A monoclonal antibody reactive with the human cytotoxic/suppressor T cell subset previously defined by a heteroantiserum termed TH2

A hybridoma-secreting monoclonal antibody was produced from the spleen cells of a mouse immunized with human thymocytes. This hybridoma antibody, termed OKT5, was reactive by indirect immunofluorescence with 80% of human thymocytes but only 20% of peripheral blood T cells. Moreover, OKT5 was unreactive with normal B cells, null cells, and macrophages at any dilution tested. A similar pattern of reactivity was seen with an equine antiserum to human thymocytes termed anti-TH2. Fluorescence-activated cell sorting demonstrated that the OKT5 antibody reactivity on peripheral T cells was restricted to the majority of the previously defined TH2+ subpopulation. In functional studies, the OKT5+ subset, like the TH2+ subset, proliferated well to the mitogen Con A and to alloantigens, and contained cytotoxic effector cells after sensitization in MLC, and suppressor effector cells after activation with Con A. In addition, like the TH2+ T cell, the OKT+ T cell was virtually unresponsive to soluble antigen. Thus, the OKT5 monoclonal antibody is reactive with the cytotoxic/suppressor T cell subset. OKT5 should provide an important probe to assess the status of suppressor cells in human disease.     

Cyclosporine-induced tolerance in experimental organ transplantation. Evidence of diminished donor-specific cytotoxicity relative to donor-specific proliferative response

Alloreactivity of intragraft and peripheral blood lymphocytes from tolerant canine lung allograft recipients was examined. Tolerance was induced by variable periods of treatment with cyclosporine. Analysis of effector cells from lung allografts (obtained by bronchoalveolar lavage) revealed the absence of specific cytolytic T lymphocyte (CTL) activity and the presence of a low level of cytolytic activity detected in a lectin-dependent cell-mediated cytotoxicity assay. In contrast, high levels of specific CTL activity and lectin-dependent activity were detected in cell preparations from lung allografts undergoing rejection. Tolerant recipients retained normal ability to generate specific CTL activity to third party alloantigens in mixed lymphocyte cultures (MLC) but had diminished ability to generate CTL to donor alloantigens in recipient X donor MLC. Addition of exogenous interleukin 2 to these MLC was unable to restore donor-specific CTL activity. Lymphocytes from tolerant recipients were, however, capable of generating proliferative responses and lectin-dependent cytotoxicity on exposure to donor alloantigens in MLC. Evidence presented in this report suggests that the lectin-dependent cytolytic activity generated in these MLC is mediated by lymphokine-activated killer cells. Such cells are likely to be activated by interleukin 2 released in the proliferative response. The results support the proposal that the cyclosporine-induced tolerant state is characterized by the relative inability to respond against major histocompatibility complex class I antigens in contrast to class II antigens and/or minor histocompatibility antigens since MLC-induced CTL are directed, for the most part, against class I molecules.     

Human T cell subpopulations defined by a monoclonal antibody. I. A small subset is responsible for proliferation to allogeneic cells or to soluble antigens and for helper activity for B cell differentiation

A monoclonal antibody (5/9) was obtained that reacts with the majority of alloactivated human T cells but only with 15 to 20% peripheral T cells. The populations reactive with the 5/9 antibody were separated from the remaining T cells by rosetting techniques using 5/9-coated ORBC. 5/9+ and 5/9- populations were analyzed for different in vitro activities. The helper activity of PWM- driven B cell differentiation appeared to be restricted and highly enriched in the 5/9+ population. In addition, 5/9+ cells contained all the cells capable of proliferation to tetanus toxoid and allogeneic cells. Thus, in vitro activities commonly used for evaluation of T cell function seem to be confined to a small fraction of peripheral T cells.     

Generation of cytolytic T lymphocytes in vitro. VIII. failure of anti-RS antisera to inhibit the generation of cytolytic T lymphocytes or cytolytic T lymphocyte activity.

Antibody reactive with "recognition structures" (RS) of mouse lymphoid cells for alloantigens (anti-RS) was prepared by immunization of F1 hybrid mice with parentalstrain lymphoid cells or with antibody produced in one parental strain against alloantigens of the other parental strain. Such antisera prevented generation of the "product of antigenic recognition" (PAR) that is produced within a few hours in cultures prepared with a mixture of lymphoid cells from genetically disparate mice. However, treatment of responding lymphoid cells with anti-RS sera and complement did not inhibit generation of cytolytic T lymphocytes (CTL) in mixed lymphocyte cultures (MLC). Treatment of cells obtained from MLC with anti-RS sera and complement failed to inhibit cytolytic activity of such cells for specific alloantigens.     

