Surface markers on the T cells that regulate cytotoxic T-cell responses I. The Ly phenotype of suppressor T cells changes as a function of time, and is distinct from that of helper or cytotoxic T cells

Regulatory T cells can be obtained from primary mixed lymphocyte cultures of CBA spleen cells responding to BALB/c stimulators. At day 3 of culture, T cells are generated which can either help or suppress the generation of cytotoxic T cells in a second primary MLC culture. The regulatory activity observed depends on the conditions employed in the assay system allowing independent assay of different functional cell types which coexist in the cultures. Both the helper activity and the suppressor activity are mediated by differentiated antigen-specific T cells whose function is radioresistant. The Ly phenotype of these regulatory cells was tested. At day 3 of the first-step culture, the phenotype of the helper cells is Ly 1.1+ Ly 2.1-, whereas the inhibitory cells are Ly 1.1 Ly 2.1+. At day 5 of M LC culture, suppressor activity and helper activity are also observed. However, at this point, a suppressor cell which is Ly 1.1-Ly 2.1+ represents the major inhibitory activity. It is not clear whether this change in suppressor cell phenotype as a function of time in culture represents one differentiation pathway or cells derived from two different precursor cells. The Ly phenotype of helper or cytotoxic T cells did not change as a function of time in culture. In day 5 first-step cells, the cytotoxic cells were typed as Ly 1.1+ 2.1+, whereas the inhibitory cells present in aliquots of the same treated cell population expressed the Ly 1.1- Ly 2.1 phenotype. Taken together, these observations show that the antigen-specific suppressor cells and helper cells which regulate the generation of cytotoxicity, and the cytotoxic cells themselves represent physically distinct subclasses of T cells.     

Analysis of the negative allogeneic effect using B cells and helper T cell lines as an indicator system: B cells identified as target cells for suppression

The target cells for H-2b T lymphocytes mediating a negative allogeneic effect in vitro were analyzed by using carrier-specific helper T cell lines of H-2b, H-2d, or F1 origin and hapten-primed T-depleted spleen cells also expressing one or both of these haplotypes. The helper T cell lines were shown to be carrier specific and H-2b or H-2d restricted. Most importantly, the lines derived from H-2b homozygous mice were devoid of alloreactivity against H-2d and vice versa. Titration of naive H-2b T lymphocytes to the indicator cultures resulted in suppression of the secondary anti-DNP response of the indicator cells whenever the B cells expressed H-2d antigens. The lack of suppression observed in mixtures in which only the helper T cell lines expressed H-2d antigens was not reversed by the increased addition of naive H-2bxd cells, indicating that an insufficient amount of H-2d antigens present on the low number of helper T cells used did not account for this finding. Moreover, the polyclonal plaque-forming cell responses of F1 spleen cells to LPS were also suppressed by naive parental T cells. From these findings it is concluded that the suppressor T cells directly recognize and inhibit allogeneic B cells without the involvement of helper T cells. In addition, it was shown that the suppression of secondary anti-hapten responses by naive allogeneic T cells is blocked by monoclonal anti-Lyt-2 antibody added at the onset of culture. Addition late in culture had no effect, pointing to a functional role of the Lyt-2-bearing structure at an early stage of the suppressive events resulting in the negative allogeneic effect.     

Generation of specific helper cells and suppressor cells in vitro for the IgE and IgG antibody responses.

