T helper cells in immune mice amplify the primary anti-tumor cytotoxic response

In this report we examine the influence of splenic helper cells in the primary cytotoxic T lymphocyte (CTL) response against syngeneic murine leukemia virus-(MuLV) induced tumor cells. We identify an Lyt-1+ 800 R radiation-resistant helper T cell that will amplify the in vitro generation of CTL against syngeneic tumor cells from nonimmune spleen cells.     

Regulation of the immune response against UV-induced skin cancers: specificity of helper cells and their susceptibility to UV-induced suppressor cells

The purpose of this study was to determine the role of T helper (Th) cells in the immune response to UV-induced tumors. Repeated exposure of mice to UV radiation results in the production of suppressor T lymphocytes that facilitate tumor growth by inhibiting host immunity. To investigate whether the suppressor T cells inhibit the response to UV tumors by blocking the generation of Th, we employed an indirect method for measuring helper cell activity. We found that Th were produced in normal mice after immunization with UV-induced tumors. These Th appeared to be specific for the immunizing tumors, in contrast to the UV-induced suppressor cells, which recognize UV-induced tumors as a group. The suppressor T cells responsible for inhibiting tumor rejection have no effect on tumor-specific helper cell activity in vitro. However, UV-induced suppressor T cells transferred into unirradiated mice seem to block the generation of helper cell activity after immunization with UV-produced tumors. These results suggest the UV-induced suppressor cells may prevent tumor rejection by blocking the generation of Th.     

The nature of the interaction between suppressor and helper T cells in the response to LDHB

Purified Lyt-1+2+ T cells were depleted of alloreactive cells by BUdR and light treatment, and then were primed in vitro against LDHB presented on allogeneic APC. Such cells could be restimulated by LDHB on the same allogeneic APC, but not by LDHB on APC syngeneic with the T cells. The restimulated T cells suppressed the proliferative response of Lyt-1+2- T cells primed and restimulated by the same antigen. The suppression, which was antigen specific, occurred after a 6-hr co-culture of the suppressor (Tse) and proliferating helper (Th) cells. The successful interaction (as measured by suppression) between allogeneic Th and Tse cells was found to be determined by the restriction specificity but not the MHC haplotype of Th cells, and the MHC haplotype but not the restriction specificity of Tse cells. Thus, suppression occurred only when the Tse cells carried genes controlling the MHC molecules that served as restriction elements for antigen recognition by the Th cells. No evidence could be obtained for the participation of APC in the Tse-Th interaction. The data suggest the interaction is based on the recognition by the Th cell of the antigen presented in the context of MHC molecules controlled by the Tse cell.     

Regulation of the primary in vitro response to TNP-polymerized ovalbumin by T suppressor cells induced by ovalbumin feeding

Spleen cells from DBA/2 mice that received a single feeding of 20 mg of ovalbumin (OVA) 7 days previously were specifically hyporesponsive to primary in vitro challenge with the thymic-dependent antigen TNP-polymerized ovalbumin (TNP-POL-OVA). The tolerance observed in spleen cells from OVA-fed animals was dependent upon OVA-specific T suppressor cells, because splenic T cells from OVA-fed mice suppressed the primary response to TNP-POL-OVA of cultures containing normal T and B cells. The tolerance and suppression was OVA specific, because spleen cells from OVA-fed animals responded well to other antigens (including TNP on another carrier), and splenic T cells from OVA-fed mice did not affect the response of normal T and B cells to sheep erythrocytes. These data confirm the existence of T suppressor cells after OVA feeding and provide a direct means of assaying their activity in a primary in vitro response.     

Regulation of the immune response to tumor antigens. X. Activation of third-order suppressor T cells that abrogate anti-tumor immune responses

The experiments described further define the suppressor T cell pathway in the S1509a tumor system. We demonstrated previously that S1509a-induced Ts1, TsF1, and Ts2 specifically suppress in vivo Ly1+2- T cell-dependent responses to S1509a and that Ts1 suppress in vivo Ly-1+2- T cell-mediated proliferative responses to S1509a. We have now shown that in vivo administration of either S1509a-induced TsF1 or TsF2 suppresses both in vivo and in vitro Ly-1+2- T cell-mediated responses to S1509a. Furthermore, we revealed the existence of Ts3, which are activated by S1509a tumor antigen and TsF2, in this murine tumor system. Finally, we demonstrated that cyclophosphamide abrogates the suppressive effect of TsF2 but not that of Ts3. These results are discussed with respect to T cell-mediated suppression in other murine tumor systems and the possible pivotal role for a tumor antigen-presenting cell in activating Ts3 in the S1509a tumor system.     

