Protection against metastasis by immunization with an allogeneic lymphocyte antigen

Q5 antigens are expressed on the surface of various experimental murine tumor cells. They share partially common antigenicity with Qa-2 alloantigens expressed on normal lymphocytes. For that reason we tested the immunoprotection by anti-Qa-2 immunization of mice against a Q5+ tumor. Nerve fibrosarcoma (NSFA) tumor, which specifically develops metastasis in the lung, has been reported to be poorly immunogenic. However, expression of the Q5 antigen was evident on the surface of NFSA cells. After immunizing (C3H/He x B6.K1)F1 (Qa-2-) mice with B6 (Qa-2+) lymphocytes, the protection against the proliferation of the semi-syngeneic NFSA tumor was examined First, immunization of normal mice induced resistance to NFSA cell transplants. Second, when the tumor cells were transplanted to the hind foot of a mouse and the resulting tumor was removed by amputating the leg, the mice were protected against the development of lung metastasis after immunization by intraperitoneal inoculation of B6 cells 3 days after tumor removal. Immunization with attenuated NFSA cells in this system failed to protect the mice from lung metastasis. On the other hand, inoculation of the mice with B6 cells without removal of the original tumor on the foot showed little effect on the progression of the tumor. Thus, cytotoxic T lymphocytes (CTL), which seemed to be present in an inactive form in the mice from which the tumor had not been removed, were induced in the mice after the removal of the major tumor followed by immunization with B6 lymphocytes. The induction of CTL by the immunization was suppressed in mice bearing large tumors. Cells stimulated by the tumor antigen seemed to be involved in the suppression. It was also shown that the Q5 antigen is the direct recognition target of the CTL since the activity of Q5-specific CTL clones in lysing tumor cells was inhibited by a monoclonal antibody specific for the Q5 antigen. In contrast to immunization with attenuated tumor cells, our novel allogeneic lymphocyte immunization procedure offers high CTL activation, by-passing the induction of T cell unresponsiveness.     

Protection against metastasis by immunization with an allogeneic lymphocyte antigen

Q5 antigens are expressed on the surface of various experimental murine tumor cells. They share partially common antigenicity with Qa-2 alloantigens expressed on normal lymphocytes. For that reason we tested the immunoprotection by anti-Qa-2 immunization of mice against a Q5+ tumor. Nerve fibrosarcoma (NSFA) tumor, which specifically develops metastasis in the lung, has been reported to be poorly immunogenic. However, expression of the Q5 antigen was evident on the surface of NFSA cells. After immunizing (C3H/He x B6.K1)F1 (Qa-2-) mice with B6 (Qa-2+) lymphocytes, the protection against the proliferation of the semi-syngeneic NFSA tumor was examined First, immunization of normal mice induced resistance to NFSA cell transplants. Second, when the tumor cells were transplanted to the hind foot of a mouse and the resulting tumor was removed by amputating the leg, the mice were protected against the development of lung metastasis after immunization by intraperitoneal inoculation of B6 cells 3 days after tumor removal. Immunization with attenuated NFSA cells in this system failed to protect the mice from lung metastasis. On the other hand, inoculation of the mice with B6 cells without removal of the original tumor on the foot showed little effect on the progression of the tumor. Thus, cytotoxic T lymphocytes (CTL), which seemed to be present in an inactive form in the mice from which the tumor had not been removed, were induced in the mice after the removal of the major tumor followed by immunization with B6 lymphocytes. The induction of CTL by the immunization was suppressed in mice bearing large tumors. Cells stimulated by the tumor antigen seemed to be involved in the suppression. It was also shown that the Q5 antigen is the direct recognition target of the CTL since the activity of Q5-specific CTL clones in lysing tumor cells was inhibited by a monoclonal antibody specific for the Q5 antigen. In contrast to immunization with attenuated tumor cells, our novel allogeneic lymphocyte immunization procedure offers high CTL activation, by-passing the induction of T cell unresponsiveness.     

