fig. 2A demonstrates that B-1 cells reconstitute 24 hour CS in nid mice. Fig. 2B demonstrates that B-1 cells reconstitute chemotactic activity in 24 hour CS ear responses. Fig. 2C demonstrates impaired IFN-y production in CS ears of xid mice.

Figure 3. Complement-dependent chemotactic activity in two hour CS ear extracts.

Fig. 3A demonstrates C5-dependent elaboration of 2 hour macrophage chemotactic activities. Fig. 3B shows neutralization of 2 hour ear extracts chemotactic activity using anti-C5a antibody (Ab). Fig. 3C demonstrates chemotaxis of peritoneal exudate macrophages (PEC) from C5aR :' mice against 2 hour ear extracts. Fig. 3D displays antigen-specific elaboration of macrophage chemotactic activity in 2 hour CS ears.

Figure 4. B-1 cells are demonstrated to be essential for early two hour activation of complement in CS. Fig. 4A shows impairment of early, 2 hr CS ear swelling responses in B-1 cell deficient xid mice. Fig. 4B presents B-1 cell reconstitution restoring 2 hour chemotactic activity.

Figure 5. Complement is involved in early phase of eliciting CS and DTH. In Figure 5A, CBA/J mice (4/group) were actively contact-sensitized with PC1 applied topically to the shaved chest and abdomen. Four days later, the mice were skin challenged to elicit CS by painting both ears with PC1. The resulting CS ear swelling responses were measured with a dial caliper by comparing thickness before versus 2-and 24-h after challenge. Net ear swelling was calculated by subtracting ear thickness before challenged from that at 2 and 24 hours.

In Figure 5B, TNP-SRBC (105 RBC) were injected i. v. to immunize BDF, mice (4/group) which were challenged s. c. 5 days later in both hind footpads with 10g TNP- SRBC. Footpad swelling was measured with a dial caliper before and 24 hour after challenge. In both Figs. 5A and 5B, sCRl was injected (750 Fg/mouse) i. p. 30 minutes -before antigen challenge (Groups B and D), or 3 h after challenged (Group E). *P<0. 05,

**P<0.01, compared with PBS injected and immunized and antigen challenge controls (Group C).

Figure 6. CS and DTH were inhibited by local depletion of complement in mice ears by CVF injection. In Figure 6A, CBA/J mice (4/group) were actively contact- sensitized with PC1 applied topically to the shaved chest and abdomen. Four days (d) later, the mice were skin challenged to elicit CS responses by painting both ears with PC1 (Groups C and D). Subsequent CS ear swelling responses were measured with a dial caliper by comparing thickness versus 2-and 24-h after challenge. Net ear swelling was calculated by subtracting ear thickness before challenge from that at 2-and 24-h. PBS or cobra venom factor (CVF) (5 ag/20 ul) was injected directly into the ears s. c. with a 30 gauge, 1/2" needle, 48 h before challenge (Groups B and D). In Figure 6B, TNP-SRBC (105 cells) were injected i. v. to immunize BDF, mice (4/group), which were challenged s. c. 5 d later in both hind footpads with 108 TNP-SRBC. Footpad swelling was measured with a dial caliper before and 24-h after challenge. PBS or cobra venom factor (CVF) (1 pg/40 Ill) was injected locally s. c. 48 h before footpad challenge. *P<0.05, **P<0.01, compared to 2-and 24-h positive immune and antigen-challenged controls which were injected with PBS (Group C).

Figure 7. C5 depletion by anti-C5 mAb-inhibited CS. CBA/J mice (4/group) were actively contact sensitized with PC1 applied topically to the shaved chest and abdomen.

Four days later, mice were skin challenged to elicit CS by painting both ears with PCI. CS ear swelling responses were measured with a dial caliper by comparing thickness before versus 2 and 24 hour after challenge. Net ear swelling was calculated by subtracting ear thickness before challenge from that at 2-and 24-h after challenge. In Figure 7A, systemic anti-C5 mAb (1 mg/mouse) was injected i. v. 24 h and 4 h before ear challenge with antigen.

In Figure 7B, local anti-C5 mAb s. c. into ears 24 h before ear challenge. *P<0.05, **P<0.01, compared with an IgG, isotype control group (Group C).

Figure 8. Increased C5-titer in 24-h CS ear extracts. Contact sensitized mice were skin challenged topically on day 4 to elicit CS by painting both ears with PCI. Twenty-four hours later, the challenged ears were extracted with PBS, as described in Tsuji et al., (1997).

In Figure 8A, total protein concentration (mg/ear) and Figure 8B C5-titer per ear were determined. Closed and open circles represent two-and four-fold dilution of ear extracts with gelatin-veronal buffer, respectively. Each value per ear was expressed as a separate dot. The number of ear extracts in each group was 12, and was derived from 6 mice.

Figure 9. Anti-Complement treatment resulted in decreased chemotactic activity in 24-h CS ear extracts. Two different complement inhibitors, anti-C5 mAb (Fig. 9A) and sCRl (Fig. 9B) were used. Mice were ear challenged 4 d after PCI contact sensitization.

Punch biopsies from each ear were collected 24 hour later and then were extracted with PBS. In vitro chemotaxis assays were performed on the ear extracts using target J774A. 1 macrophage cells which did not migrate against the RPMI 1640-0.25% gelatin medium.

Human recombinant (r) rC5a (100 ng/ml) and ZAMS (1: 20) prepared from normal mouse serum were used as positive chemoattractant controls. In Figure 9A, mice were injected i. v.

24 h and 4 h before ear challenge with either anti-C5 (1 mg/mouse) or with isotype (IgGl) matched control anti-human C8 (1 mg/mouse). In Figure 9B, mice were injected i. p. with 125 pg/mouse sCRl 30 min before ear challenge.

Figure 10. Cell infiltration in 24-h CS ears was decreased by systemic treatment with anti-C5 mAb. Mice were immunized by painting with vehicle (Groups A and B) or 5% PCI (C-F) on day 0, and challenged on the ears with PCI on day 4. Then saline (Groups A, C and E) or 1 mg anti-C5 mAb (Groups B, D, and F) was injected i. v. at 4-and 24-h before ear challenge. 5-am sections of ears were obtained 24 h after antigen challenge, and were stained with hematoxylin and eosin. Magnification is 200 (Groups A-D) or 1,000 (Groups E and F). Leukocyte infiltration in CS ear responses (Group C) was decreased by anti-C5 mAb treatment (Group D). Intraepidermal abscesses of leukocytes were observed -frequently in ears from immune and challenged mice treated with saline (Groups C and E),

but not in ears of mice treated with anti-C5 mAb (Groups D and F). Arrowheads in (Group C) denote intraepidermal abscess.

Figure 11. Ouantitation of decreased cell infiltration in CS ears of mice treated with anti-C5 mAb. Sections of ears, as in Figure 8, were histologically evaluated blindly In Figure 11 lA, infiltration of leukocytes was graded from one to four, where one through four are, respectively, [1] no infiltration, [2] slight infiltration, [3] modest infiltration, and [4] significant infiltration. In Figure 11B, formation of intraepidermal abscesses was graded from one to three, where they are respectively, none seen, few seen, and many seen.

Numbers in parentheses denote mean SE of histologic scores for each portion of the figure. *P<0.02 and **P<0.002, compared to positive controls (5% PCl-immunized, PBS- injected, ear-challenged group).

Figure 12. Reconstitution of CBA/N-xid Mice utilizing Pure Pentameric Anti-TNP IgM (32. 17 ! Antibody.

Figure 13. Reconstitution by IgM Fragments of T Cell Ear CS Reaction in CBA/N- xid B-1 Cell Deficient Mice.

Figure 14.13.4 IgM Antibody and IgM Fragment Affinity for Target Cells. The upper panel tested affinity using intact pentameric 13.4 IgM. In the middle panel, isolated and purified heavy and light chains reduced under harsh conditions (20 mM DTT) was analyzed for its ability to bind to target cells. The lower panel demonstrates the ability of 13.4 palmier IgM reduced using 0.25 mM DTT.

Figure 15. Antigen Binding Capability of Reduced. Alkylated IgM Fragments for TNP (Antigen ! Via ELISA Assay.

Figure 16. Asthma Initiation via Administration of B-1 Cell DTH-Initiating IeM.

Airway reactivity is expressed as the increase in baseline lung resistance (RL) (on the

vertical y axis) induced by inhalation of increasing doses of aerosolized methacholine (on the horizontal x axis). Mice that were passively sensitized with 100 pg 32.17 anti-TNP IgM mAb, and then were airway challenged with the relevant hapten TNBSA, had hyper- responsive airways, with increased resistance over several methacholine doses; especially lower doses (a characteristic of clinical asthma), as compared to three different control groups shown (n= 4-5 mice in each group) (P<0.05).

DETAILED DESCRIPTION I. Definitions As used herein,"delayed-type hypersensitivity"or"DTH"refers to a heightened and inappropriate immune response to an antigen or allergen. DTH reactions typical involve the reaction of an antigen with CD4+ TDTH-sensitized cells. By"contact sensitivity"or"CS"is meant an experimental response that is initiated by exposure to an antigen or allergen which results in a DTH reaction that is localized, for example, by the skin surface which comes in contact with said antigen or allergen.

As used herein, an"early phase step"in DTH initiation refers to the heretofore unknown initial immunochemical and cellular steps of a DTH reaction, as described herein, that typically occur within about the first one to four hours, often within about the first one to two hours following exposure to an antigen that triggers a DTH or CS reaction. Such early phase steps include, but are not limited to, antigen reaction with B-1 cell derived IgM or antigen binding fragments of B-1 cell derived IgM, as well as other immunochemical reactions involving and associated with the classical complement cascade.