Functional and morphologic characterization of human T lymphocytes expressing the TCR gamma /delta

A minor subset of T lymphocytes express a TCR composed of gamma and delta chains. This subset differs from conventional T cells for a number of phenotypic and functional characteristics. TCR gamma/delta+ cells simultaneously lack both CD4 and CD8 antigens. Cloning of CD4-8- peripheral blood lymphocytes, under limiting dilution conditions, revealed that they are homogeneously composed of cytolytic cells which efficiently lyse tumor target cells. Formal proofs have been provided that TCR gamma/delta+ cells are able to recognize antigens. For example, they proliferated in response to allogeneic mixed lymphocyte culture (MLC); in addition, MLC-derived TCR gamma/delta+ cells specifically lysed PHA-induced blast cells bearing the stimulating alloantigens. The selection of monoclonal antibodies specific for TCR gamma/delta molecules allowed to identify two distinct subsets of TCR gamma/delta+ cells. Both of these mABs, termed BB3 and delta TCS-1 respectively, induced specific activation of cloned cells expressing the corresponding antigenic determinants (as assessed by measurements of intracellular Ca++ and/or lymphokine production or cytolytic activity). Analysis of the distribution of subsets expressing different forms of TCR gamma/delta, showed that the BB3-reactive form is prevalent in the peripheral blood. In contrast, delta-TCS-1-reactive cells are relatively unfrequent in peripheral blood but represent the majority of TCR gamma/delta+ cells in tissues.     

Growth and characterization of T cell colonies from human thymus

A semisolid microculture system was used to study T cell colonies grown from human thymocytes. Colony growth was absolutely dependent upon media conditioned by peripheral blood leukocytes (PBL) in the presence of phytohemagglutinin. Plating efficiency was further enhanced by the addition of a non-T, adherent, radiation-resistant (7500 rad) PBL subpopulation, but was not enhanced by culture supernatants of these cells. The T colony precursor cell in the thymus occurred with a frequency of 8.0 X 10(-3) and had a surface receptor for the OKT3 monoclonal antibody. Thymocyte colony cells were functionally distinct from PBL and the major thymocyte population. The colony cells proliferated in response to T cell mitogens, but only in the presence of exogenous growth factors. The cells stimulated normal PBL in mixed leukocyte culture (MLC), but did not respond to alloantigens in MLC or in assays of spontaneous cytotoxicity. This culture system should prove helpful in the study of human thymocyte differentiation.     

Limiting dilution analysis of alloantigen-reactive T lymphocytes. III. Effect of priming on precursor frequencies.

The effect of specific priming with alloantigens on the frequency of cytolytic T lymphocyte precursors (CTL-P) has been investigated. Alloimmune lymphoid cells were obtained from the spleen of C57BL/6 (H-2b) mice primed with DBA/2 (H-2d) tumor cells or from 14-day unidirectional mixed leukocyte cultures (C57BL/6 anti-DBA/2). CTL-P frequencies directed against H-2d alloantigens were estimated by limiting dilution analysis in a sensitive micro MLC system. Under these conditions, an apparent increase of 3 to 4-fold in CTL-P frequency was observed in alloimmune (as compared with normal) C57BL/6 spleen cells. Evidence was obtained suggesting that this increase was specific for the priming alloantigens. A much greater increase in CTL-P frequency (25 to 100-fold) was observed after alloimmunization of C57BL/6 spleen cells in unidirectional MLC. Under the latter conditions, 5 to 20% of the surviving splenic MLC cells could be identified operationally as CTL-P. A similar enrichment in CTL-P frequency was obtained when lymph node, peripheral blood, or thymus cells were cultured for 14 days in MLC. These studies provide direct evidence that the pool of specific CTL-P can be expanded after alloimmunization. Furthermore, the very high frequencies observed after in vitro priming indicate that this system should be particularly useful for future studies of the progeny of individual CTL-P.     