An MHC class Ib-restricted TCR that cross-reacts with an MHC class Ia molecule

TCR transgenic 6C5 T cells recognize an insulin B chain epitope presented by the nonclassical class I MHC molecule, Qa-1(b). Positive selection of these T cells was shown previously to require Qa-1(b). Despite dedicated specificity for Qa-1(b), evidence presented in the current study indicates that 6C5 T cells can cross-recognize a classical class I molecule. Clonal deletion was observed unexpectedly in 6C5.H-2(bxq) mice, which do not express I-E MHC class II molecules and thus should not be subject to superantigen-mediated negative selection. 6C5 T cells were observed to respond in vivo and in vitro to spleen cells from allogeneic H-2(q) mice, and specificity was mapped to D(q). Evidence was obtained for direct recognition of D(q), rather than indirect presentation of a D(q)-derived peptide presented by Qa-1(b). Polyclonal CD8(+) T cells from class Ia-deficient K(b)D(b-/-) mice reacted in vitro to allogeneic spleen cells with an apparent frequency comparable to conventional class Ia-restricted T cells. Our results provide a clear example of a Qa-1-specific TCR that can cross-react with a class Ia molecule and evidence supporting the idea that this may be a common property of T cells selected by class Ib molecules.     

Syngeneic tumor immunizations produce Qa antibodies. Discovery of a new Qa antigen,Qa-6

BALB/cBy (Qa-2^–) mice injected with the syngeneic tumor, ORA I-a (Qa-2⁺), produced antibodies to Qa-2 and a newly discovered antigen, Qa-6. Specific antisera against Qa-6, in the presence of complement, lyses approximately 40% of lymph-node lymphocytes and splenocytes. Strain distribution analyses indicate that Qa-6 is specified by a gene within the TL subregion of the major histocompatibility complex. Thus, Qa-6 is the third member of the Qa/TL subset of cell surface antigens which is anomalously expressed on certain tumor cells. This finding suggests that the Qa and TL molecules may have a unique, functional role on the cell surface.     

Qa alloantigen expression on functional T lymphocytes from spleen and thymus

The cell-surface expression of the class I alloantigen Qa-2 was analyzed on resting and activated spleen and thymus cells using cytotoxic elimination and immunofluorescence and flow cytometry. Spleen cells activated by mitogens or alloantigen were homogeneously positive for cell surface Qa-2, but activated splenic T cells expressed only about one-third as much Qa-2 per cell as did nonstimulated T cells. These data correlated with the ability to perform cytotoxic elimination with Qa-2-specific monoclonal antibodies (mAbs) in that cytotoxic T lymphocyte (CTL) activity was completely abrogated by pretreatment of spleen cells prior to in vitro culture but was only partially eliminated by treatment of CTL effectors. Qa-2-positive cells constituted only a small subpopulation of fresh normal thymocytes, but were enriched (>40% positive) among cortisone-resistant thymocytes (CRT). These Qa-2-positive CRT contained mature thymocytes as defined by Ly phenotype Ly-2^–, Ly-1^hi. When normal thymocytes were treated with Qa-2-specific mAb and complement prior to in vitro sensitization for generation of allogeneic CTL, CTL activity was completely abrogated despite the fact that the fraction of cells eliminated were undetectable as assessed by cell recovery. CTL effectors from alloantigen-stimulated thymocytes were also susceptible to cytotoxic elimination with Qa-2-specific mAb. These data suggest that the Qa-2 molecule may serve not only as a marker on resting and activated peripheral T cells, but also as a unique marker for functionally mature T cells in the thymus.     