Regulation of the immune response to tumor antigen. VI. Differential specificities of suppressor T cells or their products and effector T cells.

Suppressor T cells arising during the development of certain murine methylcholanthrene-induced fibrosarcomas have previously been shown capable of limiting only those effector responses generated against the homologous tumor. Thus, S1509a-induced suppressor T cells inhibit immune reactivity only to the S1509a tumor in S1509a immune mice and have no effect on the rejection of SAI tumors in SAI-immune animals. In contrast to this is the cross-reactivity of effector cells in this system, whereby animals rendered immune to either the S1509a or SAI sarcoma are equally capable of rejecting a challenge of the opposite tumor. The specificity of suppression has been further defined in the present study, which demonstrates that S1509a-induced suppressor cells can inhibit responsiveness only to the S1509a sarcoma, even in the simultaneous presence of both the S1509a and SAI tumors. Furthermore, the suppressor factor that is obtainable from suppressor T cells demonstrates a similar precise specificity in its ability to limit selectively reactivity only against the inducing tumor, regardless of the simultaneous expression of antigens on other tumors recognized by cross-reactive effector cells. These results suggest that the antigenic determinants recognized by effector and suppressor T cells are different, and may provide a model for further dissection of suppressor cell function in vivo.     

Augmentation by serum-induced helper cells of T cell-mediated cytotoxic response against tumor associated antigens and alloantigens

Summary In vitro T cell-mediated cytotoxic responses to tumor associated antigens or alloantigens can be augmented by the addition of small amounts (0.1 to 1%) of syngeneic (mouse) or xenogeneic (rabbit) serum in the standard lymphocyte culture medium. Further studies showed that the augmentation is mediated by helper cells, which are induced by culturing the spleen cells or lymph node cells in the presence of these sera. In the syngeneic system performed with mixed lymphocyte tumor cell cultures (MLTC), the serum-induced helper cells are found to be resistant to the lysis of anti-Thy 1.2 antibody and are radioresistant; thus they have the characteristics of macrophages. In the allogeneic system performed with mixed lymphocyte culture (MLC), the serum-induced helper cells are also found to be resistant to the lysis of anti-Thy 1.2 antibody but are radiosensitive. In the latter case, however, removal of T cells abolishes the helper cell generation and only the T cell-enriched fraction provides for the generation of helper cells, indicating that the helper cells for MLC are probably derived from T cells but lose their susceptibility to anti-Thy 1.2 antibody lysis upon culturing in vitro. A study of the mode of action of the helper cells for MLC showed that they are probably needed at a later stage of cytotoxic response for the amplification of the killing efficiency of the T effector cells whereas the helper cells for MLTC are needed in the early induction phase of the immune response. These results indicate that although serum can augment the cytotoxic responses both in the syngeneic and in the allogeneic systems, the mechanism for the augmentation differs: macrophagelike helper cells are responsible for the augmentation of cytotoxic response to tumor associated antigens, whereas augmentation of cytotoxic response to alloantigens appears to be mediated by a subpopulation of T helper cells. Supported by a grant from the Japan Society for the Promotion of Science (T. I.).     

Regulation of myelin basic protein-specific helper T cells in multiple sclerosis: generation of suppressor T cell lines.

Suppressor T cell (Ts) lines specific for myelin basic protein (MBP)-reactive helper T cell (Th) clones were generated from two patients with multiple sclerosis (MS) following a primary culture of peripheral blood mononuclear cells (PBMC) with MBP and cyclosporine A (CsA). These suppressor T cell lines were maintained in culture by alternate stimulation with MBP and antigen-presenting cells (APC). The Ts lines expressed preferentially the CD4 phenotype (5/6 Ts lines tested) and exhibited potent antigen-specific suppressor activity on the proliferation of MBP-specific Th clones and not on the T cell lines with other antigen specificity. For some Ts lines, a Ts-to-Th ratio of 1 was sufficient to inhibit the proliferation of MBP-specific T cells by 90%. The suppressor T cells obtained were weakly responsive to MBP and required the presence of the autologous PBMC for proliferation. Furthermore, proliferation of these suppressor T cell lines was restricted by HLA-DR molecules (for CD4+ Ts lines) and HLA class I (for a CD8+ Ts line). The suppressor T cell lines generated and the techniques described in this study may be helpful in our understanding of the events involved in the immune regulation in MS and other autoimmune diseases.     

