Regulation of the immune response to tumor antigen. IV. Tumor antigen-specific suppressor factor(s) bear I-J determinants and induce suppressor T cells in vivo.

The nature and function of suppressor factor(s) elaborated by suppressor T cells in response to certain chemically induced tumors have been further defined. Thus, suppressor factor(s) specific for the S1509a methylchol-anthrene-induced fibrosarcoma have been shown to bear determinants encoded by the I-J subregion of the murine MHC since suppressive activity is removed by passage of the factor through an immunoadsorbent composed of anti-I-Jk coupled to Sepharose. No loss of activity was observed after passage of factor through control columns composed of normal mouse globulin. Furthermore, activity could be recovered from the relevant immunoadsorbent by elution with high salt. The administration of crude suppressor factor(s) to normal animals for 4 days resulted in the development of a population of suppressor cells that act in a manner analogous to the suppressor cell population used for production of factor. These factor-induced suppressor cells are T cells and exhibit an antigen specificity similar to that displayed by the tumor-induced suppressor cells. Thus, tumor-specific suppressor factor(s) bear I-J determinants and are capable of inducing the appearance of suppressor T cells in the nontumor-bearing host, which may then act in a specific manner to limit host responsiveness to tumor antigen.     

Delayed hypersensitivity and immune protection against herpes simplex virus: suppressor T cells that regulate the induction of delayed hypersensitivity effector T cells also regulate the induction of protective T cells

We have been studying delayed hypersensitivity (DH) to herpes simplex virus (HSV) in order to examine the role of this response in host defense against acute and recurrent HSV infections. In previous reports the basic parameters of DH to HSV have been characterized by using a murine ear swelling model, and also the regulation of DH to HSV induced by i.v. injection of the virus. In this paper, we describe a murine protection system and our use of the ability to specifically regulate DH to HSV to examine the correlation between T cells that transfer DH (TDH) and cells that transfer protection from acute HSV infection. Both DH and protection can be transferred with lymph node cells from mice immunized subcutaneously 4 days previously. The effector cell appears to be a T cell, because serum from these donors confers no protection and treatment of immune cells with anti-Thy-1.2 plus complement reduced their ability to protect. Tolerance of DH to HSV was induced by i.v. injection 7 days before subcutaneous immunization. Tolerized mice were unable to generate protective cells. Furthermore, tolerized mice contained suppressor T cells that suppressed not only DH but also the development of protective cells. Regulation of protective cells was shown to be virus specific, because mice tolerized with vesicular stomatitis virus (VSV) were not impaired in their ability to generate T cells that protected from HSV infection. The correlation between the TDH cell and cells that transfer protection from acute HSV infection is discussed.     

Immunoregulation after thermal injury: sequential appearance of I-J+, Ly-1 T suppressor inducer cells and Ly-2 T suppressor effector cells following thermal trauma in mice

Immunoregulation as a consequence of thermal injury was investigated by using a murine model involving a 30% surface area full thickness burn. Both allogeneic mixed lymphocyte reaction (MLR) and in vitro anti-SRBC responses were depressed from days 3 to 25 post-burn. Suppressor T cells could be identified in both systems between days 5 and 15. On day 5 post-burn, an Ly-1+,2-, I-J+ T cell is responsible for the majority of the suppression observed. This cell behaves like a T suppressor inducer T cell in that it must interact with an Ly-2+ cyclophosphamide-sensitive cell to manifest suppression. On day 7 post-burn, only Ly-1-,2+ suppressor T cells are found which can directly suppress the activity of Ly-1+,2- helper T cells. Thus, these cells behave as T suppressor effector cells. We suggest that feedback suppression is in operation after thermal injury, with functional suppressor inducer cells appearing on day 5 post-burn, leading to the appearance of T suppressor effector cells by 7 days post-burn. Recovery from post-burn immunosuppression occurs by day 25 post-burn and is associated with the appearance of V. villosa-adherent T cells, whose activity antagonizes that of the day 7 post-burn suppressor effector. These cells may represent contrasuppressor T cells, which could play a role in the restoration of immunocompetence after burn injury.     

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.     

Anti-idiotypic B cells are required for the induction of suppressor T cells

A nylon wool-adherent, B cell-enriched population is required during the in vitro induction of third order effector suppressor T cells (Ts3). This B cell population expresses IgM and IgD and is devoid of conventional T cell markers such as Thy-1, L3T4, and Lyt-1. Treatment of the B cell population with anti-NP antibodies expressing the NPb idiotype and complement specifically eliminated the ability to generate Ts cell activity, suggesting that the critical B cells expressed anti-idiotypic receptors. To independently verify the role of anti-idiotypic B cells in the generation of Ts cells, B cells were panned on antibody-coated plates. The results demonstrated that only NPb idiotype-binding B cells could induce effector suppressor cells from naive T cell populations. The combined data demonstrate the role of Ig network interactions in the generation of Ts cells.     

