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.     

Studies on the specificity of in vitro induced lymphocytotoxicity to SV40-transformed fibroblasts.

Cytotoxic effector lymphocytes were induced by in vitro immunization of lymph node and spleen cells from AKR-mice (H-2k) and from BALB/c-mice (H-2d) to syngeneic SV40-transformed fibroblasts. The T cell-dependent cytotoxicity was specific for target cells expressing the same H2-specificity as the immunizing cells. Nontransformed fibroblasts as stimulator cells did not induce efficient cytotoxicity to transformed or nontransformed target cells. Incubation with phytohemagglutinin during the sensitization period modified the specificity of the T cell-mediated lysis of syngeneic SV40-transformed fibroblasts: allogeneic as well as syngeneic target cells were destroyed by these effector cells. However, the polyclonal stimulant activates preferentially cytotoxicity to H2-matched target cells. The in vitro generation of cytotoxic effector cells was restricted to living SV40-transformed fibroblasts as immunizing cells; it was not possible to immunize lymphocytes in the presence of membrane proteins prepared from the SV40-transformed cells. The cytotoxicity of the in vitro immunized lymphocytes was inhibited by incubation with membrane protein preparations from syngeneic or allogeneic SV40-transformed fibroblasts.     

Murine T-cell-mediated cytotoxicity against syngeneic and allogeneic cell lines induced by fetal calf serum

Mouse spleen cells from normal animals developed easily measurable cytotoxicity against various cell lines when cultured in vitro without deliberate sensitization. Cytotoxicity, measured by a 3-hr ⁵¹Cr-release assay, was maximum on Days 3 and 4 of culture and was dependent on the presence of fetal calf serum. Neither cell recovery nor blastogenesis, however, invariably correlated with the amount of cytotoxicity generated. Nylon wool adsorption of effector cells cultured 3 days had only a marginal effect on cytotoxicity, whereas cytolysis was markedly reduced (but not totally eliminated) by treatment with anti-T-cell serum and complement. When target cells were in relative excess to effector cells, ⁵¹Cr release was proportional to effector cell number and proceeded for at least 22 hr. The cytotoxicity was not tumor or H-2 specific, and targets without known C-type viral antigens (gp71) were killed as readily as those with easily measurable viral antigens. Nontumorigenic fibroblasts were lysed, but concanavalin A-induced blast cells were not. Cytotoxicity was not augmented in cultures of spleen cells from mice injected with fetal calf serum or with tumor fragments exposed to fetal calf serum. Mercaptoethanol was not necessary for the generation of cytotoxic activity, but T cells and Sephadex G-10 or nylon wool-adherent cells were necessary and the function of the adherent cell could not be replaced by mercaptoethanol. Removal of plastic adherent cells had no effect. Fetal calf serum retained its activity when heated for 45 min at 56 °C or when dialyzed. Dilution and reconcentration by Amicon filtration revealed that the mass of the active material was between 30,000 and 100,000 daltons.The early appearance, transient nature, and nonspecificity of this cytotoxicity distinguish it from antigen-specific reactions. The effector's stability at 37 °C and its relatively easily detectable T-cell markers distinguish it from natural killer and cytotoxic cells. This activity is like lectin-induced cytotoxicity but differs because allogeneic blast cells are not lysed. The observed cytotoxic activity may be of in vivo relevance (vis-à-vis natural killer cells) or, more likely, an in vitro expression of a stage of cell differentiation that T cells may normally pass through during their response to antigen.     

Regulatory mechanism of delayed-type hypersensitivity in mice. II. Effect of suppressor cells on the development of memory cells for delayed-type hypersensitivity

Murine lymph node cells (LNC), which we showed previously to noncompetitively inhibit antibody-dependent cellular cytotoxicity (ADCC) to an erythrocyte target, were tested for their ability to inhibit ADCC to a tumor target, EL-4. Both a 4-hr ⁵¹Cr-release cytotoxicity assay and an overnight ¹²⁵IUdR (iododeoxyuridine) postlabeling cytostasis assay were used. Normal autologous lymph node cells inhibited spleen cell-mediated ADCC in both assays. Inhibition by LNC was dose dependent, but comparable numbers of sheep erythrocytes did not inhibit, indicating that LNC-mediated inhibition was not simply a matter of crowding. Inhibitory activity was enriched in LNC after removal of Fc receptor-bearing cells on EA monolayers.     