By"DTH-initiating factor"or"DIF"is meant the immunoglobulin M (IgM) molecule produced by B-1 cells that induces or modulates the signal cascade resulting in DTH or CS. By"DTH-initiating IgM"is meant the immunoglobulin molecule (or fragment thereof) produced by a B-1 cell within about one day (often, in actuality, within about one to four hours), in response to an allergen or antigen stimulus that triggers a -delayed type hypersensitivity response further mediated by CD4+ TDTH-sensitized eells. The DTH-initiating IgM also encompasses DTH-participating IgM molecules that may be found

in the general circulation prior to the particular exposure to an allergen or antigen stimulus that is the trigger for a DTH response.

By"B-1 cell"is meant a subset of B cells that is CD5+ and which (or a subpopulation of which) produces an IgM molecule that participates in the early phase steps of the initiation of a DTH or CS response. An"isolated"or"purified"B-1 cell is a cell population that is enriched in CD5+ cells relative to CD5-B cells and other cells in the source material from which the purified B-1 cell is isolated or purified. As used herein,"B- 1 a Cells"also refers to cells that are CD5+ and which (or a subpopulation of which) produce an IgM molecule that participates in the early phase steps of the initiation of a DTH or CS response. Isolation and purification of B-1 and B-l a cells can occur using FACS sorting (A. B. Kantor et al., 1997) or other cell separation techniques known to those skilled in the art. As used herein, B-lb B cells are those B cells that are CD5-.

"B-1 cell mediated conditions"are those conditions, such as delayed-type hypersensitivity and contact sensitivity, that require the action of the B-1 cell (defined herein as being CD5+ B cells) or of B-1 (CD5+) cell-derived IgM (or various fragments thereof as discussed herein). The conditions include but are not limited to pathogen infection, autoimmune diseases, diabetes, multiple sclerosis and allergic asthma.

As used herein,"immunoglobulin M fragments"or"IgM fragments"refer to fragments or portions of the IgM pentamer, such as halfmers (joined heavy and light chains) and separated IgM heavy and light chains. Ideally, such fragments retain sufficient portions of the native IgM molecule to have both the functional capacity to bind to a DTH-triggering antigen and the functional capacity to participate in the complement-mediated early phase steps of DTH initiation.

"B-1 cell agonists"include those agents, compounds, compositions, etc., which when administered to a subject or brought into contact with B-1 cells up-regulate (i. e., increase, promote or otherwise elevate) the level of DTH-related B-1 cell activity, including but not limited to the B-1 cell synthesis of IgM or IgM fragments. As used herein, "agonists"also refer to those agents, compounds, compositions, etc., which enhance the -binding and DTH-related interaction of B-1 cell derived IgM molecules with their-antigen and complement (or other) substrates.

"B-1 cell antagonists"include those agents, compounds, compositions, etc. which when administered to a subject or brought into contact with B-1 cells down-regulate (i. e., cause the inhibition, depression, prevention, reduction, etc. of) DTH-related B-1 cell activity, including but not limited to B-1 cell synthesis of IgM or IgM fragments. As used herein,"antagonists"also refer to those agents, compounds, compositions, etc., which decrease the binding and DTH-related interaction of B-1 cell derived IgM molecules with their antigen and complement (or other) substrates.

"Pharmaceutically acceptable"refers to molecular entities and compositions that are generally physiologically tolerable by a subject to whom they are administered, and, for example, do not typically produce an allergic or other unacceptable or untoward reaction, such as gastric upset, dizziness and the like. The"subject"or"patient"of the more preferred embodiments of the present invention are vertebrates or mammals, and in the most preferred embodiments are human beings.

As used herein,"effector molecule"refers to a molecule which interacts with DTH- initiating IgM to effectuate a DTH reaction. Effector molecules include, but are not limited to, components of the classical complement cascade.

An"epitope"refers generally to a specific feature of an antigen or hapten molecule that is recognized by a host's or subject's immune system. Epitopes generally are formed, at least in part, by a particular topological orientation of functional groups of the antigen.

According to the invention, a molecule contains an epitope, or shares an epitope of a second molecule, if the first molecule specifically binds or competitively interacts with the specific binding of the second molecule. There is no requirement that shared epitopes be chemically identical; however, shared epitopes must be topologically similar (i. e., have a topological arrangement of chemical functional groups that is similar in each molecule, in order to interact competitively with a target molecule. This invention contemplates multiple epitopes for any of the classical complement components or IgM antibodies that would modulate CS and DTH responses.

By"antibody"is meant a polyclonal or monoclonal antibody which is capable of -binding to an antigen that elicits a CS-or DTH-response. The term"antibody"therefore encompasses monoclonal and polyclonal antibodies and fragments thereof which posess this

function. Such antibodies can be produced either by raising them in a suitable vertebrate host, by expression in a hybridoma cell line or other cell culture system, or by recombinant methods. Polyclonal antibodies can be raised in chickens, rabbits, goats, horses or other suitable vertebrate species. Methods of preparing, isolating and purifying such antibodies are described in E. Harlow and D. Lane, ANTIBODIES: A LABORATORY MANUAL (Cold Spring Harbor Press, Cold Spring Harbor NY, 1988), as well as other references known to the skilled artisan.

II. B-1 Cell IgM Isolation and Purification B-1 cell IgM characterization will be useful in identifying additional agents that can modulate (particularly that can inhibit) IgM initiated or mediated DTH. However, in subjects in whom the upregulation of B-1 cell IgM production is desirable (e. g., to ward off parasitic infections in immune-compromised patients), development of drugs or of purified and isolated pentameric IgM as well as IgM fragments that stimulate a beneficial DTH- response may be useful. In diseases such as allergic asthma, tuberculosis and other DTH- mediated conditions, it would be useful to down regulate the B-1 cell production of IgM, thereby inhibiting or blocking B-1 cell IgM-initiation of DTH. IgM molecules synthesized by B cells, other than by B-1 or B-1 a cells, are also contemplated for use in the same manner as described for B-1 and B-la cells.

As B-1 cells are the source of IgM-initiating DTH molecules, isolation and further characterization of B-1 cells (and subpopulations thereof) may be helpful in identifying additional markers that distinguish the B-1 cells that produce DTH-initiating IgM molecules. B-1 cells and relevant subtypes can be isolated using FACS sorters as described in A. B. Kantor et al. (1998). B-1 cells have been characterized as having the following markers: CD5+, B220+ (CD45RA+), CD3-, Macl+, CD44+, HSA+ and FcyRII+. CS-initiating cells in mice have been demonstrated to possess the following phenotype: CD5+, B220+ (CD45RA+),Thy-l+, CD3-, CD4-, CD8-, aßTCR-, yoTCR-, CD23+, Macl+, IL-3R+, IL-2R-, Pgp-l+ (CD44+), J1 ld (HSA+) and FcyRII+ (Herzog et al., 1989; Ishii et al., 1994; and Ishii -etal., 1995).

The DTH-initiating IgM antibody produced by B-1 and B-la cells is also termed PC1-F. Reconstitution of DTH-initiation can occur in deficient hosts by a DTH-initiating IgM monoclonal antibody, for instance an anti-TNP IgM, from an established B cell hybridoma. The relevant B-1 cell population releases a polyclonal DTH-initiating IgM that bears determinants of IgM p-chain and is antigen specific in DTH initiation.

1. DTH-Initiating IgM Producing Hvbndomas Production of DTH-initiating IgM producing cell lines will be, for example, beneficial as a source of DTH-initiating molecules (pentameric IgM, IgM halfmers and isolated and purified IgM heavy and light chains) to stimulate complement cascades in immune deficient individuals. IgM antibody can be obtained from established hybridomas, such as anti-TNP specific B-1 cell hybridomas called Sp6 (G. Kohler et al., (1980) Eur. J.

Immunol. 10: 467-76; A. Ochi et al. (1983) Nature 302: 340-2; A. Ochi et al., (1983) Proc.

Nat'l Acad. Sci. USA 80: 6351-5; and R. G. Hawley et al., (1982) Proc. Nat'1 Acad. Sci.

USA 79: 7425-29) and 13.4 (M. G. Scott et al., (1982) J. Immunol. 128: 2622-28), as well as from new anti-TNP B-1 cell hybridomas generated from CS mice. These B-1 cell hybridomas are valuable for providing a monoclonal source of DTH-initiating molecules to facilitate biochemical characterization of the IgM molecule. In addition, these hybridomas will also be the source of cDNA that will be used for transfections and creation of transgenic mice. Prior to fusion, specific B-1 cells such as anti-TNP B-1 cells from CS- immune mice are enriched by FACS sorting using specific markers (e. g., B220+, CD5+, Thy-l+, CD3-) that will be 92% B-1 cells. Then immune B-1 cells and the non-secreting myeloma cell fusion partner are then fused using polyethylene glycol, as previously described (W. M Yokoyama, (1992) Curr. Protocols Immunol. 1: 2.5.1-2.1.8). The hybridoma lines thus obtained may be screened for antigen specific, IgM producing cells employing an assay, such as two site sandwich ELISA assay on supernatants (P. W.

Askenase et al. (1970) Immuno. Chem. (Molecular Immunol.) 7: 29).

-2. Purification of DTH-initiating IgM

DTH initiating IgM may be purified using commonly available antigen-specific and antigen-nonspecific procedures to purify antibodies. For instance, enrichment can be performed to purify anti-TNP monoclonal antibodies, using TNP-BSA Sepharose-4B antigen affinity columns. The enriched preparation of, for example, anti-TNP IgM can be further purified via an anti- agarose affinity column, with final purification via fractionation on FPLC (P. Clezardin, (1986) J. Chromat. 354: 425-33) using an anion exchange Mono-Q (5 x 50 mm) column (Pharmacia Fine Chemicals, Piscataway, NJ), and eluting bound proteins using a linear salt gradient of 0-0.5 M NaCl in a 20 mM L-histidine buffer at pH 6.0. Fractionation will be followed for mouse IgM by direct capture ELISA.