Induction of helper and suppressor T cells by nonoverlapping determinants on the large protein antigen, beta-galactosidase

The fine specificity of the T cell repertoire directed against T helper (Th)-inducing and T suppressor (Ts)-inducing determinants was examined with cyanogen bromide and tryptic peptides of Escherichia coli beta-galactosidase (GZ), a large tetrameric protein (monomer molecular weight = 116 kDa). Immunization with cyanogen bromide fragment 2 [CB-2, amino acids (a.a.) 3-92] induced both specific Th and Ts cells. Study of the induction of these functionally opposite T cell subpopulations with tryptic peptides of CB-2 indicated that Th and Ts were activated by separate, nonoverlapping determinants. Th-inducing activity resided in a nonapeptide, T6 (a.a. 44-52), whereas T4 (a.a. 27-37) induced Ts cells. The presence of distinct helper and suppressor determinants suggests that the specificity repertoire in these T cell subpopulations may differ, perhaps owing to the expression of antigen-recognizing receptors that are coded by unique gene families. Alternatively, antigen presentation structures may be physicochemically quite different, and bind to distinct parts of the peptide antigen.     

The role of antigen in the activation of regulatory T cells by immune B cells

The transfer of B cells from mice immunized with Type III pneumococcal polysaccharide (SSS-III) results in the activation of suppressor and amplifier T cells that control the magnitude of the antibody response in recipient mice, immunized subsequently with SSS-III. Prior treatment of transferred B cells with an excess of enzyme (polysaccharide depolymerase) capable of hydrolyzing SSS-III, does not alter the capacity of these cells to activate regulatory T cells. These findings indicate that the activation of regulatory T cells by immune B cells is not mediated by residual antigen on the surface of transferred cells.     

Regulation of granulomatous inflammation in murine schistosomiasis. III. Recruitment of antigen-specific I-J+ T suppressor cells of the granulomatous response by I-J+ soluble suppressor factor

Down-modulation of the schistosome egg-induced granulomatous response involves various interacting subsets of T suppressor (TS) lymphocytes. In the present study the inductive phase of the process of modulation was analyzed. A soluble, I-J+ granuloma TS cell recruiting factor (Gr-TSRF) derived from spleen cells of chronically infected mice is described. This factor eluted from immunoabsorbent columns coupled with anti-I-Jk alloantisera induced the recruitment and expansion of antigen-specific I-J+ TS cells from a TS precursor cell population in the spleens of acutely infected mice. The recruited TS cells suppressed the granulomatous response of normal recipients in a 2-day adoptive transfer model. The antigenic specificity of the recruited TS cells was demonstrated by their inability to suppress KLH-induced artificial granulomatous response. This mechanism of recruitment described in the current study and illustrated by adoptive transfer experiments is likely to be active in vivo in initiating the process of spontaneous modulation. The I-J+ Gr-TSRF and the I-J+ TS cell described in this paper, together with the previously described H-2 restricted I-C+ factor and the subsets of TS cells (THs, TSe, TSpr), indicate the existence of an intricate, regulatory pathway(s) that operates during the modulation of the granulomatous response.     

The role of macrophages in anti-idiotypic antibody and T suppressor factor induction of timothy grass pollen antigen B-specific T suppressor cells

Cultures of normal spleen cells with anti-idiotypic antibody (anti-Id) or antigen B (AgB)-specific T suppressor factor (Tsf1) in mini-Marbrook chambers for 4 days at 37 degrees C lead to the in vitro induction of AgB-specific T suppressor (TS) cells. These TS cells significantly suppress a secondary AgB-specific IgE response, but they do not affect a secondary AgB-specific IgG response. Depletion of both B cells and macrophages from normal spleen cells by panning on anti-Ig-coated petri dishes provides an enriched T cell population. These enriched T cells when cultured with anti-Id or Tsf1 in mini-Marbrook chambers do not produce AgB-specific TS cells, and mice treated with cells harvested from the mini-Marbrook chambers have normal secondary AgB-specific IgG and IgE responses. The addition of as few as 1000 bone marrow-derived macrophages (BMDM) to cultures of the enriched T cells with anti-Id, or Tsf1 restores the ability of these cultures to produce significant levels of AgB-specific TS cells. Further studies reveal that the macrophage population must be histocompatible and express a cell surface I-J antigen. Attempts to pulse BMDM with anti-Id or Tsf1 at 4 degrees C and to culture in mini-Marbrook chambers 10(3) pulsed BMDM with enriched T cells were unsuccessful in producing AgB-specific TS cells. However, pulsing BMDM with anti-Id or Tsf1 at 37 degrees C, and adding 10(3) of these pulsed BMDM to enriched T cells in culture led to the formation of significant levels of AgB-specific TS cells.     

