Specificities of killing by T lymphocytes generated against syngeneic SV40 transformants: studies employing recombinants within the H-2 complex.

T lymphocyte effectors to syngeneic SV40-transformed cells, generated by secondary in vitro sensitization of immune spleen cells, lyse SV40 transformed targets that are syngeneic at the H-2 locus. In this study we have employed recombinants within the H-2 region to examine in detail this H-2 specificity. H-2b effectors were found to lyse SV40-transformed targets from recombinants bearing either H-2Kb or H-2Db.H-2k effectors recognized only SV40-transformed H-2Kk, and not H-2Dk target cells. By using the same protocol for sensitization, no effector cells could be detected in H-2d mice. Effectors generated in H-2 recombinant mice showed that the response capacity resides with K and D. For example, HTG, which is H-2d except at the D locus (H-2Db), produced effector cells specific for SV40-transformed H-2Db targets. Thus, the secondary in vitro response to SV40 transformants was found to depend only on the K and D alleles and not to be modified by the I region to any measurable extent.     

Generation of cytotoxic lymphocytes by SV40-induced antigens

In order to study the correlation of in vivo tumor transplantation immunity and in vitro immunologic assays, cell-mediated cytotoxicity against SV40-transformed cells was studied in AL/N strain mice by using 51Cr-release assay. Killing of SV40-transformed AL/N fibroblast cells was observed by spleen cells of AL/N mice immunized with syngeneic SV40-transformed cells. Immunization with the solubilized SV40 tumor-specific transplantation antigen (TSTA) that induced transplantation immunity in vivo did not elicit cytotoxic spleen cells in vitro. However, the spleen cells from mice immunized with solubilized TSTA and then sensitized in vitro with SV40-transformed cells became cytotoxic against SV40-transformed fibroblasts. Similarly, SV40 TSTA (T antigen) purified by immunoprecipitation was able to prime the lymphocytes in AL/N mice: the primed lymphocytes could differentiate into cytotoxic lymphocytes upon in vitro stimulation by SV40-transformed cells. These data indicate that SV40 TSTA (T antigen) plays a role in the induction of cytotoxic lymphocytes.     

Characterization of a progressive tumor from C3H fibroblasts transformed in vitro with SV40 virus. Immunoresistance in vivo correlates with phenotypic loss of H-2Kk

Cultured SV40-transformed fibroblasts from C3H mice (SV-C3H) were "adapted" to in vivo growth by serial passage through sublethally irradiated, syngeneic recipients. After four in vivo passages, a population of cells was obtained (V4) that was weakly oncogenic in nonirradiated mice. Cells isolated from large V4 tumors (V5) were found to be highly oncogenic, producing lethal tumors at doses of less than 10(3) cells. V5 is insensitive to SV40-specific transplantation immunity in syngeneic animals but can be rejected completely by H-2 allogeneic mice. In vitro studies revealed that although V4 and the parent SV-C3H cells can induce SV40-specific cytotoxic T cells (CTL) in vitro and are lysed by these CTL, V5 does neither. The failure of V5 to interact with CTL was traced to the loss of H-2Kk antigen expression on these cells. The correlation between H-2Kk loss and immunoresistance in vivo suggests a central role for the cytotoxic T cell in in vivo tumor elimination in this system.     

Specificities of killing by cytotoxic lymphocytes generated in vivo and in vitro to syngeneic SV40 transformed cells.

Cell-mediated immunity to SV40-transformed C3H and C3H-SW cell lines was measured by using both 51Cr and 125IUdR release assays. Killing by cytotoxic cells generated on in vitro sensitization of immune spleen cells with syngeneic SV40 cells by either assay is specific for syngeneic SV40 transformants. Cytolysis mediated by in vitro sensitized cells is ablated by treatment of the effector cells with anti-theta serum and complement. Intraperitoneal immunization with syngeneic SV40 cells yields two distinct killer-cell populations in the peritoneal exudate when assayed by 125IUdR release. The first, nylon wool nonadherent and sensitive to anti-theta and complement, is indistinguishable from the killers generated in vitro. The second population, present in larger numbers and more efficient on a per-cell basis in killing of SV40 targets than the first, is nylon adherent and is not removed by treatment with anti-theta and complement. This second population will kill any SV40 transformed target, whether syngeneic or allogeneic. The possible roles of T cell and non-T cell effectors in rejection of syngeneic SV40 tumors are discussed.     

