Suppressor cell regulation of the spontaneously induced in vitro secondary antibody response.

The cytotoxic effect of peripheral blood lymphocytes from patients with chronic active hepatitis (CAH) on Chang liver cells was studied using a chromium-51 release assay. Cytotoxic effector cell activity was unaffected or slightly enhanced after the removal of glass-adherent mononuclear cells, indicating that the major effector cell is not a classical monocyte. Lymphocytes from 12 of 18 patients with CAH were cytotoxic, and the cytotoxic cells were contained within T-cell-depleted fractions. Further studies revealed that these effector cells were K cells, not B cells. K cells were identified as Fs- and complement receptor-bearing cells without detectable surface immunoglobulin. Serial studies in CAH showed continuous cytotoxicity in contrast to transient cytotoxicity in acute viral hepatitis, suggesting that the cytotoxic activity of lymphocytes is responsible for persistence of the disease.     

Cell types involved in cytotoxicity induced by heated cells and inhibition of this cytotoxicity by anti-human B cell sera.

We have studied the induction of cytotoxic activity in human peripheral blood lymphocytes by heated allogeneic cells. By separating T and B cells from the responder and stimulator cell populations we found that cytotoxic cells are generated in responder T cell populations by both T and the B stimulator cells. Rabbit antisera to a membrane glycoprotein complex (33,000 and 27,000 m.w. by SDS-gel electrophoresis) isolated from a human B cell line were utilized to explore further the nature of the effector cells in this type of cytotoxicity. This antiserum, present during the 6-day-culture period, blocked generation of cytotoxic effector cells. Depletion of cells bearing the B cell antigen from the responder cell population by anti-B cell serum and complement (C) eliminated cytotoxicity. Furthermore, heated cell-induced cytotoxicity was blocked by simply pretreating the responder or the stimulator cell populations with anti-B cell serum in the absence of C. Apparently the human lymphocyte that functions as the effector cell in heated cell-induced cytotoxicity bears the Ia-like antigen that might be important in triggering this type of cytotoxicity.     

Secondary cell-mediated cytotoxic response to syngeneic mouse tumor challenge.

When C57BL/6 mice previously immunized with murine sarcoma virus (MSV) were challenged with a Rauscher virus-induced lymphoma, RBL-5, a secondary cell-mediated cytotoxic response could be detected by the Cr release cytotoxicity assay. The level and distribution of the secondary cytotoxic response was affected by the route of challenge. Animals injected i.p. demonstrated a high level of cytotoxicity in the peritoneal exudate cells 3 days after challenge and subsequently cytotoxicity was detected in most lymphoid organs, although at lower levels. However, when the animals were challenged intramuscularly in the leg, the response was not detected as rapidly and furthermore cytotoxic lymphocytes were found only in the draining lymph node and not in other lymphoid organs. Treatment of the effector cells with anti-theta and complement showed the secondary response to be predominately dependent on T cells. In addition, the cytotoxicity was specific in that cells lacking cross-reacting antigens were not killed by these attacker cells from mice undergoing a secondary response.     

gammadelta(+) T-Lp6phocyte cytotoxicity against envelope-expressing target cells is unique to the alymphocytic state of bovine leukemia virus infection in the natural host

Bovine leukemia virus (BLV) is a complex B-lymphotrophic retrovirus of cattle and the causative agent of enzootic bovine leukosis. Serum antibody in infected animals does not correlate with protection from disease, yet only some animals develop severe disease. While a cytotoxic T-lymphocyte response may be responsible for directing BLV pathogenesis, this possibility has been left largely unexplored, in part since the lack of readily established cytotoxic target cells in cattle has hampered such studies. Using long-term naturally infected alymphocytic (AL) cattle, we have established the existence of cytotoxic T-lymphocyte response against BLV envelope proteins (Env; gp51/gp30). In vitro-expanded peripheral blood mononuclear (PBM) cell effector populations consisted mainly of gammadelta(+) (>40%), CD4(+) (>35%), and CD8(+) (>10%) T lymphocytes. Specific lysis of autologous fibroblasts infected with recombinant vaccinia virus (rVV) delivering the BLV env gene ranged from 30 to 65%. Depletion studies indicated that gammadelta(+) and not CD8(+) T cells were responsible for the cytotoxicity against autologous rVVenv-expressing fibroblasts. Additionally, cultured effector cells lysed rVVenv-expressing autologous fibroblasts and rVVenv-expressing xenogeneic targets similarly, suggesting a lack of genetic restricted killing. Restimulation of effector populations increased the proportion of gammadelta(+) T cells and concomitantly Env-specific cytolysis. Interestingly, culture of cells from BLV-negative or persistently lymphocytic cattle failed to elicit such cytotoxic responses or increase in gammadelta(+) T-cell numbers. These results imply that cytotoxic gammadelta(+) T lymphocytes from only AL cattle recognize BLV Env without a requirement for classical major histocompatibility complex interactions. It is known that gammadelta(+) T lymphocytes are diverse and numerous in cattle, and here we show that they may serve a surveillance role during natural BLV infection.     