Modulation of immune responses by suppressor T cells.

The activity of suppressor T cells has been demonstrated in almost every phase of the immune response. These regulatory cells modulate both humoral and cell-mediated immunity utilizing antigen-specific and nonspecific mechanisms. For comparative purposes two murine models are described, the nonspecific suppressor T cell stimulated by the mitogen concanavalin A and the antigen-specific suppressor T cell stimulated by injection of the synthetic terpolymer acid 60-L-alanine30-L-tyrosine10 (GAT) in nonresponder mice. These two T cells are similar to expression of Ly alloantigens, ability to inhibit antibody responses, and the mediation of suppression, at least in part, by soluble products. However, differences in radio-resistance and antigenic specificity of the suppressor T cells, as well as differences in molecular characteristics of the soluble factors and their targets suggest that these T cells regulate the immune response by different mechanisms. The relationship of these two suppressor T cells to other nonspecific and antigen-specific suppressor T cells is discussed.     

Nature of hapten-modified determinants involved in induction of T cell tolerance and suppressor T cells to NDFB contact sensitivity

Unresponsiveness to DNFB contact sensitivity induced by DNP-modified lymphoid cells (DNP-LC) is mediated by two separable pathways: a rapidly induced, long lasting inhibition of reactive T cell clones (donor tolerance), and a transient period of suppressor T cell (Ts) activity. The present report has examined the nature of the hapten-modified determinants responsible for the induction of these pathways by utilizing soluble DNP-LC cell lysate preparations as tolerogens. The results indicate that both DNP-modified MHC and non-MHC encoded determinants can mediate donor tolerance 7 days after tolerization. On the other hand, the induction of Ts requires DNP-modified MHC determinants, since DNP-LC lysates passed over lentil lectin or specific anti-H-2 immunoabsorbent columns lost their ability to induce Ts. Additional experiments showed that the injection of DNP-LC lysate compatible with the recipient strain at the H-2K and H-2D region of the MHC was sufficient for the induction of Ts. We propose that Ts induction involves the direct presentation of DNP-H-2 determinants to Ts precursors, whereas the induction of donor tolerance may involve host processing and presentation of DNP-modified membrane determinants in conjunction with host MHC structures.     

Antigen-specific T cell-mediated suppression. II. In vitro induction by I-J-coded L-glutamic acid50-L-tyrosine50 (GT)-specific T cell suppressor factor (GT-T8F) of suppressor T cells (T82) bearing distinct I-J determinants.

The induction of new suppressor T cells (Ts2) by suppressive extracts (TsF) from L-glutamic acid50L-tyrosine50 (GT) nonresponder mice was examined. Incubation of normal spleen cells with allogeneic GT-TsF for 2 days in vitro led to the generation of Ts2 cells able to suppress subsequent responses to the immunogen GT-methylated bovine serum albumin (GT-MBSA) in vivo. This induction occurred efficiently when TsF donor and target cells differed at all of H-2, including the I-J subregion. B10.BR (H-2k) GT-TsF, adsorbed on, then acid eluted from GT-Sepharose and anti-I-Jk [B10.A (3R) anti-B10.A (5R)]-Sepharose in a sequential fashion could induce BALB/c (H-2d) spleen cells to become Ts2 only if nanogram quantities of GT were added to the purified GT-TsF. This indicates a requirement for a molecule or molecular complex possessing both I-J determinants and antigen (GT)-binding specificity, together with GT itself, for Ts2 induction. The induced Ts2 are I-J+, since their function can be eliminated by treatment with anti-I-Jk plus C. These I-J determinants are coded for by the precursor of the Ts2 and do not represent passively adsorbed, I-J coded TsF, since anti-Ijk antiserum [(3R X DBA/2)F1 anti-5R] which cannot recognize the BALB/c (I-Jd) TsF used for induction still eliminates the activity of induced A/J (I-Jk) Ts2. These data provide further evidence for and information about the minimum of two T cells involved in antigen-specific suppressor T cell systems.     