Interferon production and tumor cell killing by human lymphocytes stimulated in mixed-lymphocyte culture

The in vitro synthesis of interferon (IFN) by human lymphocytes stimulated in mixed-lymphocyte culture (MLC) was examined. The production of IFN in MLC was restricted to T lymphocytes and maximum levels of IFN were detected in supernatants from cells incubated for 5 to 7 days. The IFN produced was identified as IFN-gamma by antibody neutralization. To identify the T cell responsible for IFN production, purified T lymphocytes were separated into subpopulations after incubation in 5 mM theophylline. Theophylline-resistant (T-res) T cells retain the ability to form sheep erythrocyte (SRBC) rosettes and are depleted in IgG Fc receptor-positive T cells (T gamma cells). Theophylline-sensitive (T-sens) T cells fail to form rosettes after theophylline treatment and are enriched in T gamma cells. In addition, analyses using monoclonal antibodies showed that T-sens cells were enriched in OKM1-, HNK-1-, and 7.2-positive cells and T-res cells contained increased numbers of 9.6- and OKT4-positive cells. Following MLC stimulation, equivalent levels of IFN-gamma were produced by T-res and T-sens cells and both subpopulations maintained natural killer (NK)-like cytotoxicity against K562 target cells. Addition of partially purified IFN-gamma to unstimulated T-res and T-sens cells resulted in the maintenance of NK-like cytotoxicity in a manner analogous to that observed after MLC. Additional experiments indicated that peripheral blood lymphocytes depleted of 9.6- or OKM1-positive cells by complement-mediated lysis were devoid of cytotoxicity against K562 cells. Furthermore, MLC stimulation of 9.6- or OKM1-depleted cells failed to restore cytotoxic activity. In summary, these experiments demonstrate that the maintenance of NK-like cytotoxicity by MLC-stimulated T cells is associated with the synthesis of IFN-gamma, that MLC stimulated T-res and T-sens T-cell subsets produce equivalent amounts of IFN, and that 9.6- or OKM1-positive cells are required for the maintenance of NK-like cytotoxicity in MLC.     

Interferon-beta and recombinant IL 2 can both enhance, but by different pathways, the nonspecific cytolytic potential of T3- natural killer cell-derived clones rather than that of T3+ clones

We studied the enhancement of cytolytic activity of T3- natural killer cell-derived clones, of T3+ T cell activated killer (AK) clones, and of fresh peripheral blood lymphocytes (PBL) by various crude and recombinant interferon (r-IFN) as well as IL 2 preparations. It was found that IFN-beta had the highest cytotoxicity inducing potency as compared to crude or r-IFN-alpha or -gamma preparations. This enhancement was blocked by anti-IFN-beta antibodies but not by anti-IFN-gamma antibodies. IL 2 also strongly enhances cytolytic activity in cloned T3- killer cells that express the IL 2 receptors as determined with the anti-Tac monoclonal antibody (MAb) at concentrations of IL 2 (25 U/ml) which induced one-half of the maximal proliferation capacity in human T cells and murine CTLL cells. For enhancement of cytolytic activity in fresh NK cells, a much higher concentration of IL 2 is required. In addition, the enhancement of cytolytic activity by r-IL 2 but not that by IFN-beta can be reduced by anti-Tac MAb, suggesting that the IL 2 receptor is involved in the enhancement by IL 2, but not by IFN. Both IFN-beta and IL 2 were able to enhance (over threefold) the cytolytic activity of T3- cloned killer cells against a variety of tumor target cell types. Another remarkable observation was that K562 cells, the most commonly used target cell for determining NK cell cytolytic activity, are not the most suitable targets to assess enhancement of nonspecific lytic activity as compared to Daudi or lung tumor-derived cell lines. No enhancement of anti-body-dependent cellular cytotoxicity was observed. Finally, the effects of these biological response modifiers were much more pronounced on "fresh" and cloned T3- natural killer cell-derived than on T3+-activated killer mature T cell-derived clones.     

Multiple cytolytic mechanisms displayed by activated human peripheral blood T cell subsets.