Defective T helper activity in the spleen of BALB/c mice immune to a syngeneic fibrosarcoma

Summary BALB/c mice were immunized with the syngeneic 3-methylcholanthrene-induced fibrosarcoma CA-2 by the growth and excision method. When lymphoid cells from different organs of these tumor-free mice were tested in a direct ⁵¹Cr-release assay, peritoneal exudate cells but not spleen cells displayed specific cytotoxicity against the syngeneic tumor target. A cytotoxic response could be obtained by tumor-immune spleen cells when cultured in a mixed lymphocyte tumor cell culture (MLTC) at high but not low density although at the same effector/stimulator ratio. Lack of cytotoxic activity in low density MLTC was not due to an impairment of cytotoxic precursors since cytotoxicity was rescued by adding exogenous interleukin-2 in experimental conditions in which no lymphokine-activated killer cells could develop relevant anti-CA-2 lysis. When low density MLTC were supplemented with either 800 R-irradiated cells or nonirradiated, negatively selected Lyt 1⁺ cells from the same immune mice, induction of a cytotoxic response against CA-2 occurred and interleukin-2 production became detectable. Additional studies indicated that spleen cells of CA-2-immune mice were also impaired in their ability to provide help to syngeneic thymocytes for the generation of cytotoxic T lymphocytes against C57BL/6J alloantigens. Dilution effect of helper cells due to immunization procedures was excluded since spleen cells of mice immunized against another BALB/c tumor, the YC8 lymphoma, or against DBA/2 minor histocompatibility antigens provided good help to thymocytes against the same alloantigens. These results indicate that tumor-immune animals may also have selective T helper defects in an important lymphoid organ like spleen.     

Lectin-dependent cell-mediated cytotoxicity in hamsters bearing syngeneic tumors

Spleen cells from LSH hamsters inoculated with xenogeneic, allogeneic, or syngeneic (PARA-7) tumor cells were assayed for their ability to mediate direct cell-mediated cytotoxicity (DCMC) and lectin-dependent cell-mediated cytotoxicity (LDCC) in a 4-hr chromium release assay. Spleen cells from animals immune to xenogeneic or allogeneic cells demonstrated specific DCMC against homologous target cells in the absence of Con A and nonspecific LDCC against both homologous and heterologous target cells in the presence of Con A. Spleen cells from animals bearing syngeneic PARA-7 tumors (TBA) failed to express DCMC against homologous or heterologous target cells; however, significant lysis of all target cells occurred in the presence of Con A. LDCC was not detectable when nonsensitized spleen cells from normal animals were employed. The LDCC reaction was dependent on the concentration of Con A and the number of effector cells present in the reaction. The development of LDCC effector cells in the TBA appeared to parallel the development of both DCMC and LDCC effector cells in immune animals.     

The influence upon mitogenic and cellular immunologic reactive systems in vitro by poly(I:C) and BCG murine interferons induced in vivo.

We have previously reported that lymphocytes from W/Fu rats immunized with syngeneic (C58NT)D tumor cells were cytotoxic against these cells in a 4-hr ⁵¹Cr release assay. We have investigated the feasibility of cryopreserving lymphocytes and target cells and have selected freezing conditions which provide good yields of viable cells and functional activity. Lymphocytes from different animals had a recovery of 60–80% viability which resulted in a corresponding 55–75% recovery of cytotoxic activity. Repeated testing of lymphocyte cytotoxicity from a pool of frozen spleen cells against either fresh or frozen (C58NT)D cells gave reproducible cytotoxicity. In addition, recovery of high levels of lymphocyte function was also demonstrated when cryopreserved cells were employed in long-term cytotoxic assays, i.e., ³H-proline and ¹²⁵IUdR release assays, in the lymphoproliferative response to mitogens (PHA and Con A)³ or tumor cells (MLTI) as measured by ³H-thymidine incorporation, and in the in vitro generation of secondary cytotoxicity.By employing these cryoprotective techniques it is possible to have: 1) a population of lymphoid cells with known functional activity and 2) a pool of target cells with known susceptibility to lysis and antigenic content. Furthermore, the use of frozen cells as internal standards in each test also permits the analysis of assay variation as well as the study of variation in various cell types.     

Xenogeneic serum-induced murine cytotoxic cells. I. The generation of effector components specific for self and allogeneic target cells.