Normal splenic lymphocytes from BDF1 mice were cultured on ovalbumin (OA)-bearing syngeneic peritoneal adherent cells for 5 days and their subsequent helper function was tested by an adoptive transfer technique. Lymphocytes harvested from the culture were mixed with DNP-KLH-primed spleen cells and transferred into irradiated syngeneic mice followed by challenge with DNP-OA. The results showed that the cultured lymphocytes has helper function for both IgE and IgG anti-DNP antibody responses. Depletion of mast cells and T cells in the peritoneal adherent cell preparations did not affect the generation of helper cells in the culture. The helper function of the cultured lymphocytes was abolished by the treatment with anti-theta antiserum and complement and was specific for ovalbumin. The OA-specific helper T cells were generated in vitro by the culture of a T cell-rich fraction of normal spleen on T cell-depleted OA-bearing peritoneal cells. If the normal splenic lymphocytes or T cell-rich fraction were cultured with 10 mug/ml of OA in the absence of macrophages, cultured lymphocytes lacked helper function. The transfer of splenic lymphocytes or splenic T cells cultured with soluble OA to normal non-irradiated mice, however, suppressed both IgG and IgE antibody responses of the recipients to subsequent immunization with DNP-OA. The suppressor cells were sensitive to anti-theta antiserum and complement and their activity was specific for OA. The cultured cells transferred into normal mice did not suppress anti-hapten antibody response to DNP-KLH. Normal lymphocytes cultured on OA-bearing macrophages and had helper function in adoptive transfer experiments failed to suppress antibody response of non-irradiated recipients to DNP-OA. The results indicate that OA-bearing macrophages primed T cells and generated helper T cells, whereas the culture of normal lymphocytes with soluble OA in the absence of macrophages generated suppressor T cells.     

The interleukin 2 secretion defect in vitro in systemic lupus erythematosus is reversible in rested cultured T cells

Interleukin 2 (IL 2) secretion in response to mitogenic stimulation in vitro is strongly reduced in circulating T lymphocytes from patients with SLE. It is still not clear how this abnormality relates to the B cell hyperactivity in the disease. Some investigators proposed that an intrinsic T helper cell defect could lead to suppressor cell dysfunction and autoimmunity. Others have found that in fact increased suppressor cell activity can cause IL 2 hyposecretion. In the present study we report that the IL 2 secretion in response to PHA plus PMA by T cells from patients with SLE, which initially was decreased by a factor of 10 as compared with the IL 2 secretion in blood donor T cells, was restored when the T cells were rested for 2 to 3 days in culture before stimulation. IL 2 hyposecretion in SLE T cells and the kinetics of normalization in culture were not changed by the addition of normal adherent cells during the stimulation with PHA/PMA, occurred in the absence of significant cell death or proliferation or change of the T4:T8 cell ratio during the resting culture, were not due to a maturation of immature T6-positive cells (less than 1.5% T6 cells in SLE T cells), and also occurred in T8-depleted T4 cells alone. Furthermore, a normalization of IL 2 secretion took place in the presence of either SLE serum or normal serum, and the addition of fresh autologous T cells to 3-day-cultured SLE T cells did not cause suppression of IL 2 secretion. These data show that some rapidly reversible defect occurs in circulating T helper cells in SLE. That this could reflect an exhaustion of T helper cells that have been activated in vivo is discussed.     

Surface markers on the T cells that regulate cytotoxic T-cell responses

Regulatory T cells can be obtained from primary mixed lymphocyte cultures of CBA spleen cells responding to BALB/c stimulators. At day 3 of culture, T cells are generated which can either help or suppress the generation of cytotoxic T cells in a second primary MLC culture. The regulatory activity observed depends on the conditions employed in the assay system allowing independent assay of different functional cell types which coexist in the cultures. Both the helper activity and the suppressor activity are mediated by differentiated antigen-specific T cells whose function is radioresistant. The Ly phenotype of these regulatory cells was tested. At day 3 of the first-step culture, the phenotype of the helper cells is Ly 1.1⁺ Ly 2.1^–, whereas the inhibitory cells are Ly 1.1⁺ Ly 2.1⁺. At day 5 of MLC culture, suppressor activity and helper activity are also observed. However, at this point, a suppressor cell which is Ly 1.1^– Ly 2.1⁺ represents the major inhibitory activity. It is not clear whether this change in suppressor cell phenotype as a function of time in culture represents one differentiation pathway or cells derived from two different precursor cells. The Ly phenotype of helper or cytotoxic T cells did not change as a function of time in culture. In day 5 first-step cells, the cytotoxic cells were typed as Ly 1.1⁺ 2.1⁺, whereas the inhibitory cells present in aliquots of the same treated cell population expressed the Ly 1.1^– Ly 2.1⁺ phenotype. Taken together, these observations show that the antigen-specific suppressor cells and helper cells which regulate the generation of cytotoxicity, and the cytotoxic cells themselves represent physically distinct subclasses of T cells.     