Purified 13.4 anti-TNP IgM produced by this method demonstrates significant DTH- initiating activity as shown in Figure 1.

Purity of monoclonal antibodies can be determined via Coomassie blue and silver staining on SDS-PAGE gels. Homogeneously pure IgM can then be reduced with 20 mM DTT (harsh reduction conditions) to separate the heavy and light chains that can be further resolved on a preparative SDS-PAGE gel under reducing conditions that can be followed by electroelution of the proteins from the gel using a mini-whole gel electro-elution apparatus (Bio Rad, CA). The protein can then be microsequenced.

3. Determination of DNA Sequence of IgM Polypeptides Using PCR and hybridoma lines such as 13.6, the cDNA sequence of the heavy p chain and the K and/orX light chains can be performed. Proper covalent linkage and folding of these subunits generally will result in a complete pentameric IgM product.

Synthetic oligonucleotides corresponding to the 3'end of the C-terminal secreted region domain that is common for all IgMs, as reported in the literature (P. H. Schreier et al., (1986) Nuc. Acids Res. 14: 2381-9) can also be prepared. The Vh (p) primer can be synthesized according to the novel N-terminal 5'sequence of the V-region of, for example, the 13.4 monoclonal IgM, or new B-1 cell hybridoma DTH-initiating IgM heavy chain, obtained by direct protein sequencing. Using primer pairs comprised of these degenerate V- -region primers, and 5'cry specific primers, one can amplify by PCR the target cDNA obtained from the 13.4 hybridoma or other B-1 cell IgM hybridoma. The unique li chain

gene sequence for B cell released 13.4"IgM"can be obtained by DNA sequencing as described by (F. Sanger et al., (1977) Proc. Nat'1 Acad. Sci. USA 74: 5463-7). This will allow determination of the complete protein sequence encoded by the cDNA as well as molecular characterization of the IgM gene and associated protein.

III. Identifying Compounds that Modulate Delayed Type Hypersensitivity Identifying agents which inhibit or promote DTH responses would be helpful for two populations of subjects: (1) subjects who suffer from inappropriate DTH responses, and (2) subjects who are incapable of mounting a DTH response. Therefore, for example, identifying agents that stimulate B-1 cell production of IgM molecules that initiate DTH would be helpful in individuals with compromised immune systems. Alternatively, inhibitory agents would be useful as therapies to prevent or block inappropriate DTH responses, such as in allergic asthma.

1. Agents that Modulate B-1 Cell Production of IgM Agents can be identified that inhibit production of IgM from B-1 cells. B-1 cell production of IgM in the presence and absence of the agent to be tested may be assayed by conventional in vitro binding studies, such as would involve labeled anti-IgM antibody.

Another method of identifying agents that may inhibit B-1 cell production of IgM would be to create commonly available gene chip assay systems (Affymetrix). For example, messenger RNA (mRNA) levels of a quiescent B-1 cell (a B-1 cell not exposed to either an agent or an allergen/antigen) is compared to the mRNA levels of an antigen-upregulated cell. This composite of mRNAs can then be assessed in light of the mRNAs obtained from a B-1 cell exposed to a compound being tested for its inhibitory abilities. From these three mRNA arrays, it can be determined whether the compound has the capacity to block or inhibit DTH-initiating IgM mRNA.

Alternatively, a compound can be tested in animal models, wherein the animal is co- administered allergen and the compound to be tested, or is administered the compound within the first two hours after allergen administration. The ability of the compound to inhibit or block initiation of DTH and the eventual DTH response in the animal as compared

to the control can then be determined using methods commonly available to the skilled artisan. Animal test systems are described in greater detail below.

The same sorts of systems can be utilized in testing agents that up-regulate B-1 cell production of DTH-initiating IgM molecules for use in subjects needing a DTH response to fight infections, etc. The present invention also includes the use of isolated DTH-initiating IgM to stimulate DTH in a subject. Isolated DTH-initiating IgM molecules contemplated as part of the present invention include pentameric IgM, IgM halfmers, and isolated and purified heavy and light chains of the DTH-initiating IgM to the extent that they possess the ability to initiate DTH. These isolated DTH-initiating IgM molecules can be obtained by recombinant means, from hybridomas or other suitable cell lines, and other means known in the art.

2. Agents that Modulate B-1 Cell IgM-Substrate Interaction DTH-initiating IgM interacts with complement inducing the classical complement cascade. Isolating agents that inhibit or enhance this interaction would be effective in blocking or promoting the classical complement cascade, to the extent that it is involved with a DTH or a CS response. The agents contemplated include, for example, antibodies capable of binding to the sites on the DTH-initiating IgM which bind to complement proteins. Other agents contemplated include, for example, anti-C5 antibodies, anti-C5a antibodies, anti-C5a receptor antibodies, anti-DTH-initiating IgM antibodies, agents including small molecules that modulate (upregulate or down regulate) interactions between IgM and members of the complement cascade, and agents that modulate the classical complement cascade. The drug screening methods described below would be useful in identifying such antibodies and other agents.

3. Drug Screening IgM or fragments thereof and complement proteins or fragments thereof, oligopeptides, polypeptides, mimetics, and other chemical compounds can be used for screening in any of a variety of drug screening techniques. The IgM or fragment employed in such a test may be free in solution, affixed to a solid support, borne on a cell surface, or

located intracellularly. The modulation of the formation IgM-complement complexes and inhibition of DTH response activity resulting from the agent may then be measured.

A technique of drug screening which provides for high-throughput drug screening of compounds having suitable binding affinity to the DTH-initiating IgM pentamer or fragments thereof can be prepared as described in detail in"Determination of Amino Acid Sequence Antigenicity,"by H. N. Geysen, WO APL. 84/03564 (1984). In summary, large numbers of small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. Alternatively, purified IgM or IgM fragments (e. g., halfmers), as described above, can be bound to a solid substrate. The peptide test compounds are reacted with whole antibodies or fragments of DTH-initiating IgM antibody and washed. Bound peptides or agents are then detected by methods known in the art.

Purified peptide test compounds can also be coated directly onto plates for use in the aforementioned drug screening techniques. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support (see P. R. Hawkins et al., (1998) U. S. Patent No. 5,712,115).

Another method of screening involves labeling the DTH-initiating IgM or complement polypeptides with any of a myriad of suitable markers, including radiolabels (e. g.,'1 or P), various fluorescent labels and enzymes (e. g., glutathione-S-transferase, luciferase, and ß-galactosidase). If desired for basic binding assays, the target polypeptide (e. g., DTH-initiating IgM or complement polypeptides) may be immobilized by standard techniques. For example, but not by way of limitation, such immobilization may be effected by linkage to a solid support, such as a chromatographic matrix, or by binding to a charged surface, such as a nylon membrane.

Competition binding assays generally take one of two forms: immobilized DTH initiating IgM or IgM fragments (e. g., halfmers) or complement proteins or fragments thereof can be used to bind potential therapeutic chemicals or polypeptides. In each case, the labeled polypeptide is contacted with the immobilized polypeptide under aqueous conditions that permit specific binding of the polypeptide (s) to form a complex (e. g., DTH- -initiating IgM-complement complex) in the absence of added agent. Particular aqueous conditions may be selected by the practitioner according to conventional methods. For

general guidance, the following buffered aqueous conditions may be used: 10-250 mM NaCI, 5-50 mM Tris-HCl (pH = 5-8), with optional addition of divalent cations and/or metal chelators and/or non-ionic detergents and/or membrane fractions. It will be appreciated by those skilled in the art that additions, deletions, modifications (such as pH) and substitutions (such as KC1 substituting for NaCl or buffer substitution) may be made to these basic conditions. Modifications can be made to the basic binding reaction conditions, so long as specific binding of DTH-initiating IgM or fragments thereof/complement complex can occur in the control reactions. Conditions that do not permit specific binding in control reactions (no agent included) are not suitable for use in performing the assays.

Preferably at least one polypeptide species is labeled with a detectable marker.

Suitable labeling includes, but is not limited to radiolabeling by incorporation of a radiolabeled amino acid (e. g,'4C-Leucine, 3H-Glycine, 35S-methionine), radiolabeling by post-translational radioiodination with"'I or.. I (e. g., Bolton-Hunter reaction and chloramine T), labeling by post-translational phosphorylation with 32p (e. g., phosphorylase and inorganic radiolabeled phosphate), or labeling by other conventional methods known in the art. In embodiments where one of the polypeptide species is immobilized by linkage to a substrate, the other polypeptide is generally labeled with a detectable marker. For additional screening methods, the methods of J. R. Bischoff et al., (1998) U. S. Patent No.

5,705,342 can be utilized with the substitution of DTH-initiating IgM and complement polypeptides for bcl-2 and r-ras.

This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding to the epitopes on the DTH-initiating IgM responsible for complement activation are tested using competition assays. In this manner, the neutralizing antibodies obtained can be used to inhibit the complement cascade that would otherwise be initiated by the DTH-initiating IgM antibody.

Using methods similar to those described above for the inhibitory agents, peptides, IgM molecules and their fragments (including IgM antibodies produced by cells other than B-1 cells), and other reagents can be analyzed for their ability to induce a DTH response or -the classical complement cascade.