Cell-mediated cytotoxicity against HLA-D-Region products expressed in monocytes and B lymphocytes

The cytotoxic effector cells that recognize HLA-D-region determinants and their precursors were characterized using monoclonal antibodies against human T lymphocytes and T-cell subsets. These studies were performed using MLC combinations giving rise to cytotoxic cells specific for both class I (HLA-A, B, C) and class 11 (HLA-D-region) antigens, and then tested against target cells displaying relevant antigens of only one class. Both class I and class II specific CTL (cytotoxic T lymphocytes) were inhibited by treatment with the OKT3 monoclonal antibody and complement, indicating that the effector cells were T lymphocytes. A major portion.of class II specific CTL, and their precursors, were inhibited by OKT4 and complement, while class I specific CTL from the same cultures were not. The T4+T8 — cell subset has previously been associated with helper or inducer functions, but not with cytotoxicity. The present findings indicate that class I and class 11 specific CTL, and their precursors, are different on the basis of the class of target antigen recognized and on the basis of surface phenotype detected by monoclonal antibodies.     

Spontaneous interstitial nephritis in kdkd mice. II. Characterization of a tubular antigen-specific, H-2K-restricted Lyt-2+ effector T cell that mediates destructive tubulointerstitial injury

In this report, we have examined the effector T cell repertoire in the spontaneous interstitial nephritis of kdkd mice. Lymph node cells from nephritic kdkd mice are capable of transferring this disease into thymectomized, irradiated, and bone marrow-reconstituted CBA/Ca recipients. CBA/Ca mice do not spontaneously develop interstitial nephritis and are normally resistant to the adoptive transfer of nephritic cells, a resistance that in the short term can be attenuated with low-dose cyclophosphamide. We therefore used delayed-type hypersensitivity responses and direct transfer of immune cells under the renal capsule to characterize nephritogenic effector cells from kdkd donor mice. Lyt-2+, L3T4- T cells from the peripheral lymphoid organs of nephritic kdkd mice, after adoptive transfer into cyclophosphamide-pretreated CBA/Ca recipients, mediate an antigen-specific delayed-type hypersensitivity response to renal tubular basement membrane antigens. These cells are restricted by gene products in H-2Kk; they are also present in nephritic, but not in control kidneys. We have also observed this same phenotypic subpopulation of kdkd lymphocytes mediate a destructive interstitial renal lesion within 7 days of being placed under the kidney capsule of CBA/Ca mice. These findings suggest that T lymphocytes reactive to a parenchymal tubular antigen are of substantial importance in the development of spontaneous interstitial nephritis in kdkd mice.     

A reappraisal of the effector cells mediating mitogen induced cellular cytotoxicity.

The ability of mitogens to induce cytotoxic effector reactions in vitro has been studied to investigate basic mechanisms of cell mediated cytotoxicity. The type of mitogen, the source of effector cells, and the nature of the target cell are all critical variables in determining the characteristics of the cytotoxic event in this system. Spleen cells and bone marrow cells from congenitally athymic nude mice as well as from their heterozygous control littermates were capable of mediating lysis of RBC targets in the presence of either PHA or Con A. Removal of macrophages from these effector populations by adherence columns, density gradient centrifugation, and carrageenan treatment failed to abrogate this cytotoxic capacity. However, purified macrophages themselves also were capable of mediating mitogen induced killing of RBC targets, although the kinetics of this cytotoxicity were substantially different from that induced by lymphocytes. In contrast to these observations, the capacity of mitogen stimulated cells to kill metabolically active complex targets like the P815 mastocytoma or cultured L cells appears to be exclusively a T lymphocyte dependent function. In addition, blastogenic transformation of the effector cells with the T cell mitogens PHA and Con A, but not with the B cell mitogen LPS, leads to enhanced killing of these complex targets. These data suggest that mitogen or lectin induced cellular cytotoxicity can detect at least three different active effector cell types (B cells, T cells, and macrophages) acting via at least four different mechanisms.     