Regulation of B cell maturation and differentiation. I. Suppression of pokeweed mitogen-induced B cell differentiation by tumor necrosis factor (TNF)

Growth and differentiation of B cells into Ig-secreting plasma cells is regulated by both T cells and macrophages and/or their secreted factors. Although the regulatory role of various cell-derived factors has been examined, the involvement of the macrophage-derived factor, TNF, in human B cell growth and differentiation has not yet been investigated. In the present study we examine the role of rTNF in polyclonal B cell response of human PBL induced by PWM. The addition of rTNF at the initiation of the culture resulted in the dose-dependent inhibition of the generation of both IgG and IgM PFC. Inhibition of PFC development followed the same dose response as rTNF-mediated cytotoxicity against a TNF-sensitive tumor target. The mechanism of rTNF-mediated suppression was examined in different experimental systems. Recombinant TNF did not affect the viability or proliferation of either the T cell or B cell subpopulations, suggesting that TNF does not mediate its suppressive effect by cytotoxic mechanisms. Kinetic studies in which rTNF was added at different times after initiation of culture indicated that inhibition can be observed as late as 4 days of culture and suggested that TNF acts at a late phase of the growth and differentiation pathway of B cells. In further studies we examined the cellular level of TNF-mediated suppression. The addition of rTNF to supernatants containing helper factors and enriched B cells resulted in no inhibition, suggesting that TNF does not act at the B cell level. This was confirmed by demonstrating that rTNF does not inhibit spontaneous PFC development by the CESS B cell line. The effect of TNF on T cell subpopulations was examined by using normal or irradiated T cells, which inactivate suppressor cells. Addition of rTNF to B cells combined with either T cell population suppressed both IgG and IgM PFC development, indicating that the target cell for suppression is the T helper cell but not ruling out an effect on macrophages or the T suppressor cells. Combined, the observed results demonstrate that rTNF suppresses PWM-induced B cell differentiation without affecting B cell proliferation. TNF appears to mediate the suppression by acting directly on T helper cells or else by regulating the production of factors controlling T cell activation and lymphokine secretion.     

Regulatory mechanism of reagin production in mice at the T cell level. I. Suggestive evidence for the generation of class specific PPD-reactive helper T cell population in Mycobacterium-primed cells.

Helper T cell activities specific for purified protein derivative (PPD) generated by immunization with Mycobacterium tuberculosis (Tbc) or PPD were investigated concerning adoptive IgE and IgG antibody responses. It is interesting that preferential triggering activity of IgG antibody response was observed when PPD-reactive cells from mice immunized with Tbc were used as a helper cell source. The selective triggering of IgG B cells by Tbc-primed cells was consistently observed using DNP-primed B cell populations from mice immunized with DNP-carrier conjugate in either ICFA or alum. T cell dependency of helper activity was demonstrated by the fact that treatment of Tbc-primed cells with anti-Thy 1 antiserum plus complement abolished their helper activity. We also demonstrated that purified T cell populations selectively triggerred IgG B cells. Selective triggering of IgG B lymphocytes by Tbc-primed T cells may not be due to the influence of suppressor T cells supposedly present in Tbc-primed cells since this selectivity was not affected by X-irradiation of Tbc-primed T cell populations which may inactivate suppressor T cells. Furthermore, passive transfer of Tbc-primed cells into normal recipient mice, the condition which may detect the suppressor T cell effect much more sensitively in IgE production, or preimmunization with Tbc 2 weeks before, did not suppress primary anti-DNP IgE antibody response to DNP-PPD. Thus, the observations presented here are favorable to the concept of the presence of IgG class-specific helper T lymphocytes. Furthermore, PPD-reactive T cells from mice immunized with PPD itself exerted their helper function for triggering B cells of both IgE and IgG classes. This may also indicate that some of the components associated with Tbc other than PPD might negatively affect the development of PPD-reactive helper T cells specific to the IgE class. The generation of such IgG-specific T cell activity in the presence of Tbc will be discussed in the light of the T cell population involved in the regulation of antibody responses of different immunoglobulin classes.     