It has been proposed that CTL-mediated cytotoxicity may involve multiple lytic mechanisms. We have examined both the antibody-redirected cytolytic potential and the direct cytotoxicity of purified human peripheral blood high buoyant density CD4+ and CD8+ T cells activated with IL-2 and anti-CD3 mAb. TNF-sensitive and TNF-resistant targets and various metabolic inhibitors were used to compare the antibody-redirected cytotoxicity of T cell subsets and discern the role of potential lytic mediators. In a 4-h assay against several different nitrophenyl-modified targets, the heteroconjugated antibody (anti-CD3-anti-nitrophenyl) redirected cytolytic potential of 72-h activated CD4+ T cells was inhibited by the continuous presence of actinomycin D, cycloheximide, and EGTA, but not mitomycin C, cyclosporin A, or cholera toxin (CT). Conversely, only CT and EGTA inhibited the antibody-redirected cytolytic potential of activated CD8+ T cells. Despite both CD4+ and CD8+ T cell subsets expressing granzymes, pore-forming protein, TNF-beta, and TNF-alpha, these T cell subsets displayed distinct pathways of antibody-redirected lysis against TNF-sensitive and TNF-resistant targets, even in the presence of anti-TNF antibodies. In addition, these same effector T cell subsets were also directly cytotoxic (in the absence of heteroconjugated antibody) against TNF-sensitive targets in an 18-h assay. Indeed, this direct cytotoxicity was completely abrogated by anti-TNF-alpha antibody and was sensitive to the metabolic inhibitors (cyclosporin A, CT, cycloheximide, and actinomycin D), all of which blocked CD4+/CD8+ T cell TNF-alpha production. Therefore, both CD4+ and CD8+ T cells were demonstrated to utilize antibody and lymphokine-mediated lytic mechanisms. CD4+ and CD8+ effector subsets were demonstrated to lyse the same TNF-sensitive target by these two different mechanisms. Although it cannot be excluded that the redirected lytic mechanisms of both CD4+ and CD8+ effectors share common elements, it is likely that other important events in this cytolytic process are fundamentally distinct between these subsets of T cells.     

Limiting dilution analysis of alloantigen-reactive T lymphocytes. IV. High frequency of cytolytic T lymphocyte precursor cells in MLC blasts separated by velocity sedimentation

A sensitive limiting dilution microculture system was used to obtain minimal estimates of the frequency of cytolytic T lymphocyte precursor cells (CTL-P) directed against DBA/2 alloantigens, after priming of spleen cells in unidirectional mixed leukocyte cultures (MLC, C57BL/6 anti-DBA/2). The mean CTL-P frequency in day 4 to 5 MLC populations was found to be approximately 50- to 100-fold greater than the frequency in normal spleen, and up to 25% of the cells present in such MLC could be identified operationally as CTL-P. Even higher frequencies (up to 50%) of CTL-P were obtained in a population of large-sized cells separated from day 4 MLC by velocity sedimentation. Furthermore, since a strikingly quantitative correlation was observed between CTL activity and CTL-P frequency in such separated MLC populations, it is likely that mature CTL in MLC are not end cells, but can further proliferate and thus behave operationally as CTL-P.     

Antigen-presenting T cells. II. Clonal responses of alloreactive and virus-specific self-restricted human cytotoxic T cell responses stimulated by T lymphoblasts

The capacity of various stimulator cell types to present alloantigens or viral antigens to resting human CD8+ cytotoxic lymphocyte precursors (CLP) was analyzed in a limiting dilution culture system. Cell sorter-separated T lymphoblasts of both CD4+ and CD8+ phenotypes but not resting T cells were found to efficiently stimulate the clonal development of allogeneic CD8+ CLP. Thus, 5000 CD4+ T lymphoblasts activated as many (one out of 200 to one out of 300) allogeneic CLP as 50,000 peripheral blood mononuclear stimulator cells. This potent stimulator activity was found in CD4+ and CD8+ T lymphoblasts activated by mitogen, anti-T3 monoclonal antibody, or mixed leukocyte reactions. Cytotoxic T cells generated in this system were highly specific for HLA class I antigens. Furthermore, T lymphoblasts infected with mumps virus efficiently induced development of autologous CLP into CTL clones that were virus specific and self-HLA restricted, as shown by split-well analysis. The possible in vivo significance of antigen-presenting T lymphoblasts is discussed.     

Generation of cytotoxic T lymphocytes in vitro. VI. Effect of cell density on response in mixed leukocyte cultures.