IV. Preparation of DTH or CS Inhibitory Antibodies Another embodiment of this invention relates to creating antibodies that modulate DTH. Two classes of antibodies (or their fragments) that expressly are contemplated inhibit DTH by binding to: (1) members of the classical complement cascade; or (2) to B-1 cell DTH initiating IgM, thereby inhibiting the early DTH initiating steps. These antibodies, by binding to their respective targets, would cause inhibition (in whole or in part) of the DTH or CS response. Another contemplated class of antibodies (or their fragments) in another aspect of the present invention would up-regulate or induce DTH. Such agents include DTH-initiating IgM molecules (IgM pentamers, halmiers and isolated IgM heavy and light chains).

Determining which regions of the complement proteins or B-1 cell IgM peptides are optimal for raising an immune response for production of antibodies can be determined using such algorithms as described by Hopp and Woods, Proc. Nat'1 Acad. Sci. USA 78: 3824 (1981) ; and Garnier et al., J. Mol. Bio. 120: 97 (1978). Additional algorithms are readily available to the skilled artisan.

One method of preparing immunizations for the creation of polyclonal antibodies utilizes approximately 1-2 mg of antigen conjugated to approximately 2 mg Imject Keyhole limpet hemocyanin (KLH) via maleimide to the extra cysteine residue located at the amino terminus of each peptide as recommended in the manufacturer's (Pierce) instructions. A coupled carrier-antigen complex (0.5 ml) is emulsified with Complete Freund's adjuvant (0.5 ml) and approximately 1.0 ml is administered in the initial injection. Animals can be subsequently boosted every few weeks, according to the boosting schedules for the vertebrate species used (e. g., every 2 weeks in chickens); in the subsequent injections, the coupled immunogen, is emulsified in Incomplete Freund's Adjuvant rather than Complete Freund's Adjuvant. Serum or eggs, if one is using avian species, is then collected.

Additional purification and concentration of the antibody can be performed as described in Harlow and Lane (1988). When preparing antibodies to these epitopes several immunoglobulin isotypes and isotype subclasses can be prepared (e. g., IgG,, IgG2, IgM, to name a few). These isotype antibody subclasses can be prepared by raising them in vertebrates, in hybridoma cell lines or other cell lines, or by recombinant means.

Monoclonal antibodies may be obtained by various techniques familiar to those skilled in the art. Briefly, spleen cells from an animal immunized with a desired antigen are immortalized, commonly by fusion with a myeloma cell (see, Kohler and Milstein, (1976) Eur. J. Immunol. 6: 511). Alternative methods of immortalization include transformation with Epstein Barr Virus, oncogenes, or retroviruses or other methods well known to those of ordinary skill in the art. Colonies arising from single immortalized cells are screened for production of antibodies of the desired antigen specificity and affinity. The yield of the monoclonal antibodies produced by such cells may be enhanced by various techniques, including injection into the peritoneal cavity of a vertebrate host.

Alternatively, peptide specific antibodies ("anti-peptide antibodies") are prepared by immunizing suitable vertebrate hosts (e. g., chickens, rabbits or other suitable vertebrates) under appropriate immunization protocols using the peptide haptens alone, if they are of sufficient length, or if required to enhance immunogenicity, conjugated to suitable carriers.

Polypeptide fragments of complement protein or B-1 cell IgM fragments can also be utilized. Methods, including one described in greater detail above, for preparing immunogenic conjugates with carriers such as bovine serum albumin (BSA), Keyhole limpet hemocyanin (KLH) or other carrier proteins are well known in the art. In some circumstances, direct conjugation using, for example, carbodiimide reagents may be effective. In other instances, linking reagents, such as those supplied by Pierce Chemical Co., Rockford, IL, may be desirable to provide accessibility to the hapten. The hapten peptides (comprising at least four consecutive amino acid residues of a complement protein or a B-1 cell IgM) or polypeptide fragments can be extended at either the amino or carboxy terminus with a cysteine (Cys) residue or interspersed with cysteine residues, for example, to facilitate linking to a carrier. Administration of the immunogens is conducted generally by injection over a suitable time period and with use of a suitable adjuvant, as is generally understood in the art. During the immunization schedule, titers of antibodies are taken to determine adequacy of antibody formation. For more information, refer to Harlow and Lane, (1988).

While the polyclonal antisera produced in this way may be satisfactory for-some applications, for pharmaceutical compositions, the use of monoclonal preparations is

preferred. Immortalized cell lines, which secrete the desired monoclonal antibodies, may be prepared using the standard method of Kohler and Milstein (1976) or with modifications which effect immortalization of lymphocytes or spleen cells, as is generally known. The immortalized cell lines secreting the desired antibodies are screened by immunoassay in which the antigen is a peptide hapten, a polypeptide of a complement protein, or B-L cell IgM. When the appropriate immortalized cell cultures which secrete the desired antibody are identified, the cells can be cultured either in vitro or by production from ascites fluid.

The desired monoclonal antibodies are then recovered from the culture supernatant or from the ascites supernatant. Additional steps to purify or further concentrate the antibody can be performed as described in Harlow and Lane (1988).

Fragments of the monoclonal or the polyclonal antisera which contain the immunologically significant portion can be used as antagonists or agonists, as well as the intact antibodies. Use of immunologically reactive fragments, such as the Fv, scFv, Fab, Fab', or F (ab') 2 fragments, is often preferable, especially in a therapeutic context, as these fragments are generally less immunogenic than the whole immunoglobulin. The antibodies or fragments may also be produced, using current technology, by recombinant means.

Regions that bind specifically to the desired binding domains on DTH-initiating IgM or complement polypeptides can also be produced in the context of chimeras ("chimeric antibodies") with multiple species origin. Alternatively, the DTH-initiating IgM or anti- complement antibody can be a humanized antibody or a human antibody, as described in, Queen et al., U. S. Patent No. 5,585,089. See also Riechmann et al., Nature 332: 323 (1988).

Anti-DTH initiating IgM antibodies (e. g., antibodies that target and bind to DTH initiating IgM antibodies) are also contemplated. Anti B-1 cell DTH-initiating IgM antibodies or fragments thereof (e. g., halfmers), as described above, are contemplated that block or disrupt B-1 cell IgM interaction with immune cells, complement or DTH-triggering antigen. Methods of preparing these antibodies are available as described for anti-IgE antibodies in D. T. Wai Fei et al., (1998) U. S. Patent No. 5,714,338; and P. Amiri et al., - (1997) U. S. Patent No. 5,656,273.

V. Combination Therapy Agents of the present invention that modulate B-1 cell activity, B-1 cell production of DTH-initiating IgM, and agents that regulate IgM interaction with its substrates, can be provided alone, or in combination with another agent or agents that modulate a particular biological or pathological process. For example, an inhibitory agent of the present invention that blocks IgM-Clg association can be administered in combination with other agents that modulate complement activity to modulate diseases or conditions mediated by B-1 cell induced delayed-type hypersensitivity. As used herein, two agents are said to be administered in combination when the two agents are administered simultaneously or are administered independently in a fashion such that the agents will act at the same time.

Another embodiment may be the administration of two or more agents that modulate B-1 cell activity, B-1 cell synthesis of IgM and/or IgM interaction with its substrates (e. g, complement).

Typical dosages of effective B-1 cell antagonists and agonists, B-1 cell DTH- initiating IgM synthesis antagonists and agonists, or IgM-ligand modulatory agents are in the ranges recommended by the manufacturer for existing therapeutic compounds with such functions. Dosages for other such agents can be optimized by evaluating in vitro responses or responses in animal models, and may be varied clinically by up to about one or two orders of magnitude in concentration or amount. The actual dosage will depend upon such factors as the judgment of the physician, the condition of a patient, and the effectiveness of the therapeutic method based on the in vitro responsiveness of the agent, or the responses observed in the appropriate animal models and in the clinic.

VI. Methods of Treating Diseases The diseases that are appropriate for treatment using the compositions disclosed or identified pursuant to the methods disclosed by the present invention are those that are mediated by B-1 cells (CD5+), and more specifically mediated, at least in part, by DTH- initiating IgM. These diseases and conditions include all those involving a delayed type -hypersensitivity (DTH) or contact sensitivity (CS) response. Such diseases include autoimmune diseases, certain types of cancer, diabetes, multiple sclerosis and allergic

asthma. The diseases or conditions contemplated share the commonality that the DTH response can be modulated to regulate progression of the condition or some other clinical manifestation of the disease. As a result, the agents contemplated may be antagonists to IgM synthesis and B-1 cell activity.

DTH-and CS-inhibitory molecules of the invention, which act on DTH-initiating IgM or the corresponding complement polypeptides with which the IgM interacts, can be administered either as primary therapy or in conjunction with other agents that known to inhibit the DTH, CS or the other conditions involving CS or DTH, such as those discussed above.

Inhibition of B-1 cell activity, B-1 cell DTH-initiating IgM production and IgM action has many important therapeutic benefits. The present invention contemplates reversing the adverse effect of DTH reactions by administering B-1 cell antagonists that would down regulate B-1 cell activity or B-1 cell synthesis of IgM. Such antagonists may include IL-12 (U. S. Patent No. 5,665,347), IFN-y (P. Velulillai et a/., 1996), IL-1Q antisense therapy (B. Peng et al., 1995), and spindle cell inhibitors (M. Zhang et a/., 1998).

The invention also includes administering the therapeutic compositions described or identified with other therapeutic compositions that inhibit B-1 cell activity, B-1 cell IgM synthesis and/or agents that block B-1 cell IgM interactions (e. g., anti-complement antibodies).

The proposed agents may also be utilized to treat asthma. Preliminary experiments suggest that pentameric IgM, or a fragment of the DTH-initiating IgM, are involved in the airway hyperactivity of a model that imitates allergic (also known as clinical) asthma.

Intravenous transfer of partially purified monoclonal anti-TNP antibody derived from a murine B-1 cell hybridoma (32.17), followed by airway TNP-hapten antigen challenge has been demonstrated to trigger increased airway reactivity in live breathing mice in response to a stimulus dose of inhaled methacholine (as discussed further in Example 11 and shown in Figure 16). As a result, agents that inhibit the early steps leading to T cell-induced inflammation may aid in the amelioration of asthmatic conditions.