Phenotypic heterogeneity of antisyngeneic tumor killer cells (ASTK) generated in allogeneic mixed lymphocyte reactions

By using monoclonal antibodies to Thy-1, Lyt-2, and Qa-5 differentiation antigens, we demonstrated a heterogeneity of cytotoxic cells developed in allogeneic mixed lymphocyte responses that lyse tumor cells syngeneic with the responder cells. There are minimally two Thy-1+ populations, one of which is Lyt-2+ and the other Lyt-2-. There is probably also a Thy-1- population. Most of the Lyt-2- tumor killer cells are Qa-5+, and most of the Lyt-2+ tumor killer cells are Qa-5-.     

Chimeric Antigen Receptor (CAR)-Specific Monoclonal Antibody to Detect CD19-Specific T Cells in Clinical Trials

Clinical trials targeting CD19 on B-cell malignancies are underway with encouraging anti-tumor responses. Most infuse T cells genetically modified to express a chimeric antigen receptor (CAR) with specificity derived from the scFv region of a CD19-specific mouse monoclonal antibody (mAb, clone FMC63). We describe a novel anti-idiotype monoclonal antibody (mAb) to detect CD19-specific CAR⁺ T cells before and after their adoptive transfer. This mouse mAb was generated by immunizing with a cellular vaccine expressing the antigen-recognition domain of FMC63. The specificity of the mAb (clone no. 136.20.1) was confined to the scFv region of the CAR as validated by inhibiting CAR-dependent lysis of CD19⁺ tumor targets. This clone can be used to detect CD19-specific CAR⁺ T cells in peripheral blood mononuclear cells at a sensitivity of 1∶1,000. In clinical settings the mAb is used to inform on the immunophenotype and persistence of administered CD19-specific T cells. Thus, our CD19-specific CAR mAb (clone no. 136.20.1) will be useful to investigators implementing CD19-specific CAR⁺ T cells to treat B-lineage malignancies. The methodology described to develop a CAR-specific anti-idiotypic mAb could be extended to other gene therapy trials targeting different tumor associated antigens in the context of CAR-based adoptive T-cell therapy.     

Cloned "anomalous" killer cells derived from allogeneic mixed leukocyte culture

In addition to allospecific cytotoxic lymphocytes, cytolytic effector cells capable of killing a broad range of targets are generated during mixed leukocyte culture (MLC). These cells, which have been previously called anomalous killer cells, are a distinct functional subset separate from natural killer cells or allospecific cytotoxic lymphocytes but display many characteristics of lymphokine-activated killers. In order to isolate anomalous killer cells for detailed analysis, we generated the cytolytic effectors from an allogeneic MLC using heat-inactivated stimulators. This treatment of the stimulator population abrogated the generation of classical allospecific cytotoxic lymphocytes but allowed the generation of anomalous killer cells which were subsequently cloned via limiting dilution. The clones derived by this method displayed the functional properties of anomalous killers seen in bulk MLCs. The clones demonstrated potent cytolytic activity against both NK-sensitive and NK-resistant tumor targets in vitro and also suppressed tumor growth in vivo. Ultrastructural studies revealed features similar to those of cloned antigen-specific cytolytic cells and clones with NK-like function. The cells expressed surface glycoproteins associated with both NK and T lymphocytes including Thy-1, Ly-2, T200, Qa-5, asialo GM1, and the antigens defined by the NK alloantisera NK-2.1 and NK-3.1. These cells may play an important role during early phases of the immune response, since cytolytic cells of broad specificity may protect the host until classical cytotoxic lymphocytes with restricted specificity are generated.     

Use of the allogeneic TCR repertoire to enhance anti-tumor immunity

It is well established that antigen-specific T lymphocytes can inhibit tumor growth in humans and in mice, leading to complete tumor elimination in some cases. However, in many cases T cell immunity is unable to successfully control tumor progression. Since tumors are derived from normal tissues, most antigens are shared with normal tissues, although expression levels are usually elevated in malignant cells. Nevertheless, low-level expression in normal cells can be sufficient to render autologous T cells tolerant and thus unable to mount effective immune responses against tumors. Here, we review how allogeneic T cells can be used to isolate T cells that effectively recognise and kill tumor cells, but not normal cells with low level of antigen expression. The TCR of allogeneic T cells can be introduced into patient T cells to equip them with anti-tumor specificity that may not be present in the autologous T cell repertoire.     