T cell tolerization in vitro: modulation of helper and suppressor cell activities

This paper analyzes the conditions for in vitro tolerization of purified whole T cell populations and the consequences on helper and suppressor T cell functions. Highly purified splenic T cells from adult DBA/2 mice were incubated in vitro for 24 hr with high doses of trinitrophenyl coupled to human gamma-globulins (TNP-HGG). A profound inhibition of the TNP-specific helper function of these T lymphocytes was observed in a cooperative culture with normal purified splenic B cells and TNP-SRBC as antigen. This state of specific unresponsiveness was maintained after trypsin treatment of the cells, at the end of the 24-hr incubation with the tolerogen. We checked that this procedure removed the vast majority of F23.1 T cell receptor determinants from the cells. This result indicates that T cell receptors for antigen were not merely blocked by the tolerogen. In addition, B cells preincubated with tolerized T cells for 24 hr remained as responsive to TNP as B cells mixed with normal T cells in similar conditions. This demonstrates that the decreased response is not the result of secondary B cell tolerization. In addition, anti-Ia monoclonal antibodies were shown to block the induction of tolerance. We also showed that tolerized T cells significantly decreased the anti-TNP response of normal T and B cells in vitro, whereas the anti-SRBC response in the same cultures was unaffected. When tolerized T cells were separated into Lyt-2- and Lyt-2+ cells, it was found that tolerized Lyt-2- cells had lost about 75% of their helper activity and that Lyt-2+ cells suppressed 70% of the response of a normal T and B cell culture. Thus, in vitro induction of T cell tolerance results in a specific T cell unresponsiveness which is due to both helper T cell inactivation and induction of specific suppressor T cells.     

Differences in the regulation of humoral responses between mice infected with Trypanosoma cruzi and mice administered T. cruzi-induced suppressor substance

In the present study, the effects of Trypanosoma cruzi and the T. cruzi-induced serum suppressor substance (SSS) on antibody responses were compared. Although infection with T. cruzi led to an alteration in T cell helper activity and a reduced specific B cell precursor frequency, SSS did not have a similar effect on either of these cell populations. The characteristics of the altered T cell helper activity was further investigated, and it was found that helper activity appeared earlier in infected mice than in normal or SSS-suppressed mice, and less antigen was required for optimal elicitation of T helper cells in infected mice. The potency of T cell helper activity also was shown to differ, and in the order T. cruzi-infected 6E normal 6E SSS-suppressed mice. It was found that spleen cells from T. cruzi-infected mice elaborated more potent specific helper factors than spleen cells from normal or SSS-suppressed mice, but did not produce a detectable nonspecific helper factor in vitro. Finally, the addition of B cells from low-dose primed, T. cruzi-infected mice to cultures of normal spleen cells resulted in subnormal responses to the priming antigen (sheep erythrocytes) but not to another unrelated antigen (trinitrophenyl-haptenated Brucella abortus), whereas similarly sensitized B cells from normal or SSS-suppressed mice caused no such effect.     

Regulation of the cytotoxic activity of alloreactive cytotoxic T lymphocytes by helper cells and lymphokines