VII. Pharmaceutical Compositions Comprising Agents that Modulate B-1 Cell Mediated DTH and CS In the treatment of the clinical conditions noted above, the compounds of this invention may be utilized in compositions such as tablets, capsules or elixirs for oral administration, suppositories for rectal administration, aerosols for administration directly into the lung, sterile solutions or suspensions for parenteral or intramuscular administration and the like. Therapeutically effective amounts of the compounds or agents of the invention can be used to modulate the DTH reactions in the subject being treated. The amount or concentration administered would vary depending on the individual's clinical manifestation of the disease being treated.

The pharmaceutical compositions of this invention can be used in the form of a pharmaceutical preparation, for example, in solid, semi-solid or liquid form which contains one or more of the compounds of the present invention as an active ingredient, in admixture with an organic or inorganic carrier or excipient suitable for external, enteral or parenteral applications. The active ingredient may be compounded, for example, with the usual non- toxic pharmaceutically acceptable carriers for tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions, aerosols, and any other form suitable for use. The carriers which can be used are water, glucose, lactose, gum acacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn starch, keratin, colloidal silica, potato starch, urea and other carriers suitable for use in manufacturing preparations, in solid, semisolid or liquid form and in addition auxiliary, stabilizing, thickening and coloring agents and perfumes may be used. The active object compound (e. g., an agent capable of modulating B-1 cell mediated DTH or CS) is included in the pharmaceutical composition in an amount sufficient to produce the desired effect upon the process or condition (e. g., regulation of the classical complement cascade stimulated by B-1 cell IgM) of the disease.

For preparing solid compositions such as tablets, the principal active ingredient is mixed with a pharmaceutical carrier (e. g., conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate -or gums) and other pharmaceutical diluents (e. g., water) to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention, or

a non-toxic pharmaceutically acceptable salt thereof. When referring to the preformulation compositions as homogenous, it is meant that the active ingredient is sufficiently dispersed throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. This solid preformulation composition is then subdivided into unit dosage forms of the type described above containing from about 0.1 mg to about 500 mg of the active ingredient of the present invention.

The tablets or pills of the novel composition can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.

The liquid forms, in which the novel composition of the present invention may be incorporated for administration orally or by injection, include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil as well as elixirs and similar pharmaceutical vehicles. Suitable dispersing or suspending agents for aqueous suspensions include synthetic natural gums, such as tragacanth, acacia, alginate, dextran, sodium carboxymethyl cellulose, methylcellulose, polyvinylpyrrolidone or gelatin.

Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for reconstitution with water or other suitable vehicles before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e. g., sorbitol syrup, methyl cellulose or hydrogenated edible fats); -emulsifying agents (e. g., lecithin or acacia); non-aqueous vehicles (e. g., almond oil, oily

esters or ethyl alcohol); preservatives (e. g., methyl or propyl p-hydroxybenzoates or sorbic acid); and artificial or natural colors and/or sweeteners.

For buccal administration, the composition may take the form of tablets or lozenges formulated in conventional manners.

The active compounds may be formulated for parenteral administration by injection, which include using conventional catheterization techniques or infusion. Formulations for injection may be presented in unit dosage form, e. g, in ampules, or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulating agents such as suspending, stabilizing, and/or dispersing agents. Alternatively, the active ingredients may be in powder form for reconstitution with a suitable vehicle, e. g., sterile pyrogen-free water, before use.

VIII. Transgenic Animals that Express DTH-initiating IgM.

Various transgenic animals may be created for research and drug screening purposes or as models that would permit the closer study of the role of DTH-initiating IgM.

Transgenic animals can be created that express DTH-initiating IgM once the cDNA for the DTH-initiating IgM is isolated using available methods. For example, anti-TNP IgM g and ic genes derived from anti-TNP B cell hybridoma Sp6 have been used to create transgenic mice (S. Rusconi et al., (1985) Science 314: 330-4). These mice express normal pentameric IgM with TNP specificity. Using cells from such mice may be helpful in adoptive cell transfers and reconstitution of normal B-1 cell deficient male CBA/N-xid mice (See for an analogous experiment M. Murakami et al., (1996) Immunol. 8: 3-9).

Additional mice can be created using the methodology of Rusconi et al., 1985.

Specifically, vectors may be constructed comprising K/R and y genomic DNA encoding a DTH-initiating IgM. Fertilized single cell eggs are then recovered from animals, such as immune competent CBA/N female mice after natural mating with CBA/N-xid males.

Approximately 1 picoliter of the vector construct containing 0.8 ug/ml of genomic DNA will be microinjected into the pronucleus of each egg.

Methods of the present invention, as described above or as discussed in the Examples below, may employ conventional molecular biology, microbiology and recombinant DNA techniques. Such techniques are commonly available in the literature.

See for example, Sambrook, Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL (Second Ed., Cold Spring Harbor Press, Cold Spring Harbor NY, 1989); DNA CLONING : A PRACTICAL APPROACH, vols. 1 and 2 (D. N. Glover ed., 1985); OLIGONUCLEOTIDE SYNTHESIS (M. J. Gait ed., 1984); NUCLEIC ACID HYBRIDIZATION (B. D.

Hames and S. J. Higgins eds., 1985); TRANSCRIPTION AND TRANSLATION (B. D. Hames and S. J. Higgins, eds, 1984); E. Harlow and D. Lane, ANTIBODIES: A LABORATORY MANUAL (Cold Spring Harbor Press, Cold Spring Harbor NY, 1988); and Ausubel et. a/., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Co. NY, 1995 to name a few.

The following working examples specifically point out preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure. Other generic configurations will be apparent to one skilled in the art.

EXAMPLES EXAMPLE 1 B-1 Cells Reconstitute Contact Sensitivitv inxidMice To determine the role of B-1 cells in inducing a DTH response, B-1 cells were administered to B-1 cell deficientxid mice, which lack the ability to mount a DTH response.

As discussed below, introduction of B-1 cells into xid mice results in the reconstitution of the ability to initiate a DTH reaction.

Methods.

Figure 2A. B-1 cell deficient xid and normal CBA/J mice were contact sensitized on day 0 with 5% PC1, and on day 4 were challenged to elicit CS by painting both ears with 0.4% PC1. Resulting CS ear swelling responses were measured 24 h later. A typical active sensitization result is shown, that was among 12 other and similar CS results. For reconstitution of xid mice, B-1 cells (CD5+, B220+) were prepared from peritoneal cells of normal CBA/J mice by depleting macrophages, and then purifying B-1 cells by FACS-

sorting (106, and 98% pure), and control B-1-deficient peritoneal cells from xid mice (3x106) were injected i. p. into CBA/J mice one day before sensitization. There separate reconstitution experiments were done with similar results. Peritoneal cells equal normal cells to control for B-1 cells.

Figure 2B. Ear extracts were prepared from xid mice and normal CBA/J mice 24 hour following antigen challenge as above, and assayed for macrophage chemotactic ability.

Perit. = peritoneal cells.

Figure 2C. 24 hour CS ear extracts were prepared and IFN-y was measured by ELISA.

Results. CS in B-1 cell deficient CBA/N-xid (xid) mice, that have X-linked immunodeficiency, were compared to normal CBA/J mice. The 24 hour CS ear swelling response was observed to be severely decreased in xid mice (Fig. 2A, Group B versus D), suggesting a requirement for B-1 cells in CS. To determine further if impaired CS in xid mice was due to absent B-1 cells, xid mice were reconstituted by FACS-sorted B-1 cell transfer from the peritoneal cavity (R. R. Hardy et al. (1994) Adv. Immunol. 55 : 297-339) of normal CBA/J mice, purified via surface co-expression of CD5 and B220. Impaired 24 hour CS ear swelling was reversed in xid mice by adoptive transfer of B-1 cells prior to sensitization (Fig. 2A, Group D versus E), but not by control peritoneal cells from xid mice, which lack B-1 cells (Fig. 1A, Group D versus F).

Importantly, defective CS in xid mice was verified biochemically by measuring chemotactic activity and IFNy in 24 hour CS ear extracts, elaboration of which was previously demonstrated to be C-dependent (Tsuji et al., 1997). Chemotactic activity due to chemokines, and IFNy in CS ear extracts were decreased significantly in xid mice 24 hours after ear challenge (Fig. 2B, Group B versus Group D and Fig. 2C, Group B versus D).

Further, impaired 24 hour chemotactic activity in xid mice was also restored by B-1 cell adoptive transfer compared to controls (Fig. 2B, Group D versus E versus F). The significant but partial reversal of CS ear swelling (Fig. 2A, Group E) may have been due to B-1 cell reconstitution intraperitoneally, but nearly complete reconstitution of CS-ear extract chemotaxis was observed (Fig. 2B, Group E). The possibility that impaired T cell function

in xid mice explained these finding was excluded by in vitro study. Thus T cells from immune xid mice stimulated by hapten-conjugated APC had normal in vitro proliferation and IFNy production (not shown). In summary, B-1 cells appear to be required for elicitation of 24 hour CS responses.

EXAMPLE 2 Contact Sensitivity-Complement Dependent Chemotactic Activity within Two Hours The role of the classical complement cascade, and specifically the roles of C5 and C5a, was examined using C5 deficient-and C5 normal-mice. After antigen challenge, macrophage chemotactic activity in these two mice strains was examined. The observed chemotactic activity could be inhibited using an anti-C5 antibody as discussed in greater detail below. The results demonstrate that T cell response in DTH is mediated in part by early state activation of the classical complement cascade at a very early stage in the initiation of a DTH reaction.