Reexposure of day 14 murine mixed leukocyte culture (MLC) populations to the original irradiated allogeneic stimulating spleen cells has previously been found to result in the ratpid generation of cytolytic T lymphocytes (CTL) associated with a net increase in cultured cell number. Under the experimental conditions used, day 5 MLC cells appeared unable to respond to the allogeneic stimulus. In order to characterize further the development of the potential for anamnestic reactivity during the course of MLC, C57BL/6 spleen cells were incubated with irradiated (1000 rads) DBA/2 spleen cells (primary MLC) for up to 3 weeks. At various time intervals after the onset of the primary MLC, the surviving cells were collected and reexposed, at varying cell concentrations, to irradiated DBA/2 spleen cells (secondary MLC). At daily intervals thereafter, CTL activity was assessed using a quantitative 51Cr-release assay system. A paradoxic effect of responding cell concentration on generation of CTL activity was observed; relatively greater increase in CTL activity was observed as the concentration of responding cells was decreased over a 100-fold range. This effect was more pronounced with responding cells reexposed to antigen after primary MLC for 20 days, but was observed even with normal cells. The apparent unresponsiveness of day 5 MLC cells to alloantigen restimulation could be overcome by simple dilution of responding cells. Cytotoxic activity at the time of restimulation with antigen seems to be a major factor determining the magnitude of the secondary response. Since intact cells bearing alloantigens are required for the generation of CTL in MLC, residual cytotoxic cells reduce the effective antigenic stimulus by destroying stimulating cells. This effect of concentration of responding cells on generation of CTL in MLC complicates interpretation of experiments investigating the role of "inhibitor" and "helper" cell in cell-mediated immune responses occurring in vitro. Under optimal conditions, the highest CTL activity and the largest increase in total cell number was observed 4 days after restimulation of day 10 MLC cells. On a per cell basis, the lytic activity was up to 4 times greater than that observed at the peak of a primary response, and the number of viable cells recovered was nearly 20 times higher than that at the onset. Such secondary MLC are thus a convenient source of lymphoid cells selected primarily on the basis of proliferation induced by alloantigens.     

Characterization of bovine mononuclear cell populations with natural cytolytic activity against bovine herpesvirus 1-infected cells

Freshly isolated or overnight cultured peripheral blood mononuclear cells from immune or nonimmune animals had natural cytolytic activity against bovine herpesvirus 1 (BHV-1)-infected tumor target cells. No lysis was demonstrated against tumor target cells alone. This natural cytolytic activity was present in mononuclear cells from the spleen, lymph node, and peripheral blood but little or no cytolytic activity was detected in bone marrow or thymus cells. When monoclonal antibodies and complement to deplete bovine mononuclear cell subpopulations from the nonadherent cells were used, results indicated the effector cell was not a T cell, B cell, or activated monocyte. From nonadherent populations separated on density gradients, it was determined that the effector cells were large, low density mononuclear cells. These results indicate the nonadherent effector cells mediating lysis of BHV-1-infected xenogeneic adherent target cells were large null lymphocytes and/or immature monocytes.     

Two distinct cytotoxic T lymphocyte subpopulations in patients with Vogt-Koyanagi-Harada disease that recognize human melanoma cells

The functional properties of cytotoxic lymphocytes from patients with Vogt-Koyanagi-Harada disease ( VKH ) specific for human melanoma cells (P-36 melanoma cell line established from a patient with malignant melanoma) were investigated by using monoclonal antibodies specific for human T cell subsets. Peripheral blood lymphocytes (PBL) from patients with VKH showed significant cytotoxic activity against the P-36 (SK-MEL-28) human melanoma cell line, but not against a human cervical carcinoma of the uterus cell line (HeLa-S3 cell line) or against a mouse melanoma cell line (B-16 cell line) originating from a C57BL/6 strain mouse or against the EL-4 mouse lymphoma cell line from a C57BL/6 mouse. The cytotoxic activity of the patients' PBL against the P-36 melanoma cell line was markedly reduced by pretreatment of the PBL with monoclonal anti-human Leu-1 antibody plus rabbit complement, but it was reduced to much less extent by pretreatment with either monoclonal anti-human Leu-2a or Leu-3a antibody plus rabbit complement. The specific cytotoxic activity of the patients' PBL against the P-36 human melanoma cell line is, therefore, mediated by T cells bearing Leu-1+ Leu-2a+ or Leu-1+ Leu-3a+ antigens. Furthermore, the cytotoxic activity was shown to be blocked not only by anti-Leu-2a antibody specific to human cytotoxic/suppressor T cells but also unexpectedly by anti-Leu-3a antibody which has previously been considered to be specific to human inducer/helper T cells. The results of this study suggest that at least two distinct subpopulations of cytotoxic T cells specific for P-36 human melanoma cells are present in the peripheral blood of VKH patients. These cytotoxic T cells have different surface antigens, Leu-2a and Leu-3a.     