Spleen cells from unimmunized mice cultured in vitro without the intentional addition of exogenous antigen generated cytotoxic effector cells which lysed tumor and mitogen-stimulated blast target cells in a 5-hr chromium release assay. Effectors were generated in fetal bovine serum but not in adult horse serum, although both serum sources supported the generation of allogeneic cytotoxic cells. No correlation was observed between the ability of a serum source to support and generate serum-induced effectors and its ability to support an allogeneic cytotoxic response. The effectors lysed targets which were H-2 matched and those which were not H-2 matched with the cultured spleen cells, although the H-2 matched targets were consistently lysed more efficiently. “Cold” target cell inhibition studies indicated that multiple clones of cytotoxic cells were generated—including effector cells with specificity for self-structures, and particular alloantigens. Possible roles for the xenogeneic serum in the generation of this response are considered, including (a) the provision of essential stimulating and target antigens; (b) the induction of the expression of neoself-determinants; and (c) the possession of mitogenic properties.     

Comparison of monocyte and alveolar macrophage antibody-dependent cellular cytotoxicity and Fc-receptor activity

The cytotoxic potential of rabbit peripheral blood monocytes and alveolar macrophages in antibody-dependent cellular cytotoxicity (ADCC) toward both erythrocyte (RBC_ox) and tumor cell (CEM T-lymphoblast) targets was examined. ADCC was measured in a 4-hr ⁵¹Cr-release assay. Alveolar macrophages were more efficient at killing the tumor cell targets (optimally sensitized with rabbit antisera) than monocytes at similar effector cell/target cell ($\text{E}\text{T}$) ratios. Tumor cell targets sensitized with seven different antisera (anti-CEM) were lysed by alveolar macrophages but not by the monocytes. In marked contrast, the monocytes were more effective at lysing the sensitized erythrocyte target cells. The degree of cytolysis of RBCox and CEM was dependent on the $\text{E}\text{T}$ ratio and the degree of sensitization of these target cells. It was demonstrated that the effector cell selectivity in ADCC was directly related to their ability or inability to bind the sensitized target cells as determined by Fc-receptor rosette formation. The transition from monocyte to macrophage may, therefore, have resulted in an alteration in the criteria necessary for Fc-receptor binding to antibody-sensitized target cells and subsequent ADCC.     

Lysis of fresh human solid tumor cells by autologous lymphocytes activated in vitro by allosensitization

Summary We have previously demonstrated that cancer patients' peripheral blood lymphocytes (PBL) allosensitized against single or pool normal donor PBL are capable of lysing fresh autologous tumor cells in a 4-h ⁵¹Cr-release assay. In this report, we present further investigations into this phenomenon. These alloactivated killer cells (A-AK cells) lysed autologous and allogeneic tumors and allogeneic but not autologous PBL. Furthermore, cold target inhibition studies demonstrated that autologous and allogeneic tumors were lysed by the same effector cells. Multiple metastases from the same patient were equivalently lysed by these A-AK cells. The presence of adherent cells and proliferation of the precursors were necessary to generate A-AK cells, although the effector cell itself was radioresistant and nonadherent. The effects of allosensitization were enhanced by the addition of lectin-free interleukin-2 preparations to the in vitro culture by partial depletion of adherent cells prior to sensitization. The A-AK effector cell was OKT3+, OKT8+, OKT4–, OKM1– and could be generated by just 3 days of allosensitization. The precursors for A-AK cells could be separated from NK cells on percoll gradients and lysis could be generated from thoracic duct lymphocytes, a population devoid of NK cells. The phenotype of the majority of the precursor cells was OKT3+, OKT4–. These allocatived PBL could be expanded in crude or lectin-free interleukin-2 without loss of cytotoxicity for fresh autologous tumor cells. Activated T cells represent a population of non-NK cells with broad lytic specificity for fresh tumor cells. Such cells may be of value in the adoptive immunotherapy of human solid tumors and may play a role in immune surveillance.     

Suppression of in vitro maintenance and interferon-mediated augmentation of natural killer cell activity by adherent peritoneal cells from normal mice