Methods.

Figure 3A. DBA, (C5-normal) and DBA2 (C5-deficient) mice were sensitized on day 0 and challenged on day 4 with 0.4% PC1. Ear extracts were prepared 2 hour after ear challenge for J774A. 1 chemotactic assay.

Figure 3B. Two hour CS ear extracts, and zymosan activated mouse serum (ZAMS) murine C5a chemotactic control were pre-incubated with rabbit anti-rat C5a serum, or control normal rabbit serum (NRS), and then J774 chemotaxis assayed.

Figure 3C. C5aR+'+ or CSaR-'~mice were injected i. p. with 3 ml thioglycolate, and PEC macrophages were prepared on day 4. Chemotaxis was performed similarly to that with J774A. 1 cells. C5aR deficient macrophages did not migrate against ZAMS (right panel).

Figure 3D. CBA/J mice were optimally immunized with 5% PCl or 3% OX. CS ear extracts were assayed for chemotactic activity.

Results. Early macrophage chemotactic activity was examined. Local chemotactic activity in ear extracts were observed as early as 1-2 hours after antigen challenge. C5a was

the main cause of this early chemotactic activity, in contrast to a predominance of chemokines at 24 hours. Thus, 2 hour chemotactic activity was absent in immunized and challenged C5-deficient mice (Fig. 3A), was largely neutralized by anti-C5a-antibody (Fig.

3B), and did not cause chemotaxis of peritoneal exudate macrophages from C5a receptor- deficient (C5aR~'~) mice (Fig. 3C). Residual 2 hour chemotactic activity was suggested to be due to chemokines like MCP-1, MIP-la, and IP-10, which was quantified by specific ELISA (not shown). However, the presence of this presumed chemokine activation was totally C5-dependent (Fig. 3A), suggesting that C5a is required for early production of chemokines. Importantly, elaboration of this C5a-dependent early chemotactic activity was antigen-specific, since only PCl-sensitized and PCl-challenged (Fig. 3D), or OX-sensitized and OX-challenged mice, had 2 hour activity in CS ears. Thus, antigen-specific early elaboration of C5a was required for local production of chemokines, that may lead to later recruitment of T cells to mediate the classical delayed aspect of CS.

The C5/C5a-dependency and antigen-specificity of chemotactic activity suggested that the classical C pathway was activated early in CS via specific antibody. Alternatively, T cell dependency was tested by employing TCRa'and TCR6'mice, and found normal chemotaxis in both (not shown). In summary, early chemotactic activity in 2 hour CS ear extracts was induced by antigen-specific and C5-dependent steps that were required for subsequent recruitment of Thl+ CS-effector cells to make IFN-y locally. These results indicate an important early and local role for C5a in CS-initiation in vivo. Therefore, CS- initiation may involve early complement (C) activation by B-1 cell-derived IgM antibody, leading to C5a generation. C5a likely then interacts with C5aR on tissue mediator cells, such as mast cells, to cause release of vasoactive mediators (P. W. Askenase,"Effector and Regulatory Mechanisms in Delayed-type Hypersensitivity (DTH), 1998) that enhance T cell recruitment.

EXAMPLE 3 B-1 Cells Are Essential for Early 2 Hour Activation of Complement in Contact Sensitivity

Given that B-1 cells were shown to be essential for initiating DTH (see Example 1), xid mice were tested to see at what point after exposure to allergen (after initial contact sensitization) B-1 cell activity was induced.

Methods.

Figure 4A. Normal CBA/J and B-1 cell deficient xid mice were contact sensitized on day 0 and ear challenged on day 4 to elicit CS by painting both ears with 0.4% PC1.

Resulting CS ear swelling responses were measured 2 h later. A typical result is shown that was selected from 12 similar 2 hour CS results. For reconstitution of xid mice, B-1 cells (CD5+, B220+) were prepared from peritoneal cells of normal CBA/J mice by depleting macrophages and then FACS sorting. B-1 cells (106 and 98% pure) and control peritoneal cells from xid mice (3x106) were injected i. p. into xid mice 1 day before sensitization.

Reconstitution was done three times with similar results.

Figure 4B. Ear extracts from PC1 immune and vehicle painted CBA/J mice or xid mice were prepared 2 hours after ear challenge and assayed for macrophage chemotactic activity.

Results. Interactions between B-1 cells and complement were examined to investigate the role of B-1 cells in early C5a-dependent CS-initiation. This was performed by measuring 2 hour ear swelling and chemotactic activity in 2 hour extracts of CS ears from xid mice. There was significant reduction of ear swelling (Fig. 4A, Group B versus D) and chemotaxis (Fig. 4B, Group B versus D) in compared to CBA/J mice. The decreased 2 hour ear swelling responses and chemotaxis in xid mice were reconstituted by adoptive transfer of FACS-sorted B-1 cells that were derived from normal CBA/J mice (Fig.

4A, Group D versus E and Fig. 4B, Group F versus G, respectively). In contrast, no change in ear swelling (Fig. 4A, Group D versus F) and chemotactic activity (Fig. 4B, Group F versus H) occurred after transfer of control peritoneal cells from xid mice. Again, significant (p<0.01) B-1 cell reconstitution of ear swelling was partial, perhaps due to the intraperitoneal transfer technique used, but the chemotaxis reconstitution was almost total.

-These results suggested that B-1 cells, that were placed in the peritoneal cavity, may be essential for the elaboration of early C5a chemotactic activity at CS sites, and for

subsequent ear swelling. Since the results above demonstrated that C5a was responsible for chemotactic activity in vitro, in may be concluded that B-1 cells were required for early complement activation at the antigen-challenged site.

EXAMPLE 4 Complement Involvement in Early Phase of Eliciting Contact Sensitivity and DTH Examination of activation of the classical complement cascade, the role B-1 cells play in activation of this cascade, and their combined roles in eliciting CS and DTH responses were examined using different strains of mice.

Methods. Specific pathogen-free female CBA/J, ICR, BDF,, C57B1/6 and B cell deficient C57Bl/6-Igh-6 (llMT) mice (6-8 weeks old) were obtained from the Jackson Laboratory (Bar Harbor, ME), and were rested at least one week before use.

Reagents. Picryl chloride (PCl) obtained from Nacalai Tesque, Inc. (Tokyo, Japan) was recrystallized twice as described R. F. Tsuji et al., (1997) J. Exp. Med. 186: 1015-26, and stored protected from light. Zymosan and CVF were purchased from Sigma Chemical Co. (St. Louis, MO). SRBC and anti-SRBC polyclonal Ab were products of Organon Teknika (Durham, NC). Trinitrobenzene sulfate sodium salt was obtained from Wako Chemicals (Osaka, Japan). Normal mouse serum drawn freshly from naive ICR mice via cardiac puncture was stored at-70°C, thawed, and then incubated with sterile 5 mg/ml zymosan at 37°C for 60 min. To activate complement, followed by centrifugation at 14,000 rpm for 10 min. Supernatant was used as zymosan activated mouse serum (ZAMS). sCRl was a gift from T Cell Sciences, Inc. (Needham, MA). Mouse anti-murine C5 mAb (BB5. 1) and isotype (IgG,)-matched mouse anti-human C8 mAb (135.8) were purified by protein A column from ascites generated in nude mice injected i. p. with appropriate hybridoma cells. These reagents are also discussed in the Examples below.

CSResponses. CBA/J mice were contact sensitized by topical application of 100 ul of 5% PCl in absolute ethanol and acetone (4: 1) applied to the shaved chest and abdomen.

Four 4ays (d) later, CS was elicited by painting both ears via topical application of 10 al of a low challenge dose of 0.4% PCl in acetone and olive oil (1: 1), compared to conventionally employed high dose of 0.8% PCI, unless otherwise noted, since an effect of complement

alteration on contact sensitivity (CS) usually was observed with a moderate dose of challenge antigen (S. J. Piddlesen et al., (1994) J. Immunol. 152: 5477-5484). B6 background mice are hyporeactive to PCl CS and thus were contact sensitized twice on days 0 and 1. Resulting thickness of antigen-challenged ears was measured on days 4 or 7 in the case of B6 background mice with a dial caliper (Ozaki MFG Co., Tokyo, Japan) before challenge at 2 and 24 h after challenge. Increased ear thickness was expressed as mean SE.

Results. When soluble recombinant C receptor-1 (sCRl) was injected systemically 30 min before antigen challenge in immunized CBA/J mice, both early (2-h) and late (24-h) components of CS ear swelling responses were significantly reduced (Fig. 5A, group D versus C). Since both 2-and 24-h ear swelling responses were affected by sCRl treatment, the decreased 24-h response may have been due to effects of sCRl on the early 2-h events of CS, which are required for elicitation of the 24-h ear swelling responses. Thus, when sCRl was given 3 hour after antigen challenge, and thus after the required early events, there was no effect on 24-h ear swelling responses (Fig. 5A, group E), suggesting that required complement acted early, before 3 hours, in the elicitation of CS.

To determine whether another system of DTH response was similarly dependent on complement acting in the early phase, BDF, mice were immunized i. v. with TNP-SRBC and then hind footpads were challenged with TNP-SRBC on day 5. Again, sCRl treatment 30 minutes before (Fig. 5B, group D versus C), but not 3 hours after antigen challenge (group E) inhibited DTH. Taken together, these results suggested that classical 24-h CS and 24-h DTH, in two different strains of mice, actually dependent on complement-mediated early events (R. F. Tsuji et al., 1997).

CS and DTH are Inhibited by Local Depletion of Complement The role of complement with respect to the induction of DTH and CS was examined using an agent (e. g., cobra venom factor) that depletes local complement. Using CVF or

similar agents, CS and DTH were inhibited indicating that localized complement played a role in mounting CS and DTH responses.