Phenotypic and functional heterogeneity of human peripheral blood T lymphocytes producing colony stimulating factor. A clonal and precursor frequency analysis

In this report we describe the precursor frequency and the subset distribution of peripheral blood human T cells producing lymphokine(s) acting on the proliferation and differentiation of bone marrow precursors of the granulocyte/macrophage series, as assessed in a liquid microculture assay. Because the sensitivity of this system was similar to that of the classic colony formation assay in semi-solid (methylcellulose) medium, it is likely that the lymphokine activity measured in this assay corresponds to the colony-stimulating factor (CSF) activity. Single human T lymphocytes, isolated from peripheral blood by E rosetting and Ficoll-Hypaque gradients, were seeded into microculture wells by limiting dilution or micromanipulation techniques and were incubated under culture conditions that allow clonal expansion of essentially all T cells. After 15 to 20 days, microcultures were stimulated with PHA and CSF activity was assayed in culture supernatants 24 hr thereafter. About 45% (1/2.3) of peripheral blood T cells were found to give rise to CSF-producing progenies. Moreover, when fluorescence-activated cell sorter-purified T4+ and T4- (or T8- and T8+) were analyzed, the frequency of the precursors of CSF-producing cells was 1/1.5 in the T4+ subset, whereas approximately one-third of the T8+ cells had this functional potential. To additionally characterize T cells responsible for CSF production, unfractionated T cells as well as T4+ and T4- cells were cloned by single cell micromanipulation. The resulting clones were analyzed simultaneously for the production of IL 2, production of CSF and for cytolytic activity in a lectin-dependent assay. It was found that 25/48 clones obtained from unfractionated T cells produced CSF, whereas 23/48 and 19/48 produced IL 2 or had cytolytic activity respectively. Six of the 25 CSF-producing clones had only this functional capability, whereas the remaining clones in addition displayed cytolytic activity (4/25), IL 2 production (10/25), or both (5/25). A similar functional heterogeneity was observed among T4+ and T4- clones, thus indicating that T cells producing CSF are functionally heterogeneous within both the T4+ and T8+ subsets.     

Activation through CD3 molecule leads to clonal expansion of all human peripheral blood T lymphocytes: functional analysis of clonally expanded cells

Monoclonal antibodies directed against CD3, a T cell-specific surface molecule essentially required for activation of these cells, are highly mitogenic for resting human peripheral blood T lymphocytes. A predetermined optimal concentration of anti-CD3 monoclonal antibody WT32 was employed to activate T cells cultured in limiting-dilution microcultures containing irradiated feeder cells and exogeneous interleukin 2. Frequencies of cells triggered into clonal expansion by WT32 under these culture conditions were 0.57 to 0.72 and 0.90 to 1.10 in peripheral blood mononuclear cells and E rosette-positive cells, respectively. It appeared that WT32 could induce virtually every human peripheral blood T lymphocyte to expand into a clonal progeny of 5 to 40 X 10(4) cells in 14 to 18 days of culture. This progeny was tested for cytolytic effector function with 51Cr-labeled murine P815 targets in the presence of PHA to detect all cytotoxic T lymphocytes (CTL) regardless of specificity, and was also assayed for natural killer like activity against K562 target cells. Frequencies of cells in the human peripheral blood T cell compartment giving rise to a clonal progeny expressing CTL function was 1/3, whereas 1/6 to 1/5 expanded into effector cell populations possessing NK activity. Frequency analysis of CD4-positive and CD8-positive populations, activated by WT32 in limiting dilution microcultures, demonstrated that 1 to 6% of the CD4-positive and 100% of the CD8-positive peripheral blood T lymphocytes expanded into CTL.     