Studies on the specificity of in vitro-induced lymphocytotoxicity to methylcholanthrene-induced fibrosarcoma cell lines

Cytotoxic effector lymphocytes were induced by in vitro immunization of lymph node and spleen cells from CS7B16(H2^b) and Balb/c(H2^d) mice to syngeneic or allogeneic methylcholanthrene-induced fibrosarcoma (MCAF) cell lines. The T cell-dependent cytotoxicity was specific to target cell lines to which the lymphocytes were immunized in vitro. Normal fibroblasts as stimulator cells did not induce lymphocytotoxicity to syngeneic MCAF cells or to normal syngeneic fibroblasts. The results indicate that the in vitro-immunized lymphocytes recognize individual specific tumor-associated antigens of the MCAF cells. In experiments in which the lymphocytes were immunized in vitro to allogeneic MCAF cells, cytotoxic reactions to alloantigens, but not to tumor-associated antigens, were detected. Incubation with phytohemagglutinin (PHA) during the sensitization period modified the specificity of the cell-mediated lysis of MCAF cells: Allogeneic as well as syngeneic target cells were destroyed by these effector cells. PHA induced a nonspecific cytotoxic effect which increased the specific lysis of target cells. The cytotoxicity of the in vitro-immunized lymphocytes was inhibited by incubation with membrane protein preparations from the syngeneic MCAF cell lines. In contrast to the specificity of the cytotoxic effect to the different syngeneic cell lines, the membrane extract of one individual syngeneic MCAF cell line was able to inhibit the lymphocytotoxicity to all other syngeneic cell lines. Membrane protein preparations from allogeneic MCAF cells or from normal syngeneic fibroblasts were not inhibitory. The in vitro-immunized cytotoxic lymphocytes did not impair the tumor growth in vivo as could be demonstrated by passive transfer of the lymphocytes in a Winn assay.     

Cell-mediated immune reaction against BK virus-transformed cells

Summary Syngeneic or allogeneic cells transformed by BK virus (BKV) were used to immunize C57BL/6J mice. After in vitro stimulation, lymphocytes prepared from the spleens of immunized mice were used in in vitro cytotoxicity tests. The results of these tests revealed the presence of a cell surface antigen, presumably corresponding to the viral transplantation antigen, common to all tested BKV- and SV40-transformed cell lines of C57BL/6J origin. An allogeneic cell line transformed by BKV also contained the same antigen. Immunization, i.e., in vivo priming, did not require syngeneic transformed cells, whereas cytolysis was only observed when the virus-specific antigen on target cells was associated with the same H2 haplotype as was expressed by effector cells. An additional unidentified antigen was shared by some of the BKV-transformed cell lines and cell lines transformed by simian adenovirus SA7.     

Evidence for the involvement of cytolytic macrophages in rejection of SV40-induced tumors

The potential role of cytolytic macrophages in in vivo resistance to tumors induced by simian virus 40 (SV40) was evaluated in two experimental systems. First, a cell line produced by sequential in vivo passage of SV40-transformed fibroblasts through syngeneic C3H/HeJ mice was found to develop both increased neoplastic character and resistance to macrophage-mediated lysis, suggesting in vivo selection pressure against the macrophage-sensitive phenotype. In the second approach, SV40-transformed cells from C3H.OL mice, a strain that fails to produce SV40-specific cytolytic T lymphocytes (CTL), were cloned, and the cloned cells were tested for susceptibility to macrophage cytolysis in vitro. Two clones SV-COL-E8 and SV-COL-F5, which represent the extremes of macrophage susceptibility and resistance, respectively, were tested for progressive growth in syngeneic C3H.OL recipients. Progression in vivo was found to correlate with resistance to macrophage cytolysis in vitro. Other in vitro measures of the neoplastic phenotype, cell division rate and anchorage-independent growth, did not predict the relative abilities of clones E8 and F5 to form tumors. Likewise, the cells were indistinguishable in their sensitivity to cytolysis by allogeneic CTL and by natural killer cells. Finally, the presence of activated macrophages in the peritoneum of mice rejecting a challenge of syngeneic SV40-transformed cells was confirmed in both CTL responder and nonresponder strains. These studies suggest that cytolytic macrophages are indeed generated during rejection of SV40-induced mouse tumors and that, in the absence of an effective anti-SV40 CTL response, resistance of the transformed cell to macrophage-mediated cytolysis can be a determining factor in in vivo tumor growth.     

