Frequency analysis of class I MHC-reactive Lyt-2+ and class II MHC-reactive L3T4+ IL 2-secreting T lymphocytes

The reactivity of Lyt-2+ or L3T4+ T cells stimulated with either mutant class I or class II MHC alloantigens was studied. Whereas stimulation with class I MHC antigens induced only Lyt-2+ T cells to proliferate and to secrete IL 2, stimulation with class II MHC alloantigens induced L3T4+ but not Lyt-2+ T cells. When the frequencies of precursors of IL 2-secreting T lymphocytes (IL 2TL-p) were determined by limiting dilution analyses, class I MHC-reactive Lyt-2+ T cells displayed frequencies (f = 1/200) as high in magnitude as those within class II MHC-reactive L3T4+ (f = 1/100). Clonally developing IL 2TL of either T cell subset were antigen-specific, as shown in split-culture experiments. Whereas L3T4+ helper TL could be induced to specific IL 2 secretion over a long time period (days 3 to 9), Lyt-2+ TL showed a marked time optimal on day 4; thereafter, the number of TL colonies inducible to secrete IL 2 decreased steadily. IL 2 production and IL 2TL-p frequencies of unseparated T responder cells were not the numerical superposition of the two individual T cell subsets (Lyt-2+ + L3T4+); the latter finding is likely to reflect regulatory influences of Lyt-2+ T cells on IL 2-secreting L3T4+ T cells.     

Specificity, phenotype, and precursor frequency of primary cytolytic T lymphocytes specific for class II major histocompatibility antigens

Most cytolytic T lymphocytes (CTL) recognize class I rather than class II MHC determinants, and relatively little is known about those CTL that do recognize class II MHC determinants. The present study was undertaken to document the specificity, phenotype, and precursor frequency of primary class II allospecific CTL. It was found that class II-allospecific CTL could be consistently generated in vitro from unprimed spleen or thymus populations in the presence of exogenously added helper factors. The class II MHC specificity of both the precursor and CTL effectors activated in primary cultures by Ia-disparate stimulator cells was documented both by blocking experiments with anti-Ia mAb and by the use of L cell transfectants. The mechanism by which primary allospecific CTL effectors lysed their targets appeared to involve direct cell-cell contact, because they failed to lyse bystander target cells. The frequency in unprimed spleen populations of precursor CTL specific for class II alloantigens was examined by limiting dilution analysis and was found to be as high as 1/15,000 splenocytes and approximately 10% of the frequency reported for primary class I allospecific CTL. Finally, the Lyt phenotype of primary class II allospecific CTL precursors and effectors was determined. It was found that anti-class II CTL derive from at least two distinct precursor subpopulations that are either L3T4+Lyt-2- or L3T4-Lyt-2+, and that the Lyt phenotype expressed by the CTL effectors are concordant with that of their precursors. No correlation was found between the I subregion gene products recognized by CTL effectors and the Lyt phenotype they expressed in that both I-A- and I-E-specific CTL were both L3T4+Lyt-2- and L3T4-Lyt-2+.     

402AX teratocarcinoma MHC class I antigen expression is regulated in vivo by Lyt 1, Lyt 2, and L3T4 expressing splenic T cells

The murine 402AX teratocarcinoma is a MHC class I antigen negative tumor of 129 strain origin. Host resistance to the 402AX tumor is genetically controlled. When passed intraperitoneally in genetically resistant mice, the tumor cells are induced to express MHC Class I antigens of the 129 genotype. When passed in genetically susceptible mice, the tumor cells remain MHC class I antigen negative. Earlier studies have demonstrated that resistance to the tumor and regulation of tumor cell MHC class I antigen expression are under the control of the host's immune system. The present studies indicate that splenic Lyt 1-, Lyt 2-, and L3T4-expressing cells regulate tumor cell MHC class I antigen expression, and that these cells require a genetically resistant host environment in which to differentiate. Splenic T cells primed to the 402AX tumor and transferred into genetically susceptible 129 mice give rise to GVHD, suggesting that immunity to the tumor involves reactivity to 129 minor histocompatibility antigens.     