The ability of adherent peritoneal cells (APC) to inhibit murine natural killer (NK) cell activity was examined. Nylon wool-nonadherent splenic effector cells were incubated overnight with or without different numbers of APC. NK activity was then measured against YAC-1 in a 4-hr 51Cr-release cytotoxicity assay. Proteose peptone-elicited or unstimulated resident APC from normal mice markedly suppressed NK activity of splenic effector cells in the presence or absence of exogenously added interferon. The suppression was dependent on the number of APC added with 10% APC, relative to the number of effector cells, resulting in a greater than 65% inhibition of cytotoxicity. The effector phase of cytotoxicity was not the target of the suppressor cells, because APC did not suppress NK activity when they were present only during the cytotoxicity assay. The addition of APC to alloimmune cytotoxic T cells under similar conditions resulted in no inhibition of cytotoxicity. Both syngeneic and allogeneic APC suppressed NK activity, but several murine macrophage-like cell lines lacked this property. In contrast to APC, incubation of effector cells with adherent spleen cells from normal mice resulted in no inhibition of NK activity. APC from mice injected with C. parvum were less inhibitory for NK activity than normal resident APC. In contrast, C. parvum APC suppressed concanavalin A-induced lymphoproliferation and were directly cytotoxic to tumor target cells in vitro, whereas normal APC lacked these properties. The results indicate that the peritoneum of untreated mice contains suppressor cells that can inhibit the in vitro maintenance and IFN-mediated augmentation of NK activity. In addition, these results indicate a broader spectrum of immune reactivities regulated by APC and suggest that, depending on their level of activation, APC can preferentially inhibit different immune functions.     

Specific immune recognition of autologous tumor by lymphocytes infiltrating colon carcinomas: Analysis by cytokine secretion

Summary Tumor-infiltrating lymphocytes (TIL) were grown in the presence of interleukin-2 from 19 colon carcinoma specimens, including 1 primary lesion and 18 metastatic lesions. These cultures showed a median proliferation of 606-fold (range 13-fold to 28 000-fold) over 49 culture days (range 26–76 days). By phenotype, mature cultures were 69%–99% CD3⁺ (mean 93%) and contained mixed populations of CD4⁺ and CD8⁺ cells (CD4>CD8 in 10 of 19 cultures). Fresh cryopreserved colon tumors were not lysed by autologous TIL in short-term⁵¹Cr-release assays, and were poorly lysed by lymphokine-activated killer cells. Ten TIL cultures were assayed for cytokine secretion in response to autologous and allogeneic tumors during a 6- to 24-h coincubation. Culture supernatants were tested by ELISA for the presence of granulocyte/macrophage-colony-stimulating factor, interferon , and tumor necrosis factor . Of 10 TIL, 4 secreted at least two of these cytokines specifically in response to autologous and/or HLA-matched fresh allogeneic colon carcinomas, but not to melanomas or HLA-unmatched colon carcinomas. Cytokine secretion was mediated by both CD4⁺ and CD8⁺ TIL, and could be inhibited by mAb directed against the appropriate class of MHC antigen. These data provide evidence for specific, MHC-restricted immune recognition of human colon carcinomas by T lymphocytes.     

Natural killer cell activity in mouse aggregation chimeras

The activity of natural killer (NK) cells in spleen against syngeneic and allogeneic tumor cells was studied by the use of tetraparental mouse chimeras. Chimeras were produced by aggregation of early embryos of histoincompatible mouse strains of “high” and “low” NK cell activity. NK activities of spleen cells were assayed in vitro by the ⁵¹Cr-release method. Coat color distribution and isozymal analysis (glucose-phosphate isomerase) of several lymphoid organs (thymus, lymph nodes, and bone marrow) revealed a predominant share of the “high”-NK-reactive genotype in the chimeras. However, the cellular NK activity against two target cell lines differing in their susceptibility to lysis was significantly lower in chimeras than in the “high”-reactive strain. Addition of “low”-NK spleen cells or of NH₄Cl-inactivated “high”-NK spleen cells to “high”-NK spleen cells inhibited their cytolytic activity. Possible mechanisms of the suppression of the cytolytic capacity of NK cells in chimeras are discussed.     

Regulatory T cells in pregnancy. VI. Evidence for T-cell-mediated suppression of CTL generation toward paternal alloantigens

The reactivity of spleen cells from allogeneically pregnant mice was assayed versus paternal strain target cells by a direct ⁵¹Cr-release assay. Despite multiple allogeneic parities, the lytic indexes of spleen cells were equivalent to those observed with nonpregnant controls. In view of previously obtained in vivo and in vitro results, spleen cells of allogeneically pregnant mice were added at the onset of MLC-CMLs³ of maternal strain responder cells versus paternal strain stimulator and target cells and studied for regulatory capacities. They did exert a suppressive effect, assessed by ⁵¹Cr release per culture. For the most, this effect was on the CTL induction, not the effector phase. The suppression of CTL generation was specific and mediated by a Thy 1⁺, Ly 2⁺ cell.     