H-2 antigen requirements in the in vitro induction of SV40-specific cytotoxic T lymphocytes

T cytotoxic cells generated to syngeneic SV40 virus transformants lyse only SV40 target cells that are syngeneic at the H-2 locus. In contrast, SV40-specific tumor transplantation immunity shows no requirements for syngeneic H-2. Inoculation of allogeneic or even xenogeneic transformants will confer immunity to a challenge of syngeneic SV40 tumor cells. The experiments described here represent an attempt to reconcile these apparently conflicting observations. In our hands, generation of SV40-specific T cytotoxic cells in vitro requires both in vivo priming and secondary in vitro sensitization. We have found that priming for a secondary syngeneic-restricted response requires only that the cell employed be SV40 transformed. That is, priming may be accomplished with syngeneic, allogeneic, or xenogeneic SV40 transformants. Thus, the apparent lack of H-2 restriction in vivo immunity does not eliminate a role for the H-2-restricted cytotoxic T cell in tumor transplantation immunity.     

Virus-specific T-cell sensitization

Syngeneic, semiallogeneic, or allogeneic spleen lymphocytes were transferred intonu/nu BALB/c mice, which were infected with vaccinia virus. Specific Sensitization of transferred thymus-derived cells was determined in vivo by mean survival time and virus titer in the spleen six days after infection, and in vitro by cell-mediated cytolysis of vaccinia virus-infected syngeneic target cells. Virus-specific Sensitization took place only after transfer of syngeneic or semiallogeneic spleen lymphocytes; allogeneic lymphocytes had no influence on mean survival time or virus titer and showed no virus-specific cytolytic activity in vitro. Infection of mice with vaccinia virus-strain WR, Elstree, DIs, or DIs-infected syngeneic fibroblasts resulted in the generation of virus-specific effector cells, while injection of a high amount of inactivated virus particles caused no Sensitization. These results suggest H-2 homology for production of virus-specific effector cells. Propagation of virus is not necessary, since early surface antigens, combined with syngeneic H-2 antigens, suffice for Sensitization of cytolytic T lymphocytes.Abbreviations used in this paper are as follows CMC cell-mediated cytolysis - CTL cytolytic T lymphocyte - LCM lymphocytic choriomeningitis - MHC major histocompatibility complex - MST mean survival time - T cell thymus-derived cell - TCID₅₀ 50 percent tissue culture infective dose     

An early event in murine cytomegalovirus replication inhibits presentation of cellular antigens to cytotoxic T lymphocytes.

Cytomegalovirus (CMV) infection of simian virus 40 (SV40)-immune mice inhibits priming of SV40-specific helper and cytotoxic T lymphocytes (CTL) in vivo (A. E. Campbell, J. S. Slater, and W. S. Futch, Virology 173:268-275, 1989; J. S. Slater, W. S. Futch, V. J. Cavanaugh and A. E. Campbell, Virology 185:132-139, 1991). We now demonstrate that murine CMV (MCMV) infection of SV40-transformed macrophages and fibroblasts prevents presentation of SV40 T antigen to SV40-specific CTL. MCMV-infected macrophages failed to stimulate SV40-immune CTL precursors in vitro. In addition, MCMV-infected, SV40-transformed macrophage and fibroblast target cells lost their susceptibility to lysis by major histocompatibility complex class I-restricted, SV40-specific CTL clones. MCMV infection did not alter the synthesis of SV40 T antigen in the target cells. MCMV early gene expression was required for inhibition of SV40 T-antigen presentation; immediate-early gene expression was insufficient for this effect. Early viral gene expression also resulted in significant reduction of H-2K and H-2D molecules on the surface of MCMV-infected fibroblasts. However, this reduction occurred independently from suppression of antigen presentation to CTL. The same target cells which were resistant to lysis by SV40 CTL were susceptible to lysis by MCMV-specific CTL. MCMV early gene products therefore interfere with the processing and/or presentation of SV40 T-antigen determinants to CTL independent of alterations in the major histocompatibility complex.     