Persistent activity of suppressor cells in T cell-mediated immune response.

Tolerance to dinitrochlorobenzene contact sensitivity induced i.v. injection of dinitrobenzenesulfonic acid in guinea pigs is a long-lasting phenomenon (up to 1 year). The tolerogen, however, was traceable in the circulation only up to 3 months after its application. In spite of that, tolerance was adoptively transferred by parabiosis 6 months after being induced. Moreover, active suppressor cells eliminated by cyclophosphamide treatment are able to regenerate in those adoptively tolerized animals. These results indicate that the tolerogenic injection stimulates precursors of suppressor cells to generate active suppressor cells and memory cells of suppression. The further formation of active suppressor cells from memory cells seems to be tolerogen independent, but the existence of specific stimulator cells for suppression may be considered. These cells may bind undetectable small amounts of tolerogen. The recovery of suppression might, however, be also due to recovery of suppressor cells which were temporarily inactivated but not destroyed by cyclophosphamide treatment.     

T helper 2 and T follicular helper cells: Regulation and function of interleukin-4

Type 2 immunity is characterized by expression of the cytokines interleukin (IL)-4, IL-5, IL-9 and IL-13, which can function in mediating protective immunity in the host or possess a pathogenic role. T helper (Th) 2 cells have emerged to play a beneficial role in mediating anti-parasitic immunity and are also known to be key players in mediating allergic diseases. In addition to the Th2 cells, recent studies have identified T follicular helper (Tfh) cells as an alternative source of IL-4 to regulate type 2 humoral immune responses, indicating that Th2 and Tfh cells exhibit overlapping phenotypical and functional characteristics. Th2 and Tfh cells appear to utilize distinct mechanisms for regulation of IL-4 expression; however unlike Th2 cells, the regulation and function of Tfh-derived IL-4 is not yet fully understood. Understanding of the molecular mechanisms for IL-4 expression and function in both cell subsets will be beneficial for the development of future therapeutic interventions.     

Activity of concanavalin A-induced suppressor cells in human MLR: differential effects on primary MLR, secondary MLR, and memory cell precursors

The relative sensitivity of the MLR responses of freshly isolated human lymphocytes to Con A-induced suppressor cells (SC) was compared to that of lymphocytes that had been primed previously in vitro. Fresh and primed cells were suppressed 72 and 17%, respectively, when cultured under similar conditions (p less than 0.0005). Titration of SC indicated that equivalent suppression in the two populations would require greater than a 50-fold excess of SC in the primed cells. Neither preincubation of the primed cells for 24 or 72 hr with the SC before restimulation, addition of fresh autologous cells to the primed cells, nor preincubation of the SC with fresh cells before addition to the primed cells increased suppression of primed cells. SC added at the beginning of a primary MLC were, however, very suppressive for both the primary response (67% suppression) and the subsequent secondary response (81% suppression). These data indicate that although the human primary MLR and the precursor of the memory cell are both sensitive to the suppressors induced by Con A, the memory cell itself possess an intrinsic resistance to such suppressors that is not related to simple kinetic phenomena nor to the loss in vitro of an intermediate regulatory cell. Cell depletion experiments suggest that resistance to nonspecific suppression may occur at the level of the helper cell.     

Regulation of IgG responses by helper and suppressor T cells activated by pneumococcal capsular polysaccharides

Type 2 antigens are usually unable to prime the helper T cells (TH) required for secondary IgG antibody responses. However, previous results from this laboratory indicated that low doses of the type 2 antigen polyvinylpyrrolidone (PVP) could activate T cells which provided help to PVP-primed B cells for the production of PVP-specific IgG antibody. Therefore, it was of interest to determine if other type 2 antigens may also be able to activate TH. Low doses of S19 or S3 (subimmunogenic for a primary IgM response) activated TH capable of providing help to S19- or S3-CRBC-primed B cells for a secondary IgG response. Higher doses of these antigens (optimally immunogenic for a primary IgM response) activated suppressor T cells (TS). Removal of these TS prior to transfer of T cells to recipient mice resulted in expression of TH function. Therefore, the preferential activation of TH versus TS was dependent on the dose of antigen used for priming. TH activated by low doses of S19 expressed Thy 1 and L3T4 and were antigen specific. In contrast to the ability of low doses of PVP to prime B cells for secondary IgG responses, low doses of S3 and S19 did not prime capsular polysaccharide-specific IgG memory B cells. High doses of S3 were able to prime B cells if TS precursors were first removed by treatment of mice with cyclophosphamide (Cy), whereas high doses of S19 did not prime B cells for secondary IgG responses in either Cy-treated or control mice. These results are discussed in relation to the general observations that type 2 antigens may not activate antigen-specific TH.     