Mutations in H-2K b influence the specificity of alloreactive effector cells included in the repertoire of H-2D b -restricted cytotoxic T lymphocytes against Moloney leukemia virus

Moloney leukemia virus-specific cytotoxic T lymphocytes (CTL), generated by secondary in vitro stimulation of spleen cells with syngeneic virus-infected cells, frequently lysed not only syngeneic virus-infected cells, but also noninfected allogeneic target cells. This phenomenon was studied with B6(H-2 ^b ) responder cells and a series of H-2K ^b -mutant responder cells. Thus, B6 Moloney-specific CTL lysed noninfected K ^b -mutant cells, but not B6 cells, whereas K ^b -mutant Moloney-specific CTL lysed noninfected B6 cells and not noninfected cells of the same mutant. Cold-target-inhibition studies showed that the CTL reactions against different allogeneic cells were mediated by different subpopulations of virus-specific CTL: lysis of allogeneic target cells was fully inhibited only by the same allogeneic and by syngeneic virus-infected cells, but not by another allogeneic cell, also lysed by the same effector-cell population. Lysis of syngeneic virus-infected cells could not be inhibited by allogeneic target cells. These data imply that a minority of virus-specific CTL shows cross-reactivity with a given allogeneic target cell. It is concluded that limited amino acid substitutions in the K^b molecule alter the repertoire of Moloney virus-specific CTL, as reflected in alloreactive CTL populations, even though the virus-specific CTL response. of B6 and all K ^b mutants is mainly D^b-restricted. Thus, the development of tolerance to self class-I major histocompatibility complex (MHC) molecules affects the repertoire of self-restricted cytotoxic T cells.     

Effect of aging on T-cell tolerance induction

In the MHC compatible rat strain combination AS → HS, the same ⁵¹Cr-labeled lymph node suspension behaves totally differently depending on whether it is injected into syngeneic or allogeneic recipients. Using ⁵¹Cr-labeled lymph node suspensions from AS or (AS × HS)F1 donors, and AS, HS, and F₁ hosts, the distribution of label over a 72-hr period was studied. Evidence has been obtained that recognition of self-components results in the homing of cells to the lymph nodes in syngeneic hosts, while recognition of foreignness impairs homing of the same cells to the lymph nodes in allogeneic hosts. The spleen is the major organ in which these recognition processes occur. Substantially more cells are destroyed by nonimmune allogeneic hosts than by syngeneic hosts, a clear difference being apparent as early as 6 hr after injection. Some lymphoid cells are obligatory spleen seekers and do not enter the lymph nodes.     

Differential sensitivity to natural cell-mediated cytotoxicity of two rat colon adenocarcinoma variants differing in their tumorigenicity: identification of the effector cells as natural killer cells

Summary DHD/K12 TRb (PROb) and DHD/K12 TSb (REGb) are two cancer cell variants originating from the same rat colon adenocarcinoma. They differ in their tumorigenicity: when inoculated into syngeneic BDIX rats, PROb cells induce progressive tumors whereas REGb cells induce tumors which always regress. As previously described, there is an inverse relation between their tumorigenicity and their susceptibility to NCMC mediated by syngeneic spleen or peripheral blood lymphocytes: PROb cells are significantly less sensitive to NCMC than REGb cells. This suggests a role for NCMC in the regression of REGb tumors. In this work the BDIX NCMC effector cells active in vitro against REGb cells were identified as NK cells according to four criteria: (1) efficacy in a 4-h ⁵¹Cr release assay, (2) sensitivity to anti-asGM1 antibody plus complement, (3) LGL morphology, and (4) ability to bind with the same affinity REGb and YAC-1 cells. In spleen, these NK cells were heterogeneous with respect to their asGM1 surface density and their morphology. PROb cells were not lysed by these NK cells in a short-term cytotoxicity assay, but only in a 16-h assay. It was shown that PROb and REGb cells were bound with the same affinity by NK cells, thus they certainly differ in their ability to resist to NK lytic mechanisms. This difference could play a role in the different tumorigenicity of the two variants. Abbreviations used: NK, natural killer; NC, natural cytotoxic; NCMC, natural cell-mediated cytotoxicity; asGM1, asialo GM1; LL, large lymphocytes; LGL, large grnular lymphocytes; LAL, large agranular lymphocytes; PBMNC, peripheral blood mononuclear cells; E:T, effector to target cell ratio; C:H, cold to hot cell ratio; FBS, fetal bovine serum     