Normal T-cell receptors for alloantigens

To study the diversity of normal mouse T lymphocytes capable of mediating allograft immunity, we modified a cell culture system so that both induction of sensitization and target cell damage could be studied in vitro. Mouse lymph node lymphocytes were sensitized in vitro against allogeneic fibroblasts. The sensitized lymphocytes produced immunospecific cytotoxic effects against target fibroblasts in vitro. We found that T lymphocytes were directly involved in both sensitization and cytotoxicity.We used this allograft system to separate nonsensitized mouse lymphocytes on the basis of their ability to bind to allogeneic fibroblasts. Adhering lymphocytes were found to be enriched in effector cells following sensitization. The nonadhering lymphocytes showed a decreased ability to undergo sensitization against fibroblasts that were syngeneic to the ones used for adsorption. However, they were able to become sensitized against unrelated fibroblasts of another H-2 phenotype.These findings indicate that specific receptors for histocompatibility antigens pre-exist on diverse populations of normal mouse T lymphocytes.     

Studies of H-2 restriction in cell-mediated cytotoxicity and transplantation immunity to Leukemia-associated antigens.

H-2 dependency of T cell-mediated cytotoxicity and transplantation immunity to leukemia-associated antigens has been investigated. Through the use of a 20-hr 125IUdR release assay, it was found that the induction of T cell-mediated cytotoxicity against Friend virus-induced leukemias of different H-2 haplotype orgins could be produced by immunization with both syngeneic and allogeneic tumor cells; the effector cells that were generated by syngeneic immunization could also provide effective killing of allogeneic tumor cells, although the killing of allogeneic targets might require a longer incubation time (20 to 40 hr). Furthermore, in vivo transplantation immunity against Friend virus-induced leukemias also was induced by immunization with both syngeneic and allogeneic tumors and syngeneic immunization could induce specific protection against the challenge with a-logeneic tumor in x-irradiated hosts. These findings clearly indicate that, both at the sensitizing phase and effector phase of the immune response, there is no strict H-2 dependency for T cell-mediated cytotoxicity or in in vivo transplantation imunity to leukemia-associated antigens.     

Immune response to a syngeneic mammary adenocarcinoma. II. In vitro generation of cytotoxic lymphocytes.

A method is described for the consistent in vitro generation cytotoxic cells by incubating Fischer 344 rat spleen cells on monolayers of a syngeneic mammary adenocarcinoma. Significant cytotoxicity by in vitro culture is generated as early as 3 days after initiation and effector cells are cytolytic only toward target cells of the sensitizing monolayer. Reciprocal sensitization with allogeneic fibroblasts as the immunizing monolayer yielded effector cells cytolytic for the fibroblasts but without effect on the mammary tumor. The consistency in the generation of cytotoxic cells by in vitro culture should permit its standardized use in following other related immune phenomena such as blocking by serologic factors and suppression, recritment of memory for cytotoxic function.     

Viral gene inhibition of class I major histocompatibility antigen expression: not a general mechanism governing the tumorigenicity of adenovirus type 2-, adenovirus type 12-, and simian virus 40-transformed Syrian hamster cells

The association between the level of class I major histocompatibility (MHC) antigen expression and the tumorigenic phenotype was determined for cells from a series of 15 lines of adenovirus type 2 (Ad2)-, Ad12-, and simian virus 40 (SV40)-transformed hamster cells and 16 lines of cells established from hamster tumors induced by SV40 mutants. These cells range from nontumorigenic to highly tumorigenic in both syngeneic and allogeneic adult hamsters. The Ad2-transformed cells--cells that were nontumorigenic in syngeneic adult hamsters--expressed either high levels or low levels of class I MHC antigens. The SV40-transformed cells--cells transformed in vitro that produced tumors with equal efficiency in both syngeneic and allogeneic adult hamsters--or cells derived from SV40-induced tumors expressed very high levels of class I MHC antigens. The Ad12-transformed cells uniformly expressed low levels of class I MHC antigens; these cells produced tumors 200- to 1,000-fold less efficiently in allogeneic adult hamsters than in syngeneic adult hamsters and produced tumors with about the same efficiency in immunoimmature newborns and immunocompetent syngeneic adult hamsters. We conclude that the expression of either high levels or low levels of class I MHC antigens is, at most, a minor factor in the differences observed among these adenovirus- and SV40-transformed cells in their tumor-inducing capacity in naive, immunocompetent hamsters.     