Methods.

For mice, reagents and CS responses, refer to Example 4. Cobra venom factor (CVF) was used to deplete complement and to determine whether CS could be inhibited.

CVF (10 ug) in PBS was injected i. p. twice per day on days 1 and 2 after immunization.

This CVF treatment regiment depleted serum of C3 and C5, because C3 was undetectable at 24 and 48 h after CVF treatment, and C5 was decreased to 12.5 and 25% of normal, respectively. Platelet counts of saline versus CVF-injected mice were not different 48 h after the final CVF injection. When sensitized mice were challenged with a conventional high dose of 0.8% PCI, there were no significant differences between saline-and CVF- injected groups (79% of saline control, P = 0.24). However, when the eliciting dose of PCl was decreased to 0.4% to elicit weaker CS, CVF diminished 24-h ear swelling responses (48% of saline control, P<0. 05).

TNP-SRBC-induced DTHResponses. DTH against TNP-SRBC (R. F. Tsuji et al., (1992) Biosci. Biotechnol. Biochem. 56: 1686-89; P. W. Askenase et al., (1975) J. Ex. Med.

141: 697-702) was induced in BDF, mice with TNP-SRBC conjugated by incubating SRBC (5x108 cells/ml) with 10 mM trinitrobenzene sulfate sodium salt in phosphate buffered saline (PBS). For immunization, mice were injected i. v. with 105 TNP-SRBC on day 0. On day 5, both footpads were challenged s. c. with TNP-SRBC (2x108 cells in 40 ul of PBS) to elicit DTH, and footpad thickness was measured before and 24 h after being challenged, as described above.

Results. CVF was locally injected into ears 48 hours before local antigen challenge, since CVF injected alone s. c. into ears caused no swelling 48 h later. Both 2-and 24-h ear swelling responses were decreased significantly by local administration of CVF in CBA mice (Fig. 6A, Group D versus C). In similar DTH experiments which used BDF, mice, local preinjection of CVF into footpads 48 h before antigen challenge also decreased DTH against TNP-SRBC (Fig. 6B, Group D versus C). Thus, these experiments showed that C

depletion with local CVF could inhibit CS and DTH and pointed to a role of local C activation in CS initiation. See R. F. Tsuji et al. (1997).

EXAMPLE 6 CS Inhibition Systemically or Locally by anti-C5 Monoclonal Antibody In order to further elucidate the role of complement in DTH reactions, an anti-C5 specific monoclonal antibody was used to deplete complement component C5. This antibody was found to inhibit a CS response both when the antibody was administered to the mice systemically and administered locally in the region of skin which received the allegen stimulus. The evidence discussed further below indicates that C5 acts early at the site of antigen stimulus.

Methods. See R. F. Tsuji et al. (1997) and the methods described in the Examples above.

C3 and C5 Titration. C3 and C5 activity were titrated by measuring Ab-dependent complement-mediated hemolysis of SRBC by C3-or C5-deficient human serum (W. H.

Churchill, et al., (1967) J. Exp. Med. 125: 657-72). In brief, SRBC (5xl08/ml) were coated with antibody in rabbit antiserum to SRBC. 100 gl of each serum sample or ear extract was mixed with 100 pl of human C3-or C5-deficient serum diluted 1: 20 with gelatin veronal buffer (pH 7.4), and with 50 ul of antibody-coated SRBC (5xlOg), followed by incubation for 60 min at 37°C. Then hemolyzed supernatants were collected and OD measured at 405 nm.

In Vitro Evaluation of Chemotactic Activity in Ear Extracts. Ears that were the site of CS reactions were removed at the base and three punch biopsies were collected from the distal portion. Biopsies were 12.5 mm2 and were frozen rapidly in liquid N2. Either the whole ear or three punch biopsies per ear were extracted in 300 or 500 ul cold PBS, with a tissue homogenizer (Biospec Products, Racine, WI) on ice, followed by centrifugation at 14,000 rpm for 15 min to obtain the supernatant of extracts. Protein concentration was determined by BCA-protein determination kit (Pierce, Rockford, IL). C5 content was evaluated by C5-titration assay.

Chemotactic activity was determined with the J774A. 1 murine macrophage cell line that responds to C5a. The cells were maintained in RPMI 1640 supplemented with 10% FBS (GIBCO BRL, Gaithersburg, MD), 25 mM Hepes, 100 U/ml penicillin, and 100 lg/ml streptomycin. The J774A. 1 cells were washed three times with RPMI 1640 without fetal bovine sera (FBS) (RPMI-gelatin) (2-5x106 cells/ml). Triplicate ear extract samples were diluted with RPMI gelatin and were placed in lower wells of 96-well chemotaxis chambers (NeuroProbe Inc. Cabin John, MD). Two-or four-fold diluted ear extracts were used according to the previous observation that J774A. Cells migrated in a dose-dependent fashion, in up to eight-fold dilution. No proteinase inhibitors were added due to their inhibitory effect on chemotaxis.

50 u. I of J774A. 1 cells was added to the upper wells, allowing cells to migrate through a polyvinyl pyrrolidone-free polycarbonate filter with 5 um pores at 37 °C for 4 h in a humidified air atmosphere, containing 5% CO2. The J774A. 1 cells attached firmly to the other surface of the filter, and then were fixed and stained with Diff Quick solution (Wako Chemicals). Migrated cells were counted at five different filter spots of each well. Among the complement components, J774A. 1 chemotaxis was specifically mediated by C5a, since there was no chemotaxis against ZAMS prepared from C5-deficient serum. J774A. 1 migration was also verified not to be due to chemokinesis by showing that addition of ear extracts to the upper wells where the cells were loaded, caused diminished migration. Thus the number of migrated J774A. 1 cells without ear extracts in the upper wells was 103.0 + 2.8 versus 27.9 2.7 (P<0. 001) with ear extracts added to the upper wells.

Results. When anti-C5 monoclonal antibody was injected i. p. 24 and 4 hours before antigen challenge, a negligible amount of C5 remained in serum, as detected by a C4 in vitro titration assay. This systemic treatment with anti-C5 mAb significantly inhibited both 2-and 24-h ear swelling responses (Fig. 7A, group D versus C). Importantly, anti-C5 was also injected locally to deplete C5 at the site of CS. This resulted in decreases in both the 2- and 24-h ear swelling responses (Fig. 7B, group D versus C). Taken together, these results indicate that complement and particularly C5, acting early at the local CS site are important for mediating elicitation of CS responses.

EXAMPLE 7 Anti-Complement Treatment Decreased Chemotactic Activity As the classical complement cascade, and specifically the C5a component of this cascade, were observed to be required in the early steps of mounting DTH reaction, anti- complement treatment of mice was performed to determine whether complement played a role in chemotaxis.

Methods. See R. F. Tsuji et al. (1997) and in the methods discussed in the Examples above.

Results. To determine the mechanism of complement participation in CS, CS ears were extracted as discussed in the previous examples, which were obtained 24 hours after antigen challenge with cold PBS. Total extractable protein was increased in CS, paralleling macroscopic ear swelling measurements 24 h after antigen challenge (Fig. 8A). C5 activity in a complement-function assay increased significantly in CS responses (Fig. 8B) but not in controls.

Local macrophage chemotactic activity was measured in vitro using migrating J774A. 1 macrophages (as described in Example 6). Increased macrophage chemotactic activity in the CS ear extracts was compared to non-immune controls (see Fig. 9A and 9B respectively). To investigate complement dependency of this chemotactic activity found in the local ear site, mice were treated systematically with anti-C5 monoclonal antibody resulting in a significant decrease in chemotactic activity (Fig. 9A, right). Furthermore, systemic treatment with sCRl also diminished macrophage chemotactic activity in CS ears (Fig. 9B, right), confirming that locally extracted macrophage chemotactic activity was complement dependent, suggesting that either C5a or other complement regulated factors were responsible for the macrophage chemotactic ability.

EXAMPLE 8 Cell Infiltration In Vivo was Inhibited bv Anti-C5 Monoclonal Antibody

Given that C5 was found to be necessary for the initiation of CS in Example 7, the effects of anti-complement agents on cell infiltration, macrophage chemotactic activity and IFN-y production were next examined.

Methods. See R. F. Tsuji et al. (1997) and the methods described in the above Examples.

Histological Evaluation of Cell Infiltration in CS Ear Responses. 5 um sections of formalin-fixed, paraffin-embedded ear tissue were stained with hematoxylin and eosin.

Semiquantitative evaluation of infiltrating cells per area (Figure 11: 1, no infiltration; 2, slight infiltration; 3, modest infiltration; 4, strong infiltration), and also formation of intraepidermal abscesses (Figure 11 : 1, not seen; 2, few seen; 3, many seen) were determined. Histology in ears was read by an observer blinded as to the experimental design and assigned a numerical grade, from which group means and SE were calculated.

In Vitro Quantitative Measurement ofIFN-y in Ear Extracts. A quantitative sandwich ELISA for IFN-y used two specific mAbs (PharMingen, San Diego, CA). In brief, wells were coated overnight with 2 pg/ml capture Ab (R4-6A2) in 0.1 M NaHCO3 (pH 8.3) at 4°C. After blocking with 1% BSA in PBS at room temperature for 2 h, samples and dilutions of standard recombinant mouse IFN-y (Genzyme Corp., Cambridge, MA) were added and incubated for 1 h at room temperature. Since there was no significant difference in IFN-y content between ears extracted with or without proteinase inhibitor cocktail (which included PMSF, EDTA, leupeptin, E-64, and pepstatin A), cold PBS alone was used to extract ears. Then, 1 Zg/ml of the other biotinylated anti-IFN-y mAb (XMG1. 2), and 1: 3,000 diluted horseradish peroxidase-conjugated streptavidin (Vector Labs., Burlingame, CA), were added to probe fore IFN-y. Peroxidase substrate (TMB tetramethylbenzidine) and TMB-one-component stop solution (Kirkegaard & Perry Labs, Inc. Gaithersburg, MD) were used for color development at 450 nm.