Activation of cytotoxic function in T lymphocytes.

The requirements for activation of cytotoxic function in mouse T lymphocytes were investigated. Initial generation of cytotoxicity in normal lymphocytes was equal in magnitude with either Con A or specific alloantigen, and in either case required DNA synthesis. Cytotoxic function in MLC-primed cells could also be regenerated by Con A, the magnitude and target specificity of the cytotoxicity thus generated being indistinguishable from that recalled by specific alloantigen. Cytotoxicity could also be regenerated by third party-stimulating cells; however, the cytotoxicity evoked by third party cells was always specific only for target cells of the original stimulating cell H-2 genotype. The data presented suggest that there are a number of ways to activate cytotoxicity in effector T cells, and are most consistent with a model for T cell triggering that minimizes a strict informational function of antigen-receptor interactions.     

Immunoregulation in cancer-bearing hosts. Down-regulation of gene expression and cytotoxic function in CD8+ T cells.

The causes of the decreased immune responsiveness in tumor-bearing hosts are incompletely understood. The impact of a decreased immune response in cancer patients on the clinical response in immunotherapy trials has not been evaluated. The present report demonstrates a marked decrease in the therapeutic efficacy of adoptively transferred T lymphocytes obtained from murine hosts bearing tumor for greater than 30 days [late tumor-bearing mice (TBM)] as compared with normal mice and mice bearing tumor for less than 21 days (early TBM). In vitro analysis of the functions of the T lymphocytes from late TBM showed an apparently normal proliferative response to anti-CD3 and IL-2 with adequate lymphokine production from CD4+ cells, but a significant decrease in the cytotoxic function of CD8+ cells. The decreased cytotoxicity was not because of cell-mediated suppression. The expression of granzyme B mRNA was significantly delayed and decreased in magnitude in CD8+ cells from late TBM. Culture supernatants from two unrelated tumor cell lines were able to inhibit the cytotoxic activity of normal CD8+ cells in vitro. The tumor-derived suppressive factor is not transforming growth factor-beta (TGF-beta), but it has not been further characterized. The data suggest that one potential mechanism responsible for immunologic defects in patients with large tumor burdens is a tumor-induced defect that compromises the function of CD8+ effector T cells.     

Thymus-dependent and thymus-independent effector functions of mouse lymphoid cells. Comparison of cytotoxicity and primary antibody formation in vitro

We have compared two effector functions, antibody formation and cytotoxic capacity in vitro, of mouse cells of various origin with special reference to the T lymphocyte dependence of these processes. We have used addition of PHA and coating of target chicken erythrocytes (CRBC) with antibody as the two means of inducing cytotoxicity. Antibody formation in vitro has been studied both against thymus-dependent sheep erythrocytes (SRBC) and thymus-independent (E. coli) antigens. Spleen cells from thymectomized, lethally irradiated bone marrow-, or fetal liver-repopulated mice were deprived of phagocytic cells by uptake of colloidal iron. They did perform better than normal spleen cells in the antibody-induced cytotoxicity and were also induced to cytotoxicity by PHA. PHA did not induce increased DNA synthesis in these T cell-deprived spleen cell preparations, which could not make primary antibodies to SRBC but were able to do so against E. coli antigens. Fresh bone marrow and fetal liver cells, deprived of phagocytic cells, were also induced into a highly efficient cytotoxicity by anti-CRBC as well as by PHA. Pretreatment of spleen cells with an alloantiserum (θ) against T lymphocytes reduced but did not abolish the PHA-induced cytotoxicity. In contrast, it did not affect the antibody-induced cytotoxicity. Such treated cells could not make antibodies to SRBC but could do so against E. coli. Pretreatment of spleen cells with a heteroantiserum (MBLA) against mouse B lymphocytes completely abolished all cytotoxic- and antibody-forming abilities of the cells, although experiments with combinations of θ-treated and MBLA-treated cells suggested that the MBLA treatment had left behind a significant portion of helper T cells needed for the in vitro antibody response. From these data we conclude, as have others, that the antibody-induced cytotoxicity is independent of T lymphocytes. It can be induced in immature precursor cells from fetal liver or bone marrow, and these cells may also become cytotoxic on interaction with PHA. However, in normal spleen cells, at least part of the PHA-induced cytotoxicity is T cell dependent. Some preliminary data suggest that this PHA-induced cytotoxicity of normal spleen cells may be a joint process between T lymphocytes and other cells.     