Cytotoxicity of autosensitized lymphocytes restricted to the H-2K end of targets

Lymphocytes from rodents cultured on syngeneic fibroblasts become cytotoxic against syngeneic but not against allogeneic target cells. We investigated whether known antigens are involved in the phenomenon and the data indicate that H-2 antigens must be shared between sensitizing fibroblasts and responder lymphocytes to generate autocytotoxic cells. Furthermore, the cytotoxicity of autosensitized lymphocytes is restricted to target cells identical with respect to theK and/orI regions. F₁ hybrid lymphocytes cultured on parental fibroblasts develop cytotoxicity towards sensitizing cells. In contrast, parental lymphocytes cultured on F₁ hybrid fibroblasts will not damage the F₁ cells, although they are cytotoxic against both syngeneic and allogeneic parental cells. In addition, parental or F₁ hybrid lymphocytes cultured on parental fibroblasts are not cytotoxic against F₁ hybrid target cells. Fibroblasts heterozygous for theK end only, are also resistant to the cytotoxic action of such lymphocytes. Thus it seems that H-2 antigens, specifically theK end, antigens have a significant role in the phenomenon of autosensitization.     

SV40 T-antigen is a histocompatibility antigen of SV40-transgenic mice

Although the extensive family of non-H-2 histocompatibility (H) antigens provides a formidable barrier to transplantation, the origin of their encoding genes are unknown. Recent studies have demonstrated both the linkage between H genes and retroviral sequences and the ability of integrated Moloney-murine leukemia virus to encode what is operationally defined as a non-H-2 H antigen. The experiments described in this communication reveal that skin grafts from an SV40 T-antigen transgenic C57BL/6 mouse strain are rejected by coisogenic C57BL/6 recipients with a median survival time of 49 days, which is comparable to those of many previously defined non-H-2 H antigens. The specificity of this response for SV40 T-antigen was demonstrated by the identification of SV40 T-antigen-specific cytolytic T lymphocytes and antibodies in multiply-grafted recipients. Although these cytolytic T lymphocytes could detect SV40 T-antigen on syngeneic SV40-transformed fibroblasts, they neither could be stimulated by splenic lymphocytes from T-antigen transgenics nor could they lyse lymphoblast targets from T-antigen transgenics. These observations suggest a limited tissue distribution of SV40 T-antigen in these transgenics. These results confirm the role of viral genes in the determination of non-H-2 histocompatibility antigenes by the strict criteria that such antigenes stimulate (1) tissue graft rejection and (2) generation of cytolytic T lymphocytes. Furthermore, they suggest that the SV40 enhancer and promoter region can target expression of SV-40 T-antigen to skin cells of transgenic animals.     

Antibody blockade of lysis by T lymphocyte effectors generated against syngeneic SV40 transformed cells.

Antibody blockade of cell-mediated lysis was used to probe the cell surface structures recognized by T lymphocytes generated to syngeneic SV40 virus-transformed mouse cells. Alloantisera to H-2 antigens were highly effective in inhibiting lysis. Anti-H2 antibody blockade of lysis was haplotype specific even on transformed F1 target cells. Inhibition occurred only when antibody was bound to the target cell. Antibody binding to the effector did not inhibit lysis. Inhibition required that antibody be bound to the H-2 molecule itself; antibody to molecules associated with H-2, such as beta2-microglobulin, had no effect. Attempts to block lysis by using antisera to known virus-coded molecules were uniformly unsuccessful. These results are discussed in light of current controversy concerning the nature of the SV40 virus-specific transplantation antigen.     

Specific T-cell-mediated killing of autologous lung tumour cells

The phenomenon that strong syngeneic T-cell-mediated cytotoxicity is observed if killer, stimulator, and target cells share H-2 histocompatibility antigens is called H-2 restriction. Here a syngeneic model system making use of hapten-coupled stimulator and target cells is used to explore whether H-2 restriction is absolute or not. Using TNP-coupled spleen or tumor cells as stimulator or target cells in syngeneic and allogeneic situations, it is shown that neither the induction step nor the effector step of TNP-dependent killing is H-2 restricted. By varying the experimental assay conditions more or less H-2-restricted, TNP-dependent killing can be observed. For instance, suboptimal coupling of TNP to targets may result in H-2-restricted killing. Similarly, the use of spleen cell targets as opposed to spleen blast cells or tumor cells may result in H-2-restricted lysis. In contrast optimal coupling of TNP to sensitive target cells and coupling of TNP to cells with certain H-2 haplotypes may lead to significant TNP-dependent killing which is not H-2 restricted. Since hapten-coupled cells lacking H-2 are neither stimulators nor targets these results suggest that the T-cell receptor recognizes TNP-modified H-2 antigens simply as nonself-H-2. Thus hapten coupling of syngeneic cells appears to lead to a histocompatibility antigen change similar to the situation in an allogeneic cytotoxic reaction. Experiments are presented which support this view showing that TNP-coupled and uncoupled syngeneic or allogeneic stimulator and target cells cross-react. For instance allogeneic sensitization may lead to killing on TNP-coupled targets syngeneic to the effector cells and TNP-coupled stimulator cells syngeneic to the effector cells may induce killing on uncoupled syngeneic targets. TNP-dependent cytotoxicity can therefore be envisaged as a kind of allogeneic reactivity due to modification of H-2 antigens by the TNP coupling. This conclusion may have bearing on other model systems in which syngeneic killing appears to be H-2 restricted. In support of this possibility it is shown that allogeneic sensitization may lead to priming of memory cells able to respond to minor histocompatibility antigens.     