The cytotoxic activity of alloreactive cytotoxic T lymphocytes (CTL) was maintained and augmented by transferring cells from a 5-day mixed lymphocyte culture MLC into a host culture (HC) containing indomethacin, freshly explanted normal spleen cells, and peritoneal cells which were syngeneic to the MLC cells. The MLC cells used in the transfer experiments were generated by culturing untreated H-2b splenic responders with irradiated H-2d stimulators, or were generated by culturing Lyt-2-depleted H-2b splenic responders with irradiated H-2d stimulators. The allo-CTL were found to be derived from the donor MLC (first culture) when unfractionated MLC cells were transferred into a host (second) culture and incubated for 5 days. In contrast, the allo-CTL were derived from host culture cells when Lyt-2-depleted MLC cells were transferred and the combined cultures incubated for 5 days. In the former case, the augmentation of MLC-derived cytotoxicity did not result from nonspecific expansion of all donor T cells; instead it was mediated by lymphokine(s), distinct from IL-2, produced by helper T cells generated in host culture, which appeared to selectively expand the antigen-specific CTL or to increase the cytotoxic activity of these CTL. The helper T cells were Thy-1+, L3T4+, and Lyt-2-. These findings indicate that antigen-nonspecific help was provided by helper cells or helper factors (lymphokines) generated in the host culture, which maintained and augmented the cytotoxic activity of the fully generated allo-CTL. This helper effect was also seen in the induction of primary allo-CTL responses which could be generated with fewer stimulating cells and with a stronger cytotoxic response at different R/S ratios tested. The generation of allo-CTL in second culture following transfer of Lyt-2-depleted MLC cells to host cultures appears to have involved antigen carryover from the MLC; however, antigen carryover alone was not sufficient. It appears that in the absence of Lyt-2+ suppressor T cells, antigen-specific help might be generated in donor cultures (Lyt-2-depleted MLC) which promoted or recruited the generation of antigen-specific CTL in host culture.     

Cellular interactions of L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT)-specific suppressor factors. I. Inhibition of the activity of GAT-specific helper T cell clones by monoclonal GAT-specific suppressor T cell factors

Considerable information concerning the serology and biochemistry of antigen-specific, T cell-derived suppressor factors has been obtained with the use of T cell hybridomas as a source of homogeneous material. Similarly, knowledge of helper T cell products and receptors is accumulating from studies of helper T cell clones and hybridomas. Our strategy for studying the mechanisms by which suppressor factors inhibit responses was to determine whether monoclonal suppressor factors could inhibit antibody responses specific for L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT) in cultures containing unprimed splenic B cells, macrophages, and GAT-specific T cell clones as a source of helper activity. The MHC-restricted, two chain suppressor factors, GAT-TsF2, inhibited these responses if the helper T cell clones and suppressor factor were derived from H-2-compatible mice. Furthermore, responses were inhibited by briefly pulsing T cell clones with GAT-TsF2 in the presence of GAT, indicating that suppressor factors need not be present continuously. In addition, helper T cell clones adsorbed syngeneic, but not allogeneic, GAT-TsF2 in the presence of GAT. Adsorption also requires a shared antigenic specificity between the H-2b-derived helper T cells and TsF2 factor. Thus, helper T cells can serve as the cellular target of antigen-specific, MHC-restricted GAT-TsF2, and cloned helper T cells can be used as a homogeneous target population for analysis of the molecular mechanisms of T cell suppression.     

The role of regulatory components from resident T lymphocytes in polyclonal B cell activation

Resident T lymphocytes have been found to exert helper and suppressor regulatory influences with regard to polyclonal activation of murine splenic B lymphocytes elicited by lipopolysaccharide. In the normal adult spleen, only T cell helper influences are exercised over polyclonal B cell activation. This activity is a property of Lyt 1+2- T cells and does not appear to be subject to MHC restriction. Suppressive influence evidently is either latent or it exists at such a low level that its effects are difficult to detect. No regulatory activity can be recovered from the supernatants of T cells, cultured either with or without LPS. However, suppressor T cell function may be evoked by activating splenic T cells with Concanavalin A or by sonicating unstimulated splenic T cells in order to liberate a suppressive potential which is not expressed by these unstimulated cells when intact. The soluble fraction of resident splenic T cell sonicates exerts both helper and suppressor regulatory influences. The soluble helper activity is derived from Lyt l+2- T cells, whereas suppressor activity is generated from Lyt 1-2+ T cells. The suppressive activity of T cell sonicates is not restricted by the MHC gene complex. Helper and suppressor activities contained in splenic T cell sonicates were separated by gel chromatography; the suppressive activity was found to elute with a molecular weight between 68,000 and 84,000 daltons, and the helper activity eluted with a molecular weight between 15,000 and 23,000 daltons. The data indicate that helper and suppressor activities are distinct molecular entities derived from distinct splenic T lymphocyte subpopulations. The possibility that these molecules are precursors to or components of antigen-specific or nonspecific helper and suppressor factors described in the literature is discussed.     