Utilization of leucine methyl ester for the generation of hybridomas producing monoclonal antibodies specific to tumor-associated antigens

In an attempt to obtain monoclonal antibodies specific to tumor-associated antigens. C3H/He mice were immunized with syngeneic MM2 tumor cells, and the primed spleen cells were fused with P3-X63-Ag8.653 myeloma cells. The outgrowth of hybridomas, however, was extremely low and monoclonal antibodies were not obtained. The reason for the low hybridoma growth was studied. It was found that MM2 cells used as the immunogen, the fusion partner myeloma cells and the resulting hybridomas shared at least one tumor-associated antigen, namely Q5 antigen. Because of this common antigen, cytotoxic cells, presumably cytotoxic T lymphocytes, which were lytic to the hybridomas, were induced during the culture for generation of the hybridomas. Removal of lysosome-rich cells, including cytotoxic T lymphocytes, from the primed spleen cells before the fusion by treatment with leucine methyl ester, a lysosomotropic agent, drastically improved the outgrowth of hybridomas. By this method, seven stable hybridoma clones producing monoclonal antibodies specific to tumor-associated antigens were obtained. Two of the seven clones were found to secrete monoclonal IgM species, which reacted with the extra-cellular region of the Q5 antigen. This procedure will be an option when production of monoclonal antibodies specific to cell-surface antigens is intended and outgrowth of hybridomas is unexpectedly low.     

Analysis of Qa-2 antigen expression by preimplantation mouse embryos: possible relationship to the preimplantation-embryo-development (Ped) gene product

The preimplantation-embryo-development (Ped) gene, a gene that controls the cleavage rate of preimplantation mouse embryos, maps to the Qa-2 subregion of the mouse major histocompatibility complex (MHC). A highly sensitive enzyme-linked immunosorbent assay (ELISA) procedure was used to detect Qa-2 antigens on mouse embryos. The use of a monoclonal antibody specific for Qa-2 antigens showed that Qa-2 antigens were present on oocytes, 2-cell, 8-cell, and blastocyst-stage embryos, with the greatest expression found on blastocysts. Expression of Qa-2 antigens by the embryos correlated completely with Ped gene phenotype. Those embryos expressing the fast Ped allele showed the presence of Qa-2 antigens (Qa-2a mice), whereas those embryos expressing the slow Ped allele showed the absence of Qa-2 antigens (Qa-2b mice). It is hypothesized that the Qa-2 antigen may be the Ped gene product.     

Virus-specific T cells engineered to coexpress tumor-specific receptors: persistence and antitumor activity in individuals with neuroblastoma

Cytotoxic T lymphocytes (CTLs) directed to nonviral tumor-associated antigens do not survive long term and have limited antitumor activity in vivo, in part because such tumor cells typically lack the appropriate costimulatory molecules. We therefore engineered Epstein-Barr virus (EBV)-specific CTLs to express a chimeric antigen receptor directed to the diasialoganglioside GD2, a nonviral tumor-associated antigen expressed by human neuroblastoma cells. We reasoned that these genetically engineered lymphocytes would receive optimal costimulation after engagement of their native receptors, enhancing survival and antitumor activity mediated through their chimeric receptors. Here we show in individuals with neuroblastoma that EBV-specific CTLs expressing a chimeric GD2-specific receptor indeed survive longer than T cells activated by the CD3-specific antibody OKT3 and expressing the same chimeric receptor but lacking virus specificity. Infusion of these genetically modified cells seemed safe and was associated with tumor regression or necrosis in half of the subjects tested. Hence, virus-specific CTLs can be modified to function as tumor-directed effector cells.     