Characterization of an endogenous Lyt2+ T-suppressor-cell population regulating autoreactive T cells in vitro and in vivo

Autoreactive T cells have been defined by their capacity to respond to self-Ia antigens expressed on non-T cells. Several recent studies have suggested that these cells may play important immunoregulatory functions. However, it is not clear what regulates the responsiveness of autoreactive T cells and why such cells are not demonstrably stimulated in vivo, where they are in the constant presence of self-Ia antigens. In the present study we examined the role of T suppressor (Ts) cells in regulating autoreactive T cells. We observed that enhanced autoreactivity occurred in vitro when Lyt2+ T cells were depleted from the responding and/or stimulating spleen cells in a syngeneic mixed-lymphocyte reaction. Similarly, addition of irradiated Lyt2+ T cells but not L3T4+ T cells inhibited the response of L3T4+ T cells to self-Ia antigens. The activity of the suppressor cells was specific to the autoreactive T cells since antigen-specific and alloreactive T-cell proliferation were not inhibited. Furthermore, depletion of Lyt2+ T cells by in vivo treatment of mice with anti-Lyt2 monoclonal antibodies caused enhanced endogenous proliferation of lymph node and splenic T cells and increased the T-cell response to self-Ia antigens in vitro. These studies, therefore, suggest that T-cell tolerance to self-Ia antigens in vivo may be maintained by naturally occurring Lyt2+ Ts. Mice having enhanced autoreactivity may provide a useful tool to address the role of autoreactive T cells in the immune response to foreign antigens and in the pathogenesis of autoimmune diseases.     

CD8 is needed for development of cytotoxic T cells but not helper T cells.

A mutant mouse strain without CD8 (Lyt-2 and Lyt-3) expression on the cell surface has been generated by disrupting the Lyt-2 gene using embryonic stem cell technology. In these mice, CD8+ T lymphocytes are not present in peripheral lymphoid organs, but the CD4+ T lymphocyte population seems to be unaltered. Cytotoxic response of T lymphocytes from these mice against alloantigens and viral antigens is dramatically decreased. Proliferative response against alloantigens and in vivo help to B lymphocytes, however, are not affected. These data suggest that CD8 is necessary for the maturation and positive selection of class I MHC restricted cytotoxic T lymphocytes but is not required on any of the intermediate thymocyte populations (CD8+CD4-TcR- or CD4+CD8+TcRlow) during the development of functional class II MHC restricted helper T cells.     

Relationship among function, phenotype, and specificity in primary allospecific T cell populations: identification of phenotypically identical but functionally distinct primary T cell subsets that differ in their recognition of MHC class I and class II allodeterminants

The goal of the present study was to evaluate the relationship among function, Lyt phenotype, and MHC recognition specificity in primary allospecific T cell populations. By using Lyt-2+ and L3T4+ T cells obtained from the same responder populations, we assessed the ability of T cells of each phenotype to generate cytotoxic effector cells (CTL) and IL 2-secreting helper T cells in response to either class I or class II MHC allodeterminants. It was found that a discordance between Lyt phenotype and MHC recognition specificity does exist in primary allospecific T cells, but only in one T cell subpopulation with limited functional potential: namely, Lyt-2+ T cells with cytotoxic, but not helper, function that recognize class II MHC alloantigens. Target cell lysis by these Lyt-2+ class II-allospecific CTL was inhibited by anti-Ia monoclonal antibodies (mAb), but not anti-Lyt-2 mAb, indicating that they recognized class II MHC determinants as their "restriction" specificity and not as their "nominal" specificity even though they were Lyt-2+. A second allospecific T cell subset with limited functional potential was also identified but whose Lyt phenotype and MHC restriction specificity were not discordant: namely, an L3T4+ T cell subset with helper, but not cytotoxic, function specific for class I MHC allodeterminants presented in the context of self-Ia. Thus, the present study demonstrates that primary allospecific T cell populations contain phenotypically identical subpopulations of helper and effector cells that express fundamentally different MHC recognition specificities. Because the recognition specificities expressed by mature T cells reflect the selection pressures they encountered during their differentiation into functional competence, these findings suggest that functionally distinct but phenotypically identical T cell subsets may be selected independently of one another during ontogeny. Thus, the existence of Lyt-2+ CTL specific for class II allodeterminants can be explained by the hypothesis that the association of Lyt phenotype with MHC recognition specificity results from the process of thymic selection that these Lyt-2+ effector cells avoid.     