Hapten-specific responses to the phenyltrimethylamino hapten. V. A single chain antigen-binding I-J+ first-order T suppressor factor requires antigen to induce anti-idiotypic second-order suppressor T cells

We have previously shown that a single i.p. injection of the monovalent antigen, L-tyrosine-p-azophenyltrimethylammonium in complete Freund's adjuvant induces a Ly-1+2-, idiotype-bearing, and antigen-binding first-order T suppressor (Ts1) population. We showed that soluble factors extracted from these cells could suppress delayed-type hypersensitivity responses if administered at the induction phase of the response. In this paper we additionally characterize the suppressor factor, TsF1, with respect to its biologic, serologic, and chemical properties. The studies show that the TsF1 is neither allotype nor H-2 restricted and can induce anti-idiotypic T suppressor cells (Ts2), but it requires the presence of antigen to do so. The factor binds antigen, bears I-J encoded determinants, is resistant to reduction and alkylation, and elutes as a single chain factor after adsorption onto monoclonal anti-I-J antibody-coupled Sepharose beads in the presence of dithiothreitol (DTT). This is in marked contrast to TsF2 (derived from Id-specific Ts2-containing spleen cells), which lost its suppressive activity after reduction and alkylation, and behaves as a two chain factor after adsorption and elution from anti-I-J-coupled beads in the presence of DTT. The TsF1 is discussed with respect to the properties of it and those of TsF1 from other similar idiotype-dominated antigen systems.     

Involvement of protein kinase C in competence induction of macrophages to generate T suppressor cells.

We have previously described an Ia-expressing macrophage hybridoma clone, termed clone 59, which attains the ability to induce Ts cells after activation with murine rIFN-gamma. In this report, we show that a protein kinase C (PKC) activator, PMA (10 ng/ml) can replace IFN-gamma in inducing this form of macrophage competence. IFN-gamma-induced cellular competence was abrogated specifically by a PKC inhibitor but not by inhibitors that have specificity for cyclic nucleotide-dependent protein kinases. Furthermore, PGE2 known to induce protein kinase A in murine macrophages also failed to induce competence. In contrast, the ability to induce Th responses was neither dependent on IFN-gamma nor inhibited by prior treatment with protein kinase inhibitors. Furthermore, PKC depletion of macrophages by treatment with high doses (100 ng/ml) of PMA abrogated their ability to induce Ts cells. In addition, PKC-depleted macrophages failed to regain the ability to stimulate Ts cells after further treatment with IFN-gamma. The ability of IFN-gamma to modulate macrophage-mediated induction of Ts cells does not clearly correlate with an increased Ia expression as inducible expression of Ia was not consistently abrogated by PKC inhibitor treatment. In addition, PKC inhibitors failed to prevent the production of the cytokines IL-1 and IL-6. However, incubation of IFN-gamma or PMA-treated macrophages with antibodies recognizing the putative IJ ligand blocked the ability to induce Ts cells, suggesting the expression of these determinants on accessory cells is responsible for Ts induction.     

The role of adherent accessory cells in the generation of effector suppressor T cells

The role of accessory cell populations in the generation of effector suppressor (Ts3) cells was studied. By using an in vitro culture system, it was previously determined that the induction of NP-specific effector suppressor activity requires T cells, antigen, and an anti-idiotypic B cell population. We now demonstrate that the generation of Ts3 cells in this system also requires accessory cells. The accessory population appears to play a role in the processing and presentation of antigen. These antigen-presenting accessory cells are required early in the induction phase of Ts3 generation. These accessory cells can present NP coupled to immunogenic or non-immunogenic polypeptide carriers, including polymers of L-amino acids. However, NP coupled to polymers of poorly metabolized D-amino acids fail to induce suppressor T cell generation. Furthermore, the data demonstrate that an H-2 homology must exist between the Ts3 precursors and the antigen-presenting cell population if suppressor activity is to be generated. We also characterize the differential genetic restrictions that govern the induction of Ts3 cells that control suppression of either T cell or B cell responses. The data suggest that although I-J region encoded gene products control the induction and effector phases of suppressor cell activity as measured on T cell responses, the suppression of B cell responses appear to be controlled by I-A gene products. Possible cellular mechanisms that might explain these findings are discussed.     