Characterization of the normal lymphocyte population cytolytic to Burkitt's lymphoma cells of the EB2 cell line.

The normal human lymphocyte population which exhibits "spontaneous" cytolysis of EB2 Burkitt's lymphoma cells has been characterized. The effector cell has EA and EAC' receptors but lacks E receptors and probably surface Ig. "Spontaneous" anti-EB2 cytotoxicity was not reduced by preincubation of the effector cells with plastic or iron carbonyl or by passage through cotton wool or agarose columns but was reduced by passage through nylon wool columns. Thymocytes were not cytotoxic to EB2 cells, and chronic lymphocytic leukaemia cells (of B cell characteristics) had reduced cytotoxicity compared with normal lymphocytes. Cells from various lymphoid organs of rats and guinea-pigs were also cytotoxic to EB2 cells with reactivity in spleen greater than or equal to blood greater than lymph nodes. Spleen cells from neonatally thymectomized rats had increased cytotoxicity compared with normal rat spleen cells, suggesting that T lymphocytes are not essential. The effector cell in rat spleen did not adhere to cotton wool or agarose columns, indicating some resemblance to its counterpart in human peripheral blood.     

Spontaneous cell-mediated cytotoxicity in patients with transitional cell carcinoma of the urinary bladder

Summary Lymphocytes isolated from the blood of TCC patients, like those of control patients, were capable of mediating spontaneous cell-mediated cytotoxicity against K-562 cells. When this natural cytotoxicity was analyzed with regard to the effector cell type it was found that in TCC patients the SLMC was mostly displayed by E-rosetting T lymphocytes, whereas compared with controls, a significant decline in the SLMC of the non-T lymphocytes was observed. The SLMC of the T lymphocytes derived from TCC patients was further demonstrated on a T leukemia target cell (Peer). When the SLMC on K-562 and on Peer target cells was compared, a specificity difference was observed between TCC and the control patients' effector cells. The SLMC activity of the TCC patients' T cells was not abolished after depletion of Fc receptor-positive cells or following treatment with monoclonal antibodies OKT 8 or OKT 4 and complement (C'). These NK-like cells are therefore distinguished from cytotoxic T lymphocytes and NK cells.     

Stage-related, cell-mediated cytotoxicity with effector cell analysis and computation of cytotoxic activity of T cells and non-T cells.

Cell-mediated cytotoxicity (CMC) by lymphocytes from patients with oral squamous cell carcinoma, as well as from nonmalignant control donors, was tested by a microcytotoxicity assay against a cultured cell line derived from an oral squamous cell carcinoma. In terms of the degree of CMC, stage-related cytotoxicity was observed. A further study of the effector cell analysis revealed that the cytotoxic effects of lymphocytes from both patients and control donors were largely attributable to non-T cells. However, the effectors were also stage related, and in early stage patients, T-cell-mediated cytotoxicity was super-imposed on non-T-cell-mediated cytotoxicity. This conclusion was further supported by the evidence of elevated cytotoxic activity of T cells in early stage patients, which was computed from simultaneous equations proposed in the present paper for computing the cytotoxic activity of T cells (CTA_T) and non-T cells (CTA_non-T).     

Cytotoxic lymphocytes induced by soluble factors derived from the medium of leukocyte cultures.