Induction of graft versus host-associated immunodeficiency by CD4+ T cell clones

Previous studies have shown that the injection of parental T cells into MHC class II mismatched F1 recipient mice can lead to graft-vs-host (GvH) reaction that manifests itself by multiple symptoms. The objective of our study was to analyze GvH reactivity induced by a single T cell clone specific for host I-A or I-E alloantigen. The T cell clones tested for GvH potential were CD4+, with or without cytolytic activity in vitro and with a lymphokine pattern that classifies them as Th1 cells. The inoculation of a single T cell clone induced a severe, but transient immunodeficiency in the host that was independent of its cytolytic activity, as demonstrated by the failure to generate a CTL response to third party allogeneic cells in vitro. Induction of immunodeficiency in the recipients required preactivation of the clones in vitro by rIl-2 and the presence of the stimulator class II alloantigen in the host. Spleen cells from these mice lacked suppressor cells, they were deficient in Il-2 secretion and exhibited a decrease in the number of CD4+ T cells. In addition, I-E expression was reduced, however, without any changes in the macrophage population and an increase in surface Ig and the B cell marker B220. Simultaneous to the immunodeficiency, the clone-injected mice produced elevated antibody titers to ssDNA.     

Perforin is present only in normal activated Lyt2+ T lymphocytes and not in L3T4+ cells, but the serine protease granzyme A is made by both subsets.

We show that among the subsets of peripheral T lymphocytes (Lyt2+ L3T4- and L3T4+ Lyt2- cells) activated in short-term cultures by stimulation with H-2 incompatible leukocytes 97% of the cytolytic activity and all detectable perforin activity resides in the Lyt2+ cells. But both populations contain approximately equal amounts of a serine protease, granzyme A, the expression of which was previously thought to be restricted to cytolytic T lymphocytes.     

The generation of cytolytic activity in mixed leukocyte cultures: cortisone-resistant thymocytes are deficient in helper T lymphocytes

Cortisone-resistant (CR) thymocytes did not generate cytolytic activity toward H-2 K or D alloantigen unless they were also stimulated by H-2 I or non-H-2 alloantigens, even though spleen cells generated brisk cytolytic activity toward H-2 K or D alone. Backstimulation by stimulating strain T lymphocytes accounted for neither the response of spleen cells toward H-2 K or D alloantigen nor the response of CR thymocytes to a full set of alloantigens. In addition, lack of non-T accessory cells did not account for the CR thymocyte pattern of reactivity. Rather, CR thymocytes appeared to be relatively deficient in helper T lymphocytes (HTL). CR thymocytes generated specific cytolytic activity toward H-2 D alloantigen when T cell growth factors (TCGF) or cloned alloreactive helper T lymphocytes were added to culture. CR thymocytes contained fewer HTL precursors detected at limit dilution than spleen cells did. Thus spleen cells generated cytolytic activity toward class I alloantigens alone, but under the same culture conditions CR thymocytes had to be stimulated by both class I and class II alloantigens. Class II alloantigens may be required to stimulate cytolytic activity only under culture conditions in which class I-reactive HTL are not sufficient to provide a minimal threshold of help.     