T cell subsets regulating antibody responses to L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT) in virgin and immunized nonresponder mice

T cell subsets from virgin and immunized mice, which are Ir gene controlled nonresponders to GAT, which regulate antibody responses to GAT have been characterized. Virgin nonresponder B10.Q B cells develop GAT-specific antibody responses to GAT, B10.Q GAT-M phi, and GAT-MBSA when cultured with virgin or GAT-primed Lyt-1+, I-J-, Qa1- B10.Q helper T cells. Virgin T cells are radiosensitive, whereas immune T cells are radioresistant (750 R); qualitatively identical helper activity is obtained with T cells from mice immunized with soluble GAT, B10.Q GAT-M phi, and GAT-MBSA. Responses to GAT and GAT-M phi are not observed when virgin or GAT-primed Lyt-1+, I-J+, Qal+ T cells are added to culture of virgin or GAT-primed Lyt-1+, I-J-, Qa1- helper T cells and virgin B cells; the GAT-specific response to GAT-MBSA is intact. The Lyt-1+, I-J+, Qa1+ T cells from mice primed with GAT, GAT-M phi, and GAT-MBSA were qualitatively identical in mediating this suppression. Virgin Lyt-2+ T cells have no suppressive activity alone or with virgin Lyt-1+, I-J+, Qa1+ T cells, whereas responses to GAT, GAT-M phi, and GAT-MBSA are suppressed in cultures of GAT-primed helper T cells containing GAT-primed Lyt-2+ T cells (with or without GAT-primed Lyt-1+, I-J+, Qa1+ T cells). Suppression of responses to GAT-MBSA in cultures of GAT-M phi-primed helper T cells requires both GAT-M phi-primed Lyt-1+, I-J+, Qa1+ T cells and Lyt-2+ T cells; the Lyt-1+, I-J+, Qa1+ T cells appear to function as inducer cells in this case. In cultures containing GAT-MBSA-primed helper T cells, either GAT-MBSA-primed Lyt-1+, I-J+, Qa1+ or Lyt-2+ T cells suppress responses to GAT and GAT-M phi; under no circumstances are responses to GAT-MBSA suppressed by GAT-MBSA-primed regulatory T cells. This regulation of antibody responses to GAT by suppressor T cells is discussed in the context of the involvement of suppressor T cells in responses to antigens under Ir control, and of the evidence that nonresponsiveness to GAT is not due to a defect in the T cell repertoire, but rather is due to an imbalance in the activation of suppressor vs helper T cells.     

Activation/suppression of the immune response in vitro by antigen and dextran sulfate: 2. The role of T-cell subsets determined by cell separation and partition analysis

Culture of spleen cells with dextran sulfate (DxS) and antigen at various different cell densities revealed a T-cell-dependent regulatory pathway not observed in conventional culture. This finding can be explained by the frequent presence in the cultures of a helper cell and the less frequent presence of a suppressor cell, both activated by antigen and DxS. The classic, radioresistant, antigen-specific, helper T cell was not regulated by this newly revealed pathway. The highly frequent, DxS-dependent helper T cell is Lyt-1⁺2^?. The suppressive effect is mediated by a Lyt-1⁺2⁺ population consisting of helpers and latent suppressors that can be made active by DxS or Lyt-1⁺ cells. The specificity of the Lyt-1⁺ helper cells was not established, but the high frequency observed implies a nonspecific mechanism. The specificity of the suppressor effect was not determined by these experiments. This regulatory mechanism is similar to the phenomena exhibited by polyclonally activated T-cell populations.     

Properties of antigen-specific suppressive T cell factor in the regulation of antibody response of the mouse. II.In vitro activity and evidence for the I region gene product.