Studies were undertaken to evaluate the cytotoxic capacity of human peripheral blood lymphocytes activated by either supernatants (CFM) derived from lymphocyte cultures or lymphocytes treated for 60 min at 45 degrees C. The effect of the addition of heat-treated cells on the cytotoxic activity of CFM-induced effector cells was also studied. CFM from either unmixed or mixed cultures of lymphocytes was capable of activating cytotoxic effector cells. These effector cells could kill any allogeneic target cells but failed to effect cytotoxicity on the target cells autologous to the responding cells. Both the heat-treated cells and CFM from cultures of these cells also activated lymphocytes to cytotoxic effector cells having specific receptors for nonself antigens. The question of whether heat-treated cells activate cytotoxic cells by themselves or through secreted soluble factor cannot yet be clearly answered. The findings of the present investigation suggest that expression of cytotoxicity induced in MLC is not necessarily restricted to the target cells syngeneic to the stimulator cells, but can be extended to any allogeneic target cells by the indirect effect of soluble factor secreted from stimulated cells that causes a polyclonal activation of cytotoxic precursors in the responding cell populations. The present findings also emphasize the need for caution in the use of heat-treated lymphocytes as innocent-bystander cells in MLC to provide additional cytotoxic specificities in the responder cells, since heat-treated cells alone can activate lymphocytes to cytotoxic effector cells that kill any allogeneic target cells.     

Cytotoxic effector cell function in organized gut-associated lymphoid tissue (GALT).

Lymphocytes from the organized gut-associated lymphoid tissues (GALT) of adult guinea pigs were examined for surface markers characteristic of T and B lymphocytes and for their capacity to function as effector cells in mitogen-induced cellular cytotoxicity (MICC) and antibody-dependent cellular cytotoxicity (ADCC) reactions. GALT lymphocytes formed rosettes with rabbit erythrocytes, a T-cell marker, and underwent proliferative responses in vitro in the presence of phytohemagglutinin (PHA), concanavalin A (Con A), and pokeweed mitogen (PWM). GALT lymphocytes were cytotoxic in vitro for erythrocyte and DBA mastocytoma targets in the presence of PHA. A population of GALT lymphocytes bound aggregated γ-globulin; however, they functioned poorly in ADCC reactions. Thus, organized GALT in the guinea pig contains lymphocytes capable of functioning in T-cell-dependent MICC reactions but either lacks the effector cell population which mediates ADCC or contains an effector cell which functions poorly in ADCC.     

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.     

Virus-induced enhancement of lymphocyte-mediated antibody-dependent cytotoxicity (ADCC) in vitro

The effect of Parotis virus on antibody-dependent cellular cytotoxicity in vitro (ADCC) of human lymphocytes was investigated in a 51Cr-release assay and, at the effector cell level, in an ADCC plaque assay. Target cells were bovine or chicken erythrocytes, which are not susceptible to natural cytotoxicity (NK) of human lymphocytes. They were not killed when incubated with virus-treated lymphocytes in the absence of antibodies. Treatment of the lymphocytes or the target cells with small amounts of virus, however, resulted in a very significant enhancement of ADCC. The same results were obtained with live or UV-inactivated virus, suggesting that enhancement was a passive phenomenon not requiring infection. Enhancement was already significant after 3 hr of incubation, indicating that it was independent of endogenously released interferon. Enhancement of ADCC by virus was due to effector cell recruitment rather than due to the increase of the cytotoxic potential of the individual K cell. The highest frequency of effector cells was present in Percoll fractions enriched in large granular lymphocytes (LGL). Virus treatment resulted in recruitment of effector cells carrying T cell markers such as the T3 antigen (OKT3+), receptors for sheep erythrocytes, or Fc receptors for IgM. In contrast, the absolute number of K cells carrying the HNK-1 marker (Leu-7) or receptors for C3 fragments was not changed by the virus. It is concluded that Parotis virus enhances ADCC by improving effector cell-target cell contacts, resulting in recruitment of effector cells with T cell characteristics. Recruitment is accompanied by a significant reduction of the antibody concentration needed for ADCC induction. This virus-mediated enhancement of ADCC may be of importance for protection of the host in the early phases of a virus infection in which the amounts of anti-viral IgG antibodies capable of inducing cellular cytotoxicity may yet be very small.     