Recognition of MHC class I allodeterminants regulates the generation of MHC class II-specific CTL

We have analyzed the signals influencing the generation of major histocompatibility complex (MHC) class II allospecific cytolytic T lymphocytes (CTL) and have found that the development of these CTL is actively regulated in primary in vitro cultures by Lyt-2+ T cells triggered in response to MHC class I alloantigens. Class II allospecific CTL can be readily stimulated in primary cultures, but the presence of a simultaneous class I MHC stimulus in these cultures causes a marked reduction of class II-specific CTL activation. This reduction can be prevented by adding to culture a dose of monoclonal anti-Lyt-2 antibody (in the absence of complement) that does not block the generation of class I-specific CTL. The role of MHC class I alloantigens in the regulation of class II allospecific responses illustrates that T cells recognizing class I and class II MHC antigens in mixed leukocyte cultures interact in a complex and nonreciprocal manner to influence the final effector T cell repertoire elicited by this complex immunogenic challenge.     

Inhibition of graft-versus-host disease by double-negative regulatory T cells

Pretransplant infusion of lymphocytes that express a single allogeneic MHC class I Ag has been shown to induce tolerance to skin and heart allografts that express the same alloantigens. In this study, we demonstrate that reconstitution of immunoincompetent mice with spleen cells from MHC class I L(d)-mismatched donors does not cause graft-vs-host disease (GVHD). Recipient mice become tolerant to skin allografts of lymphocyte donor origin while retaining immunity to third-party alloantigens. The mechanism involves donor-derived CD3(+)CD4(-)CD8(-) double-negative T regulatory (DN Treg) cells, which greatly increase and form the majority of T lymphocytes in the spleen of recipient mice. DN Treg cells isolated from tolerant recipient mice can suppress the proliferation of syngeneic antihost CD8(+) T cells in vitro. Furthermore, we demonstrate that DN Treg cells can be generated in vitro by stimulating them with MHC class I L(d)-mismatched lymphocytes. These in vitro generated L(d)-specific DN Treg cells are able to down-regulate the activity of antihost CD8(+) T cells in vitro by directly killing activated CD8(+) T cells. Moreover, infusing in vitro generated L(d)-mismatched DN Treg cells prevented the development of GVHD caused by allogeneic CD8(+) T cells. Together these data demonstrate that infusion of single MHC class I locus-mismatched lymphocytes may induce donor-specific transplantation tolerance through activation of DN Treg cells, which can suppress antihost CD8(+) T cells and prevent the development of GVHD. This finding indicates that using single class I locus-mismatched grafts may be a viable alternative to using fully matched grafts in bone marrow transplantation.     

Self-reactive T cell receptor-reactive CD8+ T cells inhibit T cell lymphoma growth in vivo

Syngenic C57BL/6 mice (H-2(b)) vaccinated with mitomycin C-treated L12R4 T lymphoma cells develop protective immunity toward the MHC class II-negative tumor cells. In the present study, we characterize the nature, mode of function, and specificity of the effector cells in this immunity. These cells are TCR-specific CD8(+) T lymphocytes with effector function in vitro as well as in vivo upon transfer to naive mice. They produce high levels of IFN-gamma and TNF-alpha, but little or no IL-4. By means of TCRbeta-negative variant L12R4 cells, P3.3, and TCR-Vbeta2 cDNA-transfected and TCR-Vbeta2-expressing P3.3 lymphoma cells, we found that a significant part of the effector T cells are specific for the Vbeta12 region. The growth inhibition of L12R4 cells in vitro was inhibited by anti-H-2, anti-K(b), and anti-D(b) mAb. Furthermore, vaccination with Vbeta12 peptide p67-78, which binds to both K(b) and D(b) MHC class I molecules, induces partial protection against L12R4 T lymphoma cells. Thus, self-reactive TCR-Vbeta-specific, K(b)-, or D(b)-restricted CD8(+) T cells mediate inhibition of T cell lymphoma growth in vitro and in vivo.     