LY-2+ effectors cytotoxic for syngeneic tumor cells: generation by allogeneic stimulation and by supernatants from mixed leukocyte cultures

The present study was aimed at gaining insight into means by which stimulation of mouse spleen cells with allogeneic normal cells in mixed leukocyte cultures (MLC) can result in the generation of effector cells cytotoxic for syngeneic tumor or transformed cells. Stimulation of lymphocytes from BALB/c or C3H mice for 5 days with cells from mice of every allogeneic strain tested, in medium containing mouse serum and lacking xenogeneic serum, resulted in the activation of effectors cytotoxic for syngeneic cells transformed spontaneously or by SV40, polyoma or adenovirus. In each experiment, all of the syngeneic transformed cell lines, as well as clones derived from these lines, were lysed to the highest degree by effectors obtained from the same culture, and therefore stimulated with cells from the same allogeneic strain. Although the particular allogeneic sensitizing strain that induced the highest cytolytic activity varied between experiments, effectors obtained from the culture with the highest cell recovery always exhibited the greatest cytotoxicity against all the syngeneic transformed cells and clones. Lysis was mediated predominantly by Ly-2+ effectors; total lytic units of cytotoxicity recovered after treatment with monoclonal anti-Ly-2 antibody and complement (C) were reduced by 85 to 90% compared to cells treated with C alone. Lysis of syngeneic tumor cells by the allosensitized effectors in cytotoxicity assays was not inhibited by the addition of unlabeled "blocking" lymphocytes from the allogeneic strain used for sensitization. In addition, it was found that lymphocytes cultured without stimulating cells for 5 days in medium supplemented with supernatants from secondary MLC that are known to contain high levels of lymphokines, mediated high levels of cytotoxicity on all the transformed cells tested, but lacked detectable cytotoxic activity for syngeneic or allogeneic Con A blasts. The MLC supernatant-activated effectors that lyse the transformed cells are phenotypically CTL, because treatment with anti-Ly-2 and C reduced lytic activity by approximately 75%. Taken together, these findings suggest that the generation in MLC of Ly-2+ effector cells cytotoxic for syngeneic transformed cell lines might not be due, in some cases, to lymphocyte responses to particular alloantigens on the stimulating cells that are cross-reactive with "alien" histocompatibility antigens on transformed cells, but rather is due to effector cell activation by lymphokines produced during allogeneic stimulation.     

H-2 I alloantigens and recall of memory cytotoxic responses

AQR mice were immunized with H-2K and H-2 I encoded alloantigens presented by (Ax6R)F₁ splenocytes. Spleen cells from these alloimmune mice were subsequently restimulated in vitro with B10.A lymphocytes and/or B10.T(6R) lymphocytes, thus presenting them with the immunizing H-2K and H-2 I alloantigens independently. When stimulated with B10.A lymphocytes, alloimmune lymphocytes develop significant cytotoxicity against the immunizing H-2K target antigens. When stimulated with a similar number of B10.T(6R) spleen cells, alloimmune lymphocytes undergo a prominant proliferative response, but develop little, if any, cytotoxicity against the immunizing H-2 K target antigens. The most efficient restimulation of cytotoxicity occurs when the alloimmune spleen cells are simultaneously restimulated by B10.A and B10.T(6R) lymphocytes. Stimulation with the immunizing H-2 I alloantigens alone is not sufficient for regeneration of detectable cytotoxic responses from alloimmune spleen populations. Stimulation with the immunizing H-2K alloantigens alone appears to be both necessary and sufficient to stimulate alloimmune cytotoxic responses. Although the immunizing H-2 I alloantigens are apparently not required to generate alloimmune cytotoxic responses, they markedly potentiate the cytotoxic responses induced by the immunizing H-2K alloantigens.