An antigen-specific suppressive T cell factor, which was extracted from carrier-primed T cells, was further characterized in an in vitro secondary antibody response. The factor was capable of suppressing secondary IgG antibody response of primed spleen cells when it was added to the culture together with relevant antigen. The suppressive T cell factor was not released from primed T cells by a short-term culture with antigen, but was kept bound to the membrane of the residual cultured cells, only the physical disruption of which can release the T cell factor. The target of the suppressive T cell factor was determined as being the helper T cell, since the factor did not exert any effect in the absence of the helper T cell with identical specificity to that of the factor. The suppressive activity was completely absorbed with alloantisera specific for products of the I region of H-2 complex, although various anti-immunoglobulin antisera failed to do so. Close analysis of the specificity of alloantisera capable of absorbing the suppressor molecule indicated that the suppressive T cell factor may, in fact, be an I region gene product probably coded for by genes in I-A and/or I-B (including I-E) subregions.     

Identification of profound peripheral T lymphocyte immunodeficiencies in the spontaneously diabetic BB rat

The BB rat is presently the best available animal model for human insulin dependent diabetes (IDD). Because of the extreme susceptibility of the strain to opportunistic infections and because current studies suggest that they have an autoimmune diathesis, of which IDD is but one result, aspects of the immune system of the BB rat were studied. Severe T lymphopenia was observed in all BB rats, irrespective of sex or the presence of IDD, while numbers of B cells and serum immunoglobulin levels were normal. Both the helper T lymphocyte and cytotoxic/suppressor T lymphocyte subsets, defined by reactions with monoclonal antibodies, were depressed, and an inversion of the helper T cell subset to cytotoxic/suppressor T lymphocyte subset ratio occurred in all BB rats with increasing maturity. Concomitantly, severe impairments of T cell-mediated immune responses were noted. BB rats poorly rejected allografts across both major and minor histocompatibility barriers, and BB splenic or peripheral blood lymphocytes had markedly defective proliferative responses to mitogens and to allogeneic cells in MLC. Irradiated and nonirradiated BB spleen cells did not inhibit WF mitogenic or MLC responses, which suggests that the T cell defect in BB rats is not solely due to increased suppressor activity. Because irradiated WF cells and Con A supernatants did not restore BB proliferative responses, and BB lymphocytes were able to produce IL-2 normally, a reduced ability of BB lymphocytes to respond to helper factors such as IL-2 is suggested. In contrast to T lymphocytes from spleen or peripheral blood, BB thymocytes responded as well as did WF thymocytes to Con A or Con A supernatants. Percentages of T lymphocyte subsets and histology of BB thymuses were also normal when compared to WF thymuses. However, spleens and lymph nodes from BB rats were severely depleted of T lymphocytes, and thymocytotoxic autoantibodies were detected in many BB rat sera. The above findings indicate that BB rats have T lymphocyte immunoincompetence, which appears to be a post-thymic or peripherally acquired maturational defect.     

Release of hemopoietic factors by normal human T cell lines with either suppressor or helper activity

We analyzed the release of activities capable of stimulating the in vitro growth of human hemopoietic progenitor cells by long-term cultured T cell growth factor (TCGF)-dependent human T lymphocytes. Seven cell lines tested produced colony-stimulating activity (CSA) as well as burst-promoting activity (BPA). The CSA stimulated primarily the growth of the cells forming colonies after 14 days of incubation. In addition the supernatants from these seven T-cell lines showed the ability to induce the in vitro growth of mixed granulocyte, erythroid, megakaryocyte, macrophage colonies (CFU-GEMM). The release of hemopoietic factors did not depend on the presence of accessory cells or phytohemagglutinin or serum during the incubation for factor production. In six of the T cell lines the majority of the cells were reactive to the OKT 8 monoclonal antibody (MoAb), whereas one cell line contained mostly OKT 4+ cells. Suppressor activity was detected in three tested OKT 8+ cell lines, while the one OKT 4+ displayed helper activity. All cell lines produced hemopoietic factors with equal efficiency. These results indicate that factors affecting human hematopoiesis are produced by normal T lymphocytes in long-term culture and this property is not related to the helper or suppressor activity of the cultured cells.     