CD8+ T cell protective immunity against Chlamydia pneumoniae includes an H2-M3-restricted response that is largely CD4+ T cell-independent

CD8+ T cells are important for immunity to the intracellular bacterial pathogen Chlamydia pneumoniae (Cpn). Recently, we reported that type 1 CD8+ (Tc1) from Cpn-infected B6 mice recognize peptides from multiple Cpn Ags in a classical MHC class Ia-restricted fashion. In this study, we show that Cpn infection also induces nonclassical MHC class Ib-(H2-M3)-restricted CD8+ T cell responses. H2-M3-binding peptides representing the N-terminal formylated sequences from five Cpn Ags sensitized target cells for lysis by cytolytic effectors from the spleens of infected B6 mice. Of these, only peptides fMFFAPL (P1) and fMLYWFL (P4) stimulated IFN-gamma production by infection-primed splenic and pulmonary CD8+ T cells. Studies with Cpn-infected Kb-/-/Db-/- mice confirmed the Tc1 cytokine profile of P1- and P4-specific CD8+ T cells and revealed the capacity of these effectors to exert in vitro H2-M3-restricted lysis of Cpn-infected macrophages and in vivo pulmonary killing of P1- and P4-coated splenocytes. Furthermore, adoptive transfer of P1- and P4-specific CD8+ T cells into naive Kb-/-/Db-/- mice reduced lung Cpn loads following challenge. Finally, we show that in the absence of MHC class Ia-restricted CD8+ T cell responses, CD4+ T cells are largely expendable for the control of Cpn growth, and for the generation, memory maintenance, and secondary expansion of P1- and P4-specific CD8+ T cells. These results suggest that H2-M3-restricted CD8+ T cells contribute to protective immunity against Cpn, and that chlamydial Ags presented by MHC class Ib molecules may represent novel targets for inclusion in anti-Cpn vaccines.     

Sufficiency of the CD8+ T cell lineage to mount an effective tumoricidal response to syngeneic tumor-bearing novel class I MHC antigens

The origins of "help" in rejection of syngeneic tumors by the CD8 T cell lineage was examined with a model tumor inappropriately expressing novel class I MHC and subject to cytolytic T cell (CTL)-mediated rejection. The requirement for CD4+ Th cells to induce CD8+ CTL effectors in vivo was investigated by using C3H mice selectively depleted of either CD4+ or CD8+ T cells. Rejection of the tumor was vigorous and indistinguishable from normal mice after depletion of CD4+ T cells in vivo. In contrast, in CD8+ T cell-depleted mice tumors grew progressively, confirming that T cells of the CD8+ lineage are required for a tumoricidal immune response, and cells of this lineage are sufficient for a primary response. Taken together, these results demonstrate that, in the absence of CD4+ T cells in vivo, unprimed cells of the CD8+ lineage are fully competent to mount an effective CTL immune response to syngeneic cells expressing novel class I Ag, consistent with the concept that only T cells with class I recognition specificity may be required to satisfy the need for both help and effector functions in the response.     

Ontogeny and function of B220+ L3T4+ T-cell subset of MRL/Mp-lpr/lpr mice

T-cell-enriched populations obtained from lymph nodes (LNs) of 4-month-old MRL/Mp-lpr/lpr (MRL-lpr), C3H/HeJ-lpr/lpr (C3H-lpr), and C3H/HeJ-gld/gld (C3H-gld) mice were studied for the expression of B220, L3T4, and Lyt 2 antigens. A new B220+ L3T4+ phenotype was demonstrated in T-cell populations of these mice by two-color flow cytometry with phycoerythrin-conjugated monoclonal antibodies (MoAb) to L3T4 and FITC-anti-B220 MoAb. The generation of the T subset was apparently under the influence of the lpr or gld gene, since it could not be demonstrated in T-cell-enriched populations of MRL/Mp- +/+ and normal C3H mice. The expression level of L3T4 antigen on the T subset was lower than that on B220- L3T4+ cells, while the level of B220 antigen was similar to that of B220+ L3T4- or B220+ Lyt 2- cells. The B220+ L3T4+ phenotype appeared in LNs and spleens, but not in thymuses, of MRL-lpr mice as early as 2 months of age. Its proportion to whole LN T cells at this age was equivalent to that observed in 4-month-old mice. Functional studies on FACS-sorted cell populations revealed that the T subset similar to B220+ L3T4- cells possessed deficiencies in the IL-2-IL-2 receptor system. The appearance of the T subset with an intermediate phenotype and its functional defects suggests that lpr and gld genes in these autoimmune mice exert their influences on the ontogeny and function of L3T4+ T cells and contribute to the induction of early lupus.     