Studies of the immunological capacity of germ-free mouse radiation chimeras. IV. Cell-mediated immunity

The cell-mediated immune (CMI) response of germ-free mouse radiation chimeras was compared with that of conventional mice. Spleen or thymus cells from chimeric or normal mice were injected intravenously into lethally irradiated, allogeneic hosts. Spleens of the irradiated hosts were assayed for effector cells using the ⁵¹Cr release assay. Spleen cells from syngeneic and allogeneic chimeras and normal mice were equally active in giving rise to effector cells. However, thymus cells from allogeneic chimeras were completely inactive within 9 months post-bone marrow transplant while thymus cells from syngeneic chimeras and normal mice still remained functional. Although allogeneic chimeras contain cells potentially reactive toward host antigens, cells cytotoxic to host antigens were not detectable. In addition, these studies indicate the helper cell and effector cell, both associated with T-derived lymphocytes, represent two different populations.     

Cell-mediated cytotoxicity against syngeneic tumors

Spleen cells from DBA/2 mice bearing the DBA/2 P815X mastocytoma for approximately 2 weeks can be stimulated in vitro by mastocytoma cells to generate cytotoxicity measured as ⁵¹Cr release from mastocytoma cells in a 4-hr assay. These cytotoxic cells will not kill allogeneic cell lines but will kill a series of first transplant generation syngeneic tumors. T cells are involved in that treatment of the responding or the cytotoxic cell populations with either anti-T or anti-theta antibody + complement will abrogate all cytotoxicity. Anti-Ly 2.1 antibody + complement treatment of either responder cells (prior to the in vitro culture with irradiated tumor cells) or effector cells after culture markedly decreases cytotoxicity whereas treatment with anti-Ly 1.1 was more effective prior to culture compared to its effect on cytotoxic cells per se. These T cells are in the small lymphocyte class and occur either singly or in aggregates. Suppression of antisyngeneic tumor cytotoxicity by antibody inhibits preferentially the expression of cytotoxicity in the aggregate fractions.     

Alloantigen-induced cytotoxicity against syngeneic tumor cells: analysis at the clonal level

It has been shown that peritoneal exudate cells (PEC) from BALB/c mice immunized with minor histocompatibility antigens presented by DBA/2 or B10.D2 spleen cells are capable of lysing syngeneic YC8 tumor cells in a 4-hr 51Cr-release assay. In this study, we employed limiting dilution analysis to determine the frequency of CTL precursors (CTL-P) reactive against both the specific DBA/2 (or P815) target and the syngeneic tumor YC8. The mean frequency of anti-DBA/2 CTL-P in PEC from BALB/c mice immunized with DBA/2 was 1/302. Between one-third and one-fifth of limiting dilution microcultures that exhibited lytic activity against DBA/2 lymphoblasts (or P815) were also able to lyse YC8. No lysis of YC8 was observed in the absence of a parallel lysis on DBA/2 lymphoblasts or P815 target cells. T cell clones, derived by micromanipulation from microcultures selected for cytotoxic activity against YC8 and/or P815, maintained either the specific anti-allogeneic or the doubly reactive ( antiallogeneic plus anti-syngeneic tumor) phenotype. Fourteen clones (six specific and eight doubly reactive) were tested for cytotoxic activity on a panel of target cells with different haplotypes. All showed H-2-restricted specificity for minor histocompatibility antigens shared by DBA/2 and B10.D2. The restriction element for some of the clones mapped in the K region of the H-2 complex, whereas for other clones the restriction element mapped in the D region; both K- and D-restricted clones were able to lyse YC8. When the clones that exhibited lysis on YC8 were tested on two other BALB/c tumor targets, LSTRA, a Moloney virus induced lymphoma, and RL male-1, a radiation induced lymphoma, two of seven were found to lyse all three syngeneic tumor targets equally well, but not syngeneic BALB/c blasts. These clones were functionally categorized as conventional CTL because they were unable to proliferate when cultured with antigen in the absence of exogenous lymphokines, and were unable to produce lymphokine with IL 2 activity when stimulated by the appropriate splenocytes. When tested in vivo in a Winn assay, a strong anti-tumor activity against YC8 was exerted by the anti-DBA/2 clones DY4 -3 and DY16 -3. These clones lysed both YC8 and the immunizing target cells in vitro. No in vivo effect in neutralizing YC8 tumor growth was observed with clone D2-1, a clone that lysed DBA/2 targets but not YC8 in vitro.