The role of suppressor cells in the induction of murine photoallergic contact dermatitis and in its suppression by prior UVB irradiation

Induction in mice of marked photoallergic contact dermatitis (PCD) to 3,3',4',5-tetrachlorosalicylanilide (TCSA) with UVA (320 to 400 nm) radiation requires pretreatment with cyclophosphamide (CY). Attempts to induce photoallergic contact dermatitis without CY result in only a small degree of sensitivity, accompanied by significant net splenic suppressor cell activity. These suppressor cells are antigen specific, inhibit the induction but not the elicitation of photoallergic contact dermatitis to TCSA, and are T lymphocytes. Exposure of mice to UVB (280 to 320 nm) radiation at a site distant from that of sensitization, before CY administration and sensitization, inhibits the development of photoallergic contact dermatitis. This is analogous to the suppression of allergic contact dermatitis (ACD) observed in mice after exposure to UVB radiation; such suppression is accompanied by the formation of antigen-specific splenic suppressor cells. However, in contrast to the findings with allergic contact dermatitis, splenic suppressor cells are not detected in mice that are treated with UVB radiation before CY administration and sensitization to TCSA. This is presumably because CY prevents their formation. This provides evidence that UVB-irradiated mice have a second form of anergy that is not mediated by suppressor cells.     

Ultraviolet-induced suppressor T cells and factor(s) in murine contact photosensitivity. III. Mode of action of T-cell-suppressor factor(s) and interaction with cytokines

The mode of action of T-cell-suppressor factor (TsF) induced by ultraviolet B (UVB) preirradiation in terms of interaction with several cytokines was studied. Suppression of murine contact photosensitivity (CPS) to 3,3',4',5-tetrachlorosalicylanilide (TCSA) by preirradiation of the sensitizing site to low doses of UVB was caused by antigen-specific suppressor T cells (Ts) and was not associated with the generation of efferent limb-acting suppressor cells. TsF released by Ts inhibited the proliferation of immune lymph node (LN) cells in vitro and reduced interleukin (IL)-2 production of these cells in an antigen-specific fashion without affecting the IL-2 receptor (IL-2R) expression. Both rIL-2 and rGM-CSF have the ability to restore CPS responses in the UVB-preirradiated mice when administered after but not before photosensitization. However, rIL-2 but not rGM-CSF counteracted the in vivo inhibitory effect of TsF. rGM-CSF did not affect the density of I-A+ epidermal Langerhans cells (LCs). It was suggested that TsF inhibited IL-2-mediated immune T-cell proliferation, while rGM-CSF reconstituted the CPS by enhancing the function of photodamaged LCs. These results indicate multiple steps of the UVB-induced immunosuppression circuit, each of which seems to be controlled by different immunomodulators.     

Regulatory mechanism of delayed-type hypersensitivity in mice. IV. Effect of suppressor T cells on the development of memory T cells involved in accelerated generation of DTH-effector cells in vitro

The effect of suppressor T cells (Ts) on the induction and the subsequent development of memory T cells for delayed-type hypersensitivity (DTH) was examined. The memory cells were induced in the spleens of mice primed previously with a low dose of reduced and alkylated ovalbumin (Ra-OA), and they generated DTH-effector T cells (DTH-Te) in a significantly accelerated fashion when cultured with OA in vitro. Ts were obtained from the spleens of mice which received OA-coupled spleen cells i.v. 4 days previously, and they inhibited antigen-specifically the induction of DTH responses in the recipient mice sensitized with alum-absorbed OA only when transferred with 5 weeks before sensitization. The spleen cells from mice given Ts together with the priming antigen 7 weeks before culture failed to generate DTH-Te in an accelerated manner on restimulation with OA in vitro. The memory cells from primed mice also did not cause accelerated generation of DTH-Te, when cultured with Ts in the presence of OA in vitro. These results indicate that both the induction of the memory cells by priming with antigen in vivo and the subsequent development of memory cells to DTH-Te by restimulation in vitro are inhibited independently by Ts. This corresponded well with the effect of Ts on the development of DTH-memory in vivo.     

Ultraviolet-induced suppressor T cells and factor(s) in murine contact photosensitivity. I. Biological and immunochemical characterization of factor(s) extracted from suppressor T cells

Murine contact photosensitivity (CPS) to 3,3',4',5-tetrachlorosalicylanilide (TCSA) is a highly specific, T-cell-mediated delayed-type hypersensitivity (DTH). Preexposure of the photosensitizing site to low doses of ultraviolet B(UVB) rendered mice unresponsive to challenge reaction. This unresponsiveness was associated with the generation of antigen-specific, afferent limb-acting, Lyt-1+2-,L3T4+ suppressor T cells (Ts-cps) in the spleen, thymus, and lymph node. Cell-free extract(s) obtained by freezing and thawing of these cells contained T-cell-suppressor factor (TsF) that inhibited the development of the induction phase of the CPS response to TCSA in vivo in an antigen-specific fashion. The treatments of TsF both with immunoadsorbent columns and with reduction and alkylation showed that the factor bore photoantigen-binding site(s), was reactive with monoclonal anti-I-Jd, anti-I-E alpha but not anti-I-Ad, and behaved as a single-chain factor containing both photoantigen binding and I-J molecules. By gel chromatography the majority of the suppressive activity was eluted in the fractions corresponding to molecular weights of 60-80 and 100-200 kDa. Our present study demonstrated clearly that UVB-induced unresponsiveness in the DTH reaction was mediated by a soluble suppressive factor derived from T cells.     