Feedback mechanisms between T helper cells and macrophages in the determination of the immune response

The interactions between macrophages and T helper (Th) cells are a complex interplay of positive and negative signals. Some of the mathematical models of interactions between T helpers have indeed taken the influence of macrophages into account. In this work the macrophage is not considered as an extrinsic agent, that is duly directed by the T cells to be cytotoxic, nor is there consideration of T helper cell populations that are dominantly regulated by extrinsic properties of antigens per se, or by certain classes of presenting cells that preferentially select certain classes of lymphocytes or bias their commitment. Rather, a simplified model of feedback loops between Th cells and macrophages is formulated and analyzed. It is suggested how the mutual influence between Th and macrophages can determine the cytokine secretion pattern of these populations. The model provides a feedback scenario to account for experimental findings concerning reversal in the dominance of a specific cytokine profile in the course of some infections. A possible scenario accounting for the difference between the stability of Th1 and Th2 cytokine pattern is put forward. The model suggests explanations for the variability in the outcome of the immune response according to different body compartments. A rationale is presented that accounts for paradoxical findings indicating that Th1 cytokines are sometimes responsible for the downregulation of a Th1 dominated response.     

Regulation of hapten-specific T-cell response. II. Functional analysis of helper T cells and cytotoxic T cells in animals suppressed by azobenzenearsonate (ABA)-specific suppressor T cells

The administration of azobenzenearsonate-modified syngeneic spleen cells (ABA-SC) intravenously induces a population of first order hapten-specific inducer suppressor T cells (Ts1), which downregulate various aspects of T-cell-mediated immune responses via a well defined suppressor-T-cell pathway. In this study, we investigated the effects of these suppressor cells on the generation of ABA-specific cytolytic T lymphocytes (CTL) and helper T cells (Th) in vivo. We found evidence for functional impairment of ABA-activated Th and ABA-specific CTL precursors (CTLp) in the suppressed animals by a number of different in vitro criteria. Functional analysis of ABA-specific CTLp and ABA-activated Th in suppressed animals revealed that ABA-specific Ts inhibit the generation of CTL by impairing the antigen-specific activation of Th, which may in turn, prevent the clonal expansion of antigen-specific CTLp. The significance of these findings in relationship to our understanding of the cellular interactions necessary for the generation of CTL and the mode of action and mechanisms of suppressor T cells is discussed.     

Regulation of immune responses by T suppressor cells and by serum in chronic paracoccidioidomycosis

Regulation of cellular responses was studied during the course of chronic murine disseminated paracoccidioidomycosis. Regulation of peripheral blood lymphocyte (PBL) proliferative responses to concanavalin A (Con A) was studied in vitro by mixing PBL from infected and noninfected mice. PBL from mice infected for 18 weeks had depressed responses to Con A and they depressed the Con A responses of PBL from noninfected mice by 95% when they were mixed in a 1:1 ratio. After treatment of PBL from infected mice with anti-Lyt-2.2 antibody plus complement, the responses to Con A were increased to normal values. The percentage of T-cell subpopulations in PBL from infected mice did not differ significantly from those of normal mice. Immunoregulation of delayed-type hypersensitivity (DTH) responses to antigen by serum from infected animals was studied in mice 1 week after intranasal (i.n.) infection, a time when DTH responses were maximal. DTH responses to antigen 7 days after i.n. infection (10(7) CFU Paracoccidioides brasiliensis) were significantly reduced when 0.5 ml of immune mouse serum (ELISA antibody titer to P. brasiliensis antigens 1:10,240) was given i.v. 1 day before infection (P less than 0.01) or 1 day before skin testing (P less than 0.001). Normal mouse serum did not have this effect. The results indicate that progression of chronic disseminated paracoccidioidomycosis was associated with the development of T-cell suppressor activity for Con A responses of PBL, and that DTH responses to antigen were depressed by the administration of serum with specific high titer antibodies.     