Influence of 2'-deoxyguanosine upon the development of DTH effector T cells and suppressor T cells in vivo

Subcutaneous (s.c.) immunization of mice with allogeneic spleen cells can induce delayed-type hypersensitivity (DTH) to both major and minor histocompatibility antigens. Intravenous immunization with allogeneic spleen cells, however, induces a poor state of DTH. Furthermore, i.v. immunization with allogeneic spleen cells, especially if they have been irradiated, induces suppressor T lymphocytes. These suppressor T cells are capable of suppressing the host-vs-graft (HvG) DTH reactivity that normally arises after s.c. immunization. Moreover, they can suppress the development of anti-host DTH effector T cells during graft-vs-host (GvH) reactions. These models for HvG and GvH DTH reactivity were used to study the influence of 2'-deoxyguanosine (dGuo) and guanosine (Guo) on the generation of DTH-reactive T cells and suppressor T cells in vivo. It was found that daily i.p. administration of 0.01 mg dGuo to mice immunized i.v. partially prevented the generation of suppressor T cell activity, whereas daily administration of 0.1 or 1 mg dGuo resulted in a complete abolition. Administration of dGuo has no effect on the anti-host DTH reactivity by spleen cells from nonsuppressed donors except for when a daily dose of 10 mg is administered. This dose proved to be toxic for precursors of DTH effector T cells. Daily i.p. injection of Guo had no effect on the generation of suppressor T cells nor on the generation of DTH effector T cells. The effect of dGuo was found to be due to a direct effect on suppressor T cells and not to the induction of contrasuppressor cells. These data suggest a differential sensitivity of DTH-reactive T cells and suppressor T cells for dGuo. Because suppressor T cells and DTH-reactive T cells require proliferation for expressing maximal functional activity in the systems used, both cell types probably have different enzyme activities involved in the purine metabolism and similar deoxycytidine kinase activities, but have different nucleotidase (5'NT) activities, those in suppressor T cells being the lowest. If so, suppressor T cells will accumulate deoxyguanosine triphosphate, which causes an inhibition of the ribonucleotide reductase activity and thus of the DNA synthesis by these cells.     

Autophagy induction and autophagic cell death in effector T cells

The population size of the T cells is tightly regulated. The T cell number drastically increases in response to their specific antigens. Upon antigen clearance, the T cell number decreases over time. Apoptosis, also called type I programmed cell death, plays an important role in eliminating T cells. The role of autophagic cell death, also called type II programmed cell death, is unclear in T cells. Our recent work demonstrated that autophagy is induced in both Th1 and Th2 cells. Both TCR signaling and IL-2 increase autophagy in T cells, and JNK MAP kinases are required for the induction of autophagy in T cells, whereas caspases and mTOR inhibit autophagy in T cells. Autophagy is required for mediating growth factor withdrawal-dependent cell death in T cells. Here, we hypothesize that autophagic cell death plays an important role in T cell homeostasis.     

Contribution of regulatory T cells and effector T cell deletion in tolerance induction by costimulation blockade

Blocking of costimulatory signals for T cell activation leads to tolerance in several transplantation models, but the underlying mechanisms are incompletely understood. We analyzed the involvement of regulatory T cells (Treg) and deletion of alloreactive cells in the induction and maintenance of tolerance after costimulation blockade in a mouse model of graft-vs-host reaction. Injection of splenocytes from the C57BL/6 parent strain into a sublethally irradiated F(1) offspring (C57BL/6 x C3H) induced a GVHR characterized by severe pancytopenia. Treatment with anti-CD40L mAb and CTLA4-Ig every 3 days during 3 wk after splenocyte injection prevented disease development and induced a long-lasting state of stable mixed chimerism (>120 days). In parallel, host-specific tolerance was achieved as demonstrated by lack of host-directed alloreactivity of donor-type T cells in vitro and in vivo. Chimerism and tolerance were also obtained after CD25(+) cell-depleted splenocyte transfer, showing that CD25(+) natural Treg are not essential for tolerance induction. We further show that costimulation blockade results in enhanced Treg cell activity at early time points (days 6-30) after splenocyte transfer. This was demonstrated by the presence of a high percentage of Foxp3(+) cells among donor CD4(+) cells in the spleen of treated animals, and our finding that isolated donor-type T cells at an early time point (day 30) after splenocyte transfer displayed suppressive capacity in vitro. At later time points (>30 days after splenocyte transfer), clonal deletion of host-reactive T cells was found to be a major mechanism responsible for tolerance.     

Induction of suppressor T cells and tolerant B cells in vitro by DNP-IgG

Intravenous injection at proper time of irradiated reticulum cell sarcoma cells into SJL mice immunized with dinitrophenylated (DNP) keyhole limpet hemocyanin inhibits the production of anti-DNP IgG₁ and IgG₂ antibodies.