Statistics. Statistics in this example and in the previous examples were performed using the two-tailed Student's t test and P<0.05 was taken as a level of significance. Each experimental group consisted of four to six mice.

Results. To determine whether cell infiltration, as well as macrophage chemotactic activity (Fig. 9) and IFN-y production were decreased by C5 depletion, the histology of CS ear sections was examined. Decreased local cell infiltration in CS ears (Fig. 1 OD versus 10C) and also inhibited intraepidermal abscesses formation (Fig. 10F versus 10E) were noted in mice treated with anti-C5 mAb. Semi-quantitative histologic observation was carried out blindly and indicated that C5 depletion resulted in significant decreases in leukocyte infiltration of CS (Fig. 11A), and in decreased formation of intra epidermal abscesses (Fig. 11B).

EXAMPLE 9 Methods of Purifying IgM and IgM Fragments DTH-initiating IgM pentamers and fragments were purified to determine whether purified DTH-initiating IgM, as well as fragments thereof, could induce responses similar to what had been observed in previous examples. Specifically, it was examined whether purified DTH-initiating IgM pentamers could reconstitute a DTH response in xid mice Methods. The 32.17 IgM (or other IgM producing hybridoma such as 13.4) was grown and the supernatant from the cells collected. The supernatant was tested by ELISA to be positive for IgM. The supernatant was the purified using chromatography on an antigen affinity column using trinitrophenol-benzene sulfonic acid (TNPBSA) Sepharose 4B. The eluate was then dialyzed against PBS. FPLC gel filtration chromatography was then utilized to further purify the IgM and to concentrate it. The eluate was then applied to a Superose 6 column. The IgM thus isolated and purified was further tested using an IgM specific ELISA and on a SDS-PAGE gel under non-reducing conditions.

The 32.17 pentameric IgM antibody thus obtained was administered to CBA/N-xid mice as described in the previous examples.

Results. In Figure 12, after sensitization with PC1 or carrier only, the mice were reconstituted with purified 32.17 (as described above) IgM antibody (Group E). The -controls are Groups A, B (a positive control in CBA/J mice), and C. Group D reflects a control which used the same carrier as used for Group E. The immune B-1 cell deficient xid

mouse does not elicit a delayed hypersensitivity T cell ear skin allergy response compared to normal CBA/J mice (Group B). Administration of the purified IgM pentamer reconstituted the T cell response in the B-1 cell deficient xid mice (Group E).

EXAMPLE 10 Reconstitution bv IgM Fragments of T Cell Ear CS Reaction in CBA/N-xid B-1 Cell Deficient Mice and bv Antigen Affinity Purified IgM To further elucidate the role of B 1 cells in the initiation of DTH, DTH-initiating IgM fragments (halfmers and individual heavy and light chains) were used to reconstitute CS reaction in xid mice which lack B-1 cells and therefore do not naturally produce DTH- initiating IgM antibodies or antibody fragments.

Methods. Intact 13.4 IgM pentamers were was obtained from B cell hybridoma 13.4. The 13.4 IgM was purified using an anti-IgM affinity chromatography column.

Pentameric IgM was fragemented into halfmers and individual light and heavy chains using DTT and iodoacetiimide to reduce and alkylate the IgM into fragments. Under harsh reducing conditions (20 mM DTT) or mild conditions (0.25 mM DTT), intact IgM pentamer was reduced and alkylated to produce separated DTH-initiating IgM heavy and light chains (Figure 13, Groups E and F) or halfmers, respectively (Figure 13, Groups G and H). The IgM halfmer is joined heavy and light chains.

20 ug (Figure 13, Groups C, E, and G) or 100 llg (Figure 13, Groups D, F, and H) were administered to CBA/N-xid B-1 cell deficient mice. Groups A and B are the controls where only saline was administered to the mice. In Groups C and D, the intact, purified 13.4 IgM pentamer was administered to the animals. Methods of administration and detection of inhibitory response is as described above.

FACS sorting analysis (Figure 14) was performed using enriched mast cells of whole spleen populations. The upper panel represents intact 13.4 IgM pentameric antibody.

The middle panel represents isolated and separated heavy and light chain portions of the 13.4 IgM pentamer described above. The lower panel reflects data using the isolated 13.4 halfmer (heavy and light chain dimer).

ELISA analysis (Figure 15) of the capability of intact pentameric IgM versus IgM fragments to bind antigen was performed by binding TNP-BSA to an ELISA plate at standard concentrations using methods as described in Example 8. Reactions were allowed to proceed and the results read at 450 nm absorbance.

Results. As reflected in Figure 13, IgM pentamer (Groups C and D) was able to restore the CS response more fully than the 13.4 IgM halfmer (Groups G and H) or separated IgM heavy and light chains (Groups E and F) at both the 20 ug and 100, ug dosages. This response is clearly greater than that observed for the control. **P indicates a statistical response of P<0. 05; *P indicates P<0. 001.

In Figure 14, the IgM pentamer, halfmer and fragments were tested using FACS- sorting to determine the ability of the intact and fragment immunoglobulin to bind to target cells (e. g., mast cells). The IgM fragments are again shown to bind to target cells, however the fragments do not bind as well as the intact 13.4 IgM pentamer (Fig. 14, top panel).

Specifically the halfmer (Fig. 14, bottom panel) had one tenth to one fifteenth (30%) the binding capability of the intact 13.4 IgM pentamer. The isolated and separated heavy and light chains (Fig. 14, middle panel) had one thirtieth (4%) the binding capacity as compared to the intact 13.4 IgM pentamer.

The antigen binding affinity of the reduced, alkylated 13.4 IgM fragments was also tested via ELISA assay. TNP, the recognized antigen target of the 13.4 IgM antibody, was used at various standard concentrations in the ELISA assay to assess the binding affinity of the intact pentameric IgM, versus the fragments thereof. The results correspond to the antigen binding capability previously observed: intact IgM pentamer > IgM halfmer > isolated and purified heavy and light chains. These results are reflected in Figure 15.

EXAMPLE 11 Asthma Initiation bv a Monoclonal B-1 Cell IgM Antibody In an effort to determine whether an asthmatic response could be induced using DTH-initiating IgM antibodies administered as described above, antigen affinity purified IgM (32.17 anti-TNP) was administered to mice.

Methods. The 32.17 antigen (hapten) affinity purified IgM was given i. v. at a dose of 100 ug per mouse, and subsequently with 10 pg i. v. per recipient mouse. Then the mice were challenged via their airways with water soluble TNP hapten, TNBSA (trinitrobenzene sulfonic acid, which is also known as picryl sulfonic acid). Airway hyper-reactivity was measured in live, anesthetized and ventilated mice 48 hours after TNBSA challenge.

Results. The results in Figure 16 clearly show an elevated methacholine challenge dose response indicating an increased airway hyper-reactivity in TNP antigen challenged mice which were administered 32.17 IgM anti-TNP antibody (p) (see Figure 16). This hyper-reactivity was not present in animals that were TNBSA airway challenged alone (A), or in recipients of IgM alone that were not challenged (1 : 1), nor in control responses of untransferred, and unchallenged normals (O).

EXAMPLE 12 Use of DTH-initiating IgM pentamers and fragments to identify agents that modulate DTH orCS Purified IgM or IgM fragments (e. g., halfiners or isolated heavy and light chains), as described above, can be bound to a solid substrate and used to identify agents that bind to DTH-triggering immunoglobulins and are therefore candidates for therapeutic formulations that might also modulate DTH or CS.

Peptide compounds as well as small molecules from chemical libraries can be reacted with DTH-initiating IgM pentamers or fragments. After allowing sufficient time for possible binding to occur, the substrate can be washed to remove unbound reagents.

Complexed or bound peptides or reagents are then detected by methods well known in the art. Purified peptide test compounds can also be coated directly onto plates for use in the aforementioned drug screening techniques. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support (see P. R. Hawkins et al., (1998) U. S. Patent No. 5,712,115).

In one format, an assay similar to an antigen capture assay is used to identify agents that bind to DTH-initiating IgM antibodies or antibody fragments. Nitrocellulose reverse

dot blot assays, as described in Harlow and Lane (1988), or ELISA assays utilizing 96 well polyvinylchloride (PVC) plates may be employed to detect agents capable of binding to the IgM antibody or antibody fragment.

Briefly, 50 p1 of affinity purified DTH-initiating IgM or fragments thereof is added to the wells of a 96 well PVC plate in 20 pl/ml of PBS per well. The plate is incubated for 2 hr at room temperature or overnight at 4°C. 50 mM carbonate buffer (pH 9.0) can be substituted for PBS. The plates are washed twice with PBS. 200 ul of 3% BSA/PBS with 0.02% sodium azide is then added to each well. The plate is then incubated for at least 2 hours at room temperature and washed three times with PBS to remove excess reagents. 50 pi of 3% BSA/PBS with 0.02% sodium azide is then containing 50,000 cpm of'zsI-labeled agent to be tested is added per well. The plate is incubated for 1 hour at room temperature and then washed with PBS until the counts in the wash buffer approach background levels.

The PVC plate is then exposed to X-ray film or placed in a gamma counter and the counts analyzed. Radioisotopes can be substituted for a horseradish peroxidase type reaction and color changes can be detected using an ELISA reader as discussed in examples above.

Although the present invention has been described in detail with reference to examples above, it is understood that various modfications can be made without departing from the spirit of the invention. All references, articles, texts and patents referred to above are hereby incorporated by reference in their entirety.