Suppressive effect of human natural killer cells on pokeweed mitogen-induced B cell differentiation

The suppressive effect of human natural killer (NK) cells on B cell differentiation induced by pokeweed mitogen (PWM) was investigated. By using Percoll discontinuous density gradient centrifugation, peripheral blood nonphagocytic and nonadherent mononuclear cells were divided into low and high density fractions for which NK cells (Large granular lymphocytes, LGL) and T cells were enriched, respectively. These fractionated mononuclear cells were co-cultured with purified autologous B cells in the presence of PWM, and were examined for their helper and suppressor activities on differentiation of B cells to immunoglobulin-(IgM and IgG) producing cells by a highly sensitive reversed hemolytic plaque assay. The T cell-enriched high density fractions provided help for B cell differentiation to levels higher than that of unfractionated mononuclear cells. On the other hand, the NK-enriched low density fractions did not show helper activity, and when added to the culture of B cells plus helper T cells, they markedly suppressed B cell differentiation. This suppressive activity, as well as the NK cytotoxicity of the NK-enriched fractions, was abrogated by treatment of the cells with monoclonal antibody against human NK cells (HNK-1), but not against T cells (OKT3) in the presence of complement. NK cells also suppressed PWM-driven B cell differentiation in the presence of T4+ (helper/inducer T) but not T8+ (cytotoxic/suppressor T) cells; however, they showed no inhibition of soluble factor-induced B cell differentiation assayed in the absence of helper T cells. It is thus concluded that human peripheral blood NK cells exhibit an ability to suppress PWM-driven B cell differentiation, possibly by acting through the effect on helper T cells but not directly on B cells.     

Suppression of a polyclonal B-cell response by supernatants from the murine autologous mixed lymphocyte reaction

Previously, we have demonstrated that supernatants from autologous mixed lymphocyte (AMLR) cultures contain helper factors which can mediate the development of a cytotoxic T-cell response to hapten modified self. In the current study, the effect of AMLR supernatants on the humoral response was explored. BALB/C splenic non-T cells produced a large polyclonal antibody response to lipopolysaccharide (LPS), as measured in a Protein A SRBC plaque assay. Surprisingly, syngeneic AMLR supernatants suppressed the LPS-induced generation of plaque-forming cells. The presence of T cells in the stimulated cultures did not affect suppressor activity. The decreased response was not the result of a shift in kinetics, as maximal activity was observed on Day 4, whether or not AMLR supernatants were added. The AMLR culture supernatants were most effective in suppressing the plaque-forming cell response when added at the initiation of culture. AMLR supernatants added after 24 hr of culture resulted in only about 50% of maximum suppression. Supernatants added at 48 or 72 hr had no effect. Interferon-gamma (IFN-gamma) has been detected in AMLR culture supernatant and has been reported to suppress the development of plaque-forming cells in response to LPS. However, it is unlikely the suppressive activity observed in these studies is due to IFN-gamma. Dialysis of the AMLR culture supernatant against a pH 2 buffer for 24 hr or incubation at 70, 80, or 90 degrees C for 10 min, treatments that inactivate IFN-gamma, enhanced suppression. These results suggest that in addition to cytotoxic-T-cell helper factors, the cellular interactions in the AMLR induces the production of a stable mediator(s) which is able to directly suppress B cells at an early stage of their development into plasma cells.     

Suppressor cells in contact sensitivity. Effect on T cells helping antibody production to picryl chloride.

T cells from mice injected with picryl sulfonic acid have previously been shown to suppress the effector and possibly other phases of contact hypersensitivity reactions to picryl chloride. In this report we examine their effect on T cells helping the early direct anti-TNP plaque-forming cell response of mice painted with picryl chloride. They did not directly inhibit the activity of the helper cells but did inhibit the ability of mice to generate helper cells after skin painting. The suppressor cells were T cells as tested by passage through nylon wool columns and sensitivity to anti-θ serum. Viable syngeneic cells were required for suppression and their effect was specific. The suppressor cells could not be generated in adult thymectomized mice but could be produced in mice treated with high doses (200 mg/kg) of cyclophosphamide. These properties are distinct from those of suppressor T cells produced following immunization with picryl chloride but are the same as those of other suppressor cells induced by PSA which inhibit contact hypersensitivity.