Specific T-cell factor production and lymphocytes in the direct surroundings of a subcutaneous allogeneic tumor.

Antigen-specific T-cell factors (TCF) play a role in the initiation of cellular immune responses. In allogeneic mouse-tumor models lymphocytes from the direct tumor surroundings of both euthymic and nude mice produce TCF. These lymphocytes produce TCF when collected already 1 day after subcutaneous (sc) injection of tumor cells. In contrast to euthymic mice, draining lymph nodes and spleen of nude mice did not contain TCF-producing lymphocytes at any stage after sc tumor cell injection. In sensitized euthymic mice TCF production by lymphocytes is significantly higher in the direct tumor surroundings than in draining lymph nodes or spleen. At 2 and 5 days after tumor cell injection, the mononuclear cell infiltrate of the tissue surrounding the tumor in euthymic mice showed low expression of Thy 1, CD3, TCR alpha beta, TCR gamma delta, CD4, CD8, and asialo GM1, whereas several lymphocytes and mast cells were positive for monoclonal antibody (mAb) 14-30 (directed against TCF). In both euthymic and nude mice, sc injected tumor cells showed apoptosis. In conclusion, the direct tumor surroundings are the first (and, for nude mice, the only) site of TCF production, sc injection of tumor cells attracts mAb 14-30-positive lymphocytes and renders mast cells positive for mAb 14-30.     

Novel myeloperoxidase-derived HLA-A2-restricted peptides as therapeutic targets against myeloid leukemia

Background aimsHuman myeloperoxidase has been shown to be overexpressed in many types of leukemia, such as chronic myeloid leukemia, acute myeloid leukemia and myelodysplastic syndrome. The authors identified two myeloperoxidase-derived HLA-A2-restricted peptides, MY4 and MY8, as novel leukemia-associated antigens.MethodsEx vivo-elicited MY4- and MY8-specific cytotoxic T lymphocytes were generated, and tested for leukemia cell lysis in vitro and in NOD/SCID AML xenograft model.ResultsThese MY4- and MY8-specific cytotoxic T lymphocytes killed leukemic blasts while sparing healthy donor bone marrow cells. In addition, co-injection of MY4- and MY8-specific cytotoxic T lymphocytes into nonobese diabetic/severe combined immunodeficiency mice with acute myeloid leukemia drastically reduced tumor burden in vivo. The authors also found that MY4- and MY8-specific T cells could be detected in the peripheral blood mononuclear cells of allogeneic stem cell transplant recipients.ConclusionsThese antigen-specific T cells were significantly increased in blood samples from patients compared with healthy donors, suggesting that both MY4 and MY8 are immunogenic and that MY4- and MY8-specific cytotoxic T lymphocytes may play a role in reducing leukemia in vivo. Thus, the discovery of MY4 and MY8 as novel leukemia-associated antigens paves the way for targeting these antigens in immunotherapy against myeloid leukemia.     

Guinea pig homologues of TL and QA-2 antigens.

The H-2, thymus-leukemia (TL), and Qa-2 antigens of mice are encoded by closely linked genes on murine chromosome 17, and have structural similiarity in that each antigen is borne on a approximately 44,000 dalton molecule associated with beta2 microglobulin (beta2mu). The extensive homology of major histocompatibility complex (MHC) products that exists for the mouse and guinea pig suggested that a similar homology might exist for products of genetic regions closely linked to the MHC. By taking advantage of the selective association of beta2mu with H-2, Qa-2, and TL antigens, and by using the technique of sequential immunoprecipitation, we demonstrated two previously undescribed guinea pig molecules reactive with anti-guinea pig beta2mu. The first molecule was composed of a 36,000 dalton glycoprotein associated with beta2mu and was found on guinea pig thymocytes, but not lymphocytes. The second molecule was composed of a 40,000 dalton glycoprotein associated with beta2mu, and was found on both guinea pig thymocytes and lymphocytes. By structure, chemical composition, association with beta2mu, and tissue distribution, the first molecule is an attractive candidate for the guinea pig homologue of TL antigen, whereas the second fits the criteria for the guinea pig homologue of Qa-2 antigen.     