Heterogeneity of CD4+ T cells involved in anti-allo-class I H-2 immune responses. Functional discrimination between the major proliferating cells and helper cells assisting cytotoxic T cell responses.

MLR in various combinations with class I H-2 disparity revealed that there are three patterns of MLR in the aspect of responding T subset (CD4 vs CD8) dominance. Irrespective of the CD8 vs CD4 dominance, a single i.v. administration of class I-disparate allogeneic spleen cells resulted in almost complete abrogation of anti-class I proliferative capacity of both CD4+ and CD8+ T cells in six combinations. The suppression of proliferative responses was correlated with the striking reduction in the ability to produce IL-2 upon stimulation with the relevant class I alloantigens. In contrast, i.v. presensitized recipient mice exhibiting only marginal MLR/Il-2 production could generate comparable magnitudes of anti-allo class I CTL as well as graft rejection responses to those induced by normal unpresensitized mice. The administration in vivo of anti-CD4 antibody along with the i.v. presensitization not only suppressed the generation of CTL responses by spleen cells but also induced appreciable prolongation of allo-class I-disparate skin grafts under conditions in which neither alone did it. These results demonstrate that 1) the suppression of graft rejection responses is not necessarily reflected on the reduction of MLR; 2) CD8+ CTL precursors responsible for graft rejection can be activated by either allo-class I-reactive CD8+ or CD4+ Th cells; 3) i.v. presensitization induces functional elimination of CD8+ and CD4+ proliferative/IL-2-producing T cells but not of CD8+ CTL precursors and CD4+ Th whose capacity is expressed by assistance of CTL induction but not by their own proliferation. Thus, this study illustrates the heterogeneity of class I alloantigen-reactive CD4+ T cells in the aspect of their capacity to proliferate themselves vs contribute to CTL induction as well as graft rejection.     

Differential helper and effector responses of Lyt-2+ T cells to H-2Kb mutant (Kbm) determinants and the appearance of thymic influence on anti-Kbm CTL responsiveness