Characterization of a T suppressor cell line that downgrades experimental allergic encephalomyelitis in mice.

A T-suppressor (Ts) cell line of CD8 phenotype was isolated from spleens of SJL/J mice that had recovered from experimental allergic encephalomyelitis (EAE) induced by injection of MBP-activated T cells. The Ts cell line inhibited the proliferation of MBP-sensitized T cells in vitro. Addition of recombinant IL-2 enhanced the Ts-mediated suppression. Adoptively transferred Ts line was able to downgrade EAE in mice subsequently challenged with MBP-activated T cells. The mechanism of suppression appeared to involve neither direct cytolysis of the effector T cells nor the production of a soluble suppressor factor. The findings suggest an in vivo role for suppressor T cells in the regulation of EAE.     

The role of suppressor factors in the regulation of immune responses by ultraviolet radiation-induced suppressor T lymphocytes. I. Activity of suppressor cell culture supernatants

The purpose of this study was to determine whether soluble suppressor factors are involved in the regulation of immune responses by ultraviolet radiation-induced suppressor T lymphocytes (UV Ts). The UV Ts were induced by applying contact allergens to the ventral, unirradiated skin of mice that had been exposed 5 days earlier to UVB radiation. Supernatants from cultures that contained a mixture of UV Ts, normal responder lymphocytes, and hapten-modified stimulator cells were injected iv into normal recipients at the time of sensitization; they inhibited the induction of contact hypersensitivity (CHS) in vivo in an hapten-specific manner. The supernatants similarly suppressed the generation of specific cytotoxic T lymphocytes (CTL) in vitro. Moreover, supernatants from cultures that contained either UV Ts alone or UV Ts in combination with either the responder or the stimulator cells failed to suppress the CHS and CTL responses. These results suggest that hapten-specific inhibitory factors may participate in the regulation of immune responses by suppressor cells generated by epicutaneous sensitization of UV-irradiated mice.     

Suppressor T cell mechanisms in contact sensitivity. II. Afferent blockade by alloinduced suppressor T cells.

We investigated the mechanism(s) by which MHC-restricted suppressor T cells (Ts) induced by i.v. injection of allogeneic DNP-modified lymphoid cells (alloinduced Ts) suppress the DNFB contact sensitivity response. It was shown that alloinduced Ts acted only during the early phases (afferent limb) of sensitization. They were incapable of suppressing previously sensitized recipients or of inhibiting the expression of DNFB-immune LN cells when co-transferred into normal recipients. The target of alloinduced Ts seems to be cell proliferation, i.e., inhibition of antigen-induced cell proliferation (DNA synthesis) in Ts recipient mice. The failure of recipients of alloinduced Ts to generate DNFB-immune LN cells capable of transferring contact sensitivity to normal recipients also suggests that these Ts act by preventing the development of an expanded clone of mature immune T cells. The suppressive effects of alloinduced Ts also were inhibited by prior in vitro treatment with anti-TNP serum. The data are discussed in terms of current models of suppression, and are compared to mechanisms of suppression in other contact sensitivity models.     

Regulation of the murine IgE antibody response. I. Characterization of suppressor cells regulating both persistent and transient responses and their sensitivity to low doses of X-irradiation.

Anti-ovalbumin (OA) IgE antibody responses were measured in B6D2F1 mice as a function of time and antigen dose. One hundred to 200 microgram of OA in Al(OH)3 elicited transient responses, whereas 1 to 10 microgram of OA in Al(OH)3 elicited persistent anti-OA IgE responses of high titer. T cells isolated from the spleens of mice mounting either a persistent or a transient response strongly suppressed primary anti-DNP IgE responses in unirradiated recipient mice that were immunized with DNP-OA in Al(OH)3; it was, therefore, concluded that suppressor T cells (Ts cells) were activated during both the persistent and transient IgE responses. Nevertheless, in the present study it was not possible to completely rule out the contention that IgG antibodies may also have been suppressing the IgE response. With a modified adoptive transfer system, it was shown that these Ts cells were sensitive to low doses (250 R) of x-irradiation. The suppressive activity of long-term OA primed cells was also shown to be markedly enhanced when cultured for 24 hr with soluble OA; this finding was interpreted to indicate the presence of memory suppressor cells.     

Regulation of granulomatous inflammation in murine schistosomiasis. IV. Antigen-induced suppressor T cells down-regulate proliferation and IL-2 production

In murine schistosomiasis mansoni the cell-mediated immune response to the deposited eggs is mediated by CD4+ delayed-type hypersensitivity effector T (TDH) cells that produce vigorous granulomatous responses in the liver and intestines of acutely infected animals. The response is significantly down-modulated in chronically infected mice by Ag-specific Ts cells. The present study was undertaken to establish an in vitro