Characterization of T cell proliferative responses induced by anti-Qa-2 monoclonal antibodies.

The MHC class I Qa-2 Ag are attached to the cell surface by a glycanphosphatidylinositol (GPI) anchor. Crosslinking of Qa-2 and several other cell surface Ag attached by the GPI linkage has been shown to lead to cell activation. We have developed 10 new anti-Qa-2 mAb and characterized their capacity to induce proliferation of spleen cells. In the absence of anti-Ig-mediated crosslinking, none of the mAbs alone could induce activation. However, mAb 23.1 which reacts with the alpha 3 domain of Qa-2, when combined with most of the other mAbs (alpha 1, alpha 2 domain reactive), activated cells in the absence of anti-Ig crosslinking. The mAb pair 23.1 plus 24.16 was the most proficient and induced proliferation in the absence of any exogenous second signals. Responses were greatly enhanced and equivalent to those seen with anti-CD3 by the addition of phorbol myristate acetate (PMA). Ionomycin, rIL-2, or rIL-4 also potentiated anti-Qa-2 responses but less efficiently than PMA. Significant strain variation in the magnitude Qa-2-mediated proliferative responses was observed correlating with the levels of Qa-2 expressed on the cell surface. Crosslinking of Qa-2 molecules by the mAb combinations was required because monovalent Fab fragments failed to activate cells. F(ab')2 fragments of mAb 23.1 plus 24.16 induced vigorous proliferation indicating that accessory cell presentation of the mAb via Fc receptors was not required. Immobilized (plate bound) anti-Qa-2 mAb induced proliferation suggesting that the Qa-2 pathway may be distinct from that of other GPI molecules such as Thy-1 and Ly-6. Populations enriched for T cells (approximately 95%) responded as well as whole spleen cells, whereas B lymphocytes failed to proliferate to anti-Qa-2. Both CD4+ and CD8+ cells were activated following crosslinking of Qa-2. Finally, T cell activation mediated by Qa-2 induced elevation of [Ca2+]i, IL-2R expression, and the release of IL-2. These data demonstrate that crosslinking of Qa-2 on T lymphocytes represents a potent pathway for inducing cell activation.     

Characterization of cytotoxic cells generated from bone marrow culture

Bone marrow (BM) harbors precursors (Pre-NK) to NK cells. Recently, we devised an in vitro culture system that induces differentiation of the presumptive BM Pre-NK cells into cytotoxic cells to YAC in the presence of rat concanavalin A (Con A) conditioned medium. We have now analyzed the antigenic phenotype of the effector cells, precursor cells, and the target specificity of these cytotoxic cells. The cytotoxic cells had antigenic profiles similar to endogenous NK cells with the exception of Lyt-2 antigen. They are strongly positive for Qa-5, Thy-1, and partially positive for NK-1, Ly-5, Ly-6, Ly-10, and AsGm-1 and Lyt-2 antigens. The Pre-NK or accessory cells are positive for Qa-5, Ly-10, and Ly-20 and partially positive for NK-1, Thy-1, and AsGm-1 antigens. These Qa-5+ NK cells do not exhibit cytotoxic activity to WEHI or P815. They could also be generated from BM of nude mice as well as beige mice. We concluded from these studies that rat Con A-conditioned medium contained factors that could differentiate Pre-NK cells to mature NK cells and that these cells are heterogeneous. This in vitro culture system is useful in delineating the ontogeny of NK cells.     

Studies on the induction and expression of T cell-mediated immunity. IV. Non-overlapping populations of alloimmune cytotoxic lymphocytes with specificity for tumor-associated antigens and transplantation antigens.