Regulation of the cytotoxic T lymphocyte response against Qa-1 alloantigens

Spleen cells from B6.Tlaa (Qa-1a) mice primed against C57BL/6 (Qa-1b) splenocytes in vivo generate Qa-1-specific CTL when rechallenged with Qa-1b Ag in vitro. The addition of unirradiated Qa-1b splenocytes to these cultures inhibits the generation of Qa-1-specific CTL. By using highly purified cell populations, we demonstrate that the only cell population in resting spleen capable of causing this inhibition is NK1.1+. Although resting CD8 cells lack inhibitory activity, purified CD8 cells precultured with Con A and IL-2 inhibit anti-Qa-1 CTL. This inhibition is specific for the Qa-1b Ag expressed on the inhibitor cells, is not due to cold target competition, and is thus similar to that ascribed to veto cells. Although NK cells from resting spleen inhibit the generation of Qa-1-specific CTL, NK cells precultured in the presence of Con A and IL-2 show an approximate 30-fold increase in veto activity. Thus, NK cells represent the most likely cell population for down-regulating anti-self class I-reactive CTL.     

Regulatory T cells dynamically control the primary immune response to foreign antigen

The population dynamics that enable a small number of regulatory T (T(R)) cells to control the immune responses to foreign Ags by the much larger conventional T cell subset were investigated. During the primary immune response, the expansion and contraction of conventional and T(R) cells occurred in synchrony. Importantly, the relative accumulation of T(R) cells at peak response significantly exceeded that of conventional T cells, reflecting extensive cell division within the T(R) cell pool. Transfer of a polyclonal T(R) cell population before immunization antagonized both polyclonal and TCR transgenic responses, whereas blocking T(R) cell function enhanced those responses. These results define an inverse quantitative relationship between T(R) and conventional T cells that controls the magnitude of the primary immune response. The high frequency of dividing T(R) cells suggests degenerate TCR specificity enabling activation by a broad spectrum of Ags.     

Genetics of insulin-specific helper and suppressor T cells in nonresponder mice

The role of insulin-specific helper and suppressor T cells in the H-2-linked genetic control of antibody responses to heterologous insulins was examined in vitro. These data demonstrate that pork insulin stimulates both primed helper T cells and dominant suppressor T cells in all nonresponder strains tested. Thus, the nonresponder phenotype is attributed to the activation of specific suppressor T cells rather than to an absence of helper T cell activity. Examination of the antigenic cross-reactivity patterns of pork insulin-primed helper and suppressor T cells in various strains demonstrates that fine specificity of the helper T cells differs from that of the suppressor T cells and that the patterns of antigenic cross-reactivity of these subpopulations are controlled by the H-2 gene complex. Furthermore, in a given strain of mice variants of insulin that stimulate helper T cells that cross-react with mouse insulin also stimulate dominant suppressor T cells that cross-react with mouse insulin. Such variants of insulin are perceived as nonimmunogenic. These observations raise the possibility that nonresponsiveness that is controlled by H-2 linked genes results from the activation of regulatory mechanisms involved in maintaining self-tolerance.     

Long-term effects of in vitro sensitization on immune reactivity of lymphocytes: generation of suppressor and helper T cells.

Mouse spleen cells were cultured for 5 days with or without HRBC. Cultured cells were 'parked' in irradiated syngeneic recipients for 3 weeks and then tested for their immunologic reactivity in vitro. We found that spleen cells from recipients of HRBC-sensitized cells (S) as well as spleen cells from recipients of control unsensitized cells (U) possessed radiosensitive suppressor and radioresistant helper activities. Suppressor activity was observed by the capacity of unirradiated S and U spleen cells to inhibit the in vitro generation of IgM and IgG PFC by spleen cells primed in vivo to HRBC or to LacKLH. Helper activity was shown by the capacity of the irradiated S and U cells to restore IgM and IgG PFC responses of in vivo primed, T-depleted spleen cells to HRBC, LacHRBC, and LacCRBC. Both suppressor and helper activities were mediated by T cells. The possibilities that immunologically specific or nonspecific mechanisms account for these phenomena are discussed.