Evidence for the presence of suppressor T lymphocytes in animals treated with cyclosporin A

Mice sensitized with alloantigens and treated with cyclosporin A (CsA) were incapable of generating antigen-specific cytolytic lymphocytes (CL). Lymphocytes from these CsA-treated animals could not be reactivated upon exposure to the same alloantigens in mixed lymphocyte culture (MLC), whereas their response to a third-party antigen remained intact, suggesting a long-lasting and specific effect of CsA. After being irradiated, these lymphocytes from CsA-treated animals were added to normal MLC and were shown to prevent normal lymphocytes from becoming cytolytic in a dose-dependent and antigen-nonspecific fashion. These suppressor cells were not detected in mice receiving CsA only, indicating that CsA did not induce but rather permitted the expression of suppressor cells possibly generated by allosensitization. The suppressor cells appeared to be T lymphocytes, because treatment with anti-Thy-1.2 antibody and C abrogated their suppressive activity. The present results suggest that activation and/or sparing of suppressor cells by CsA may account for the long-lasting unresponsiveness seen in CsA-treated animals.     

Natural killer cell number and function in the spontaneously diabetic BB/W rat

The BB/W rat provides a good model of spontaneous autoimmune diabetes. Diabetes-prone (DP) rats have a virtual lack of OX 8+ OX 19+ T cytotoxic/suppressor cells in peripheral blood lymphocytes (PBL) and spleen, suggesting that the OX 8+ OX 9- natural killer (NK) cells are the predominant cytotoxic cell in this animal. In this study, we have shown that rat NK cells belong to the OX 8+ OX 19- asialo GM1 bright population, and that rat NK cell function may be depleted in vivo by administration of OX 8 antibody. Furthermore, evidence is provided to indicate that NK cell number and activity are enhanced on a per cell basis in DP rats as compared to the diabetes-resistant W line rat. DP rats had about threefold more NK cells than did W-line rats. The cytotoxic activity mediated by spleen and PBL against the YAC-1 target generally correlated with the relative number of cells having the OX 8+ OX 19- phenotype. DP lymphocytes mediated low levels of cytolytic activity against the relatively resistant NK target cell K562. To more directly compare the activity of W-line and DP NK cells, spleen NK cells were isolated by flow sorting of the OX 8+ OX 19- population. At a 5:1 E:T ratio, DP OX 8+ OX 19- cells elicited 21% +/- 3 specific lysis and W-line cells elicited 7% +/- 2 specific lysis. To determine whether the elevated levels of NK cells and NK cell activity in DP rats were a consequence of NK cell proliferation, spleen cells were size-separated by centrifugal elutriation. The NK cell activity was predominantly mediated by small to medium-size lymphocytes and not blast-size enriched populations. Moreover, when the DNA content of splenic OX 8+ cells was measured, 98% of the cells were in the G0-G1 phase of the cell cycle. These data indicate that NK cell number and activity are elevated in DP rats, and support a role for NK cells in the pathogenesis of BB/W diabetes.     

The isolation and characterization of the human suppressor inducer T cell subset

Immunization of mice with lower primate lymphoid cells has provided a useful strategy for raising monoclonal antibodies against functionally important surface determinants on human T lymphocytes. We have developed a monoclonal antibody, anti-2H4, which defines functionally unique human T cell subsets. This anti-2H4 antibody was reactive with approximately 42% of unfractionated T cells, 41% of T4+ inducer cells, and was reactive with approximately 54% of T8+ cytotoxic/suppressor population. Anti-2H4 was not reactive with human thymocytes, but reacted with subsets of peripheral blood B cells and null cells. This antibody subdivided peripheral blood T4+ cells into two functionally distinct populations. The T4+2H4+ subset proliferate well to concanavalin A (Con A) stimulation, but poorly to soluble antigen stimulation, and provides poor help to B cells for PWM-induced Ig synthesis. The T4+2H4- subset, in contrast, proliferates poorly upon stimulation with Con A, but well on exposure to soluble antigen, and provides a good helper signal for PWM-induced Ig synthesis. What is, perhaps, most important, the T4+2H4+ subset functions as the inducer of the T8+ suppressor cells. Previous attempts to define the latter subset of cells has relied heavily on the use of specific autoantibodies present in the sera of patients with juvenile rheumatoid arthritis (JRA) and systemic lupus erythematosus (SLE). The present results suggest that anti-2H4 antibody defines the human suppressor induced subset of lymphocyte previously described as T4+JRA+. Last, the results reemphasize the previously documented remarkable structural conservation of certain T cell-specific determinants on lymphocytes of phylogenetically distant primates.