Two non-overlapping populations of alloimmune cytotoxic T cells with specificity for tumor-associated antigens (TAA) and for histocompatibility antigens (H-2) were characterized by two independent methods. The heterogeneity of cytotoxic cells was demonstrated in spleen cells derived from BALB/c (H-2d) mice sensitized to EL-4 (H-2b) tumor and from C57BL/6 (H-2b) mice sensitized to G-35 (H-2d) tumor cells. Adsorption of immune lymphocytes on monolayers prepared with cells bearing the sensitizing H-2 antigens abrogated the in vitro cell-mediated cytotoxicity (CMC) directed against 51Cr-labeled normal target cells (spleen cells or ConA-activated spleen blasts), whereas significant cytolytic activity to the corresponding 51Cr-tumor cells was still retained. Likewise, in competitive inhibition assays, CMC to 51 Cr-tumor target cells was only partially inhibited by unlabeled normal cells, whereas CMC to 51Cr-normal target cells was completely abrogated. These results suggested that alloimmune cytotoxic lymphocytes are heterogeneous and can be subdivided into two independent populations of restricted specificity. Several experiments suggested that the effector cell population directed against TAA can no longer elicit a graft-vs-host (GVH) reaction in vivo. This was demonstrated by adoptive transfer into lethally-irradiated allogeneic recipients of cytotoxic or primed spleen cells fractionated on host target cell monolayers. Furthermore, these results demonstrated that both effector cells and memory cells possess high affinity binding receptors to corresponding H-2 antigens. The potential use of fractionated immune lymphocytes sensitized to tumor allografts in adoptive immunotherapy is discussed.     

Specific lymphocyte-target cell conjugate formation between tumor-specific helper T-cell hybridomas and IA-bearing RCS tumors and IE-bearing allogeneic cells. I. Role of Ia and both L3T4 and LFA-1 antigens in recognition/binding

The studies reported here describe the feasibility of using single cell techniques with nonadherent target cells for the formation of T helper lymphocyte-target cell conjugates in an Ia recognition system. We have taken advantage of four tumor-specific T cell hybridomas lines, two of which respond only to IA-bearing RCS tumor cells of SJL/J (H-2s) origin, and the other two that respond to both RCS and IA- or IE-bearing allogeneic cells of H-2k,d haplotypes. The conjugate frequency between the T cell hybridomas and target cells was scored microscopically and was facilitated by labeling the lymphocyte with fluorescein. The frequency of conjugate formation ranged from 20 to 40% above background. Conjugate formation was antigen specific and correlated well with the hybridoma specificity determined by IL 2 responses after antigenic stimulation. The cross-reactive hybridomas formed conjugates with RCS and LPS blasts derived from CBA or DBA/2 origin, but not with cells of syngeneic or other allogeneic strains. Conjugate formation with RCS was inhibited greater than 50% with mAb directed against IAs determinants on the RCS tumor cells, and conjugate formation with allogeneic cells was blocked only with mAb directed to either IA/IEk or IA/IEd specificities directed against the alpha or beta polypeptide chain. Blocking of conjugate formation was also achieved by various mAb directed against surface membrane molecules associated with the T cell hybridomas. LFA-1 mAb inhibited significantly the formation of conjugates. However, L3T4 mAb blocked only partially the conjugates. Other antibodies directed against Lyt-1 or Thy-1.2 antigens were without blocking effect. The poor blocking observed with L3T4 mAb did not correlate with the almost complete blocking observed in the IL 2 response by the same hybridomas. These studies of the syngeneic anti-RCS tumor response directed against IA-bearing RCS showed that the conjugate assay permits mapping of tumor-associated Ia epitopes. In addition, the results of these studies demonstrate the feasibility of conjugate formation in determining the antigenic specificity of the T helper system. This assay system can be used to establish the minimal frequency of antigen-reactive cells and can divide the T helper response into multiple steps (i.e., recognition/binding, activation, proliferation, and lymphokine release) and determine the surface membrane molecules involved in recognition.