The goal of this study was to assess and compare the allorecognition requirements for eliciting Lyt-2+ helper and effector functions from primary T cell populations. By using interleukin 2 (IL 2) secretion as a measure of T helper (Th) function, and cytolytic T lymphocyte (CTL) generation as a measure of effector function, this study compared the responses of Lyt-2+ T cells from wild-type B6 mice against a series of H-2Kb mutant determinants. Although all Kbm determinants stimulated B6 Lyt-2+ T cells to become cytolytic effector cells, the various Kbm determinants differed dramatically in their ability to stimulate Lyt-2+ T cells to function as IL 2-secreting helper cells. For example, in contrast to Kbm1 determinants that stimulated both helper and effector functions, Kbm6 determinants only stimulated B6 Lyt-2+ T cells to become cytolytic and failed to stimulate them to secrete IL 2. The distinct functional responses of Lyt-2+ T cells to Kbm6 determinants was documented by precursor frequency determinations, and was not due to an inability of the Kbm6 molecule to stimulate Lyt-2+ Th cells to secrete IL 2. Rather, it was the specific recognition and response of Lyt-2+ T cells to novel mutant epitopes on the Kbm6 molecule that was defective, such that anti-Kbm6 Lyt-2+ T cells only functioned as CTL effectors and did not function as IL 2-secreting Th cells. The failure of Lyt-2+ anti-Kbm6 T cells to function as IL 2-secreting Th cells was a characteristic of all Lyt-2+ T cell populations examined in which the response to novel mutant epitopes could be distinguished from the response to other epitopes expressed on the Kbm6 molecule. The absence of significant numbers of anti-Kbm6 Th cells in Lyt-2+ T cell populations was examined for its functional consequences on anti-Kbm6 CTL responsiveness. It was found that primary anti-Kbm6 CTL responses could be readily generated in vitro, but unlike responses to most class I alloantigens that can be mediated by Lyt-2+ Th cells, anti-Kbm6 CTL responses were strictly dependent upon self-Ia-restricted L3T4+ Th cells. Because the restriction specificity of L3T4+ Th cells is determined by the thymus, in which their precursors had differentiated, anti-Kbm6 CTL responsiveness, unlike responsiveness to most class I alloantigens, was significantly influenced by the Ia phenotype of the thymus in which the responder cells had differentiated.(ABSTRACT TRUNCATED AT 400 WORDS)     

Helper T lymphocytes which recognize the MHC class I alloantigens in vivo are CD4+CD8-1

To determine the CD4 or CD8 phenotype of the Th lymphocyte which recognizes in vivo the MHC class I alloantigens, B10 recombinant mice were treated with anti-CD8 or anti-CD4 mAb and immunized with lymphoid cells from donors differing in the K or D region of the MHC. Alloantibodies were evaluated by a 51Cr-release assay or by indirect immunofluorescence. The production of IgG anti-Dd and anti-Kk alloantibodies was increased by the deletion of the CD8+ and absent in mice depleted of the CD4+ subset. These experiments indicate that the helper influence elicited by the recognition of a MHC class I alloantigen in vivo is due to cells of the CD4+CD8- phenotype.     

Distinct proliferative T cell clonotypes are generated in response to a murine retrovirus-induced syngeneic T cell leukemia: viral gp70 antigen-specific MT4+ clones and Lyt-2+ cytolytic clones which recognize a tumor-specific cell surface antigen

After immunization of B6 mice with the syngeneic retrovirus-induced T cell leukemia/lymphoma FBL-3, two major tumor-specific proliferative T cell clonotypes were derived. T cell clones derived from long-term lines propagated by in vitro culture with irradiated tumor cells and syngeneic spleen cells were exclusively of the Lyt-2+ phenotype. Such clones were cytolytic, retained their proliferative phenotype indefinitely when expanded by repeated cycles of reactivation and rest, and recognized a tumor-specific cell surface antigen in association with class I MHC molecules. This tumor cell antigen was not present on nontransformed virus-infected cells. Class II MHC-restricted MT4+ clones specific for the viral antigen gp70 were derived from lymph node T cells of FBL-3 tumor-immune mice only by in vitro culture with purified Friend virus in the presence of syngeneic splenic APC. Once derived, however, such clones could be stimulated in the presence of FBL-3 tumor cells and syngeneic spleen cells, demonstrating the reprocessing of tumor-derived gp70 antigen by APC in the spleen cell population. In contrast, no reprocessing of the tumor cell surface antigen by splenic APC for presentation to the class I MHC-restricted T cell clones could be demonstrated. Evidence is presented that FBL-3 T leukemia/lymphoma cells function as APC for Lyt-2+ class I MHC-restricted clones, and that no concomitant recognition of Ia molecules is required to activate these clones. Both Lyt-2+ and MT4+ clones were induced to proliferate in the presence of exogenous IL2 alone, but this stimulus failed to result in significant release of immune interferon. In contrast, antigen stimulation of both clones resulted in proliferation as well as significant immune interferon release. Immune interferon production is not required for the generation of MHC-restricted cell-mediated cytolytic function.