Mechanism of cell-mediated cytotoxicity at the single-cell level: VII. Trigger of the lethal hit event is distinct for NK/K and LDCC effector cells as measured in the two-target conjugate assay

Normal human peripheral blood lymphocytes (PBL) express several in vitro cytotoxic functions, among which are natural killer (NK), antibody-dependent cellular cytotoxicity (ADCC), and lectin-dependent cellular cytotoxicity (LDCC). The relationship of these various cytotoxic functions and the identity of cells involved has been a subject of controversy. Recently it was reported that NK and K for ADCC can be mediated by the same cell, suggesting that they constitute in large part a single subpopulation with multiple cytotoxic functions. The ability of this NK/K effector cell to mediate LDCC was examined here using the two target conjugate assay. The effector cells were Ficoll-Hypaque PBL or LGL-enriched fractions. The targets used were K562 or MOLT for NK, RAJI coated with antibody for ADCC, and RAJI coated with PHA or Con A or modified by NaIO₄ for LDCC. In the two-target conjugate assay, one of the targets is fluorescein labeled for identification. The results show that (a) LDCC copurifies with NK/K and is enriched in the LGL fraction, as measured in both the ⁵¹Cr-release assay and the single-cell assay for cytotoxicity; (b) single effector cells simultaneously bind to NK or ADCC and LDCC targets, revealing that single cells bear binding receptors for all targets; and (c) single lymphocytes were not able to kill both bound NK/K and LDCC targets. However, significant two-target killing was obtained when both targets were NK targets, ADCC targets, LDCC targets, or one NK and one ADCC target. These results demonstrate that the NK and LDCC effector cells are distinct subpopulations copurified in the LGL fraction. In addition, the results show that lectin is unable to trigger globally an NK effector cell to mediate cytotoxicity against a bound NK insensitive target. Thus, although both NK and LDCC effector cells are present in the LGL fraction and can bind to both types of targets, the trigger of the lethal hit event is the function of specialized effector cells.     

Identification of effector cells for TNFalpha-mediated cytotoxicity against WEHI164S cells

WEHI164S cells were found to be very sensitive targets for in vitro killing in a 6-h culture when liver or splenic lymphocytes were used as effector cells in mice. Of particular interest, a limiting cell-dilution analysis showed that effector cells were present in the liver with a high frequency (1/4,300). In contrast to YAC-1 cells as NK targets, perforin-based cytotoxicity was not highly associated with WEHI164S killing. The major killer mechanism for WEHI164S targets was TNFalpha-mediated cytotoxicity. By cell sorting experiments, both NK cells and intermediate T cells (i.e., TCR(int) cells) were found to contain effector cells against WEHI164S cells. However, the killer mechanisms underlying these effector cells were different. Namely, NK cells killed WEHI164S cells by perforin-based cytotoxicity, TNFalpha-mediated cytotoxicity, Fas ligand cytotoxicity, and other mechanisms, whereas intermediate T cells did so mainly by TNFalpha-mediated cytotoxicity. These results suggest that TNFalpha-mediated cytotoxicity mediated by so-called natural cytotoxic (NC) cells comprised events which were performed by both NK and intermediate T cells using somewhat different killer mechanisms. Intermediate T cells which were present in the liver were able to produce TNFalpha if there was appropriate stimulation.     

Regulation of cell-mediated cytotoxicity. III. Synergism of cortico-resistant with anti-thymocyte serum-resistant splenic T cells in the generation of cytotoxic lymphocytes in graft-vs-host reactions.

Lethally irradiated F1 hybrid mice were given an i.v. injection of parental strain spleen cells. Six days later, their spleen cells were used as the effector cells to measure the in vitro cell-mediated cytotoxicity (CMC) of the parental cells. The treatment of the donors with hydrocortisone resulted in a marked decrease of the capacity of their spleen cells to produce a CMC reaction, whereas the treatment with antithymocyte serum (ATS) resulted in an almost complete loss of such activity. The mixing of spleen cells from hydrocortisone-treated parental donors with the spleen cells from ATS-treated parental donors before injection resulted in a synergistic amplification of the cytotoxic response. The anti-Thy-1 serum treatment of either spleen cell population abolished the synergism completely. These results indicate that cortico-resistant T cells act as precursors of cytotoxic lymphocytes and that ATS-resistant T cells produce an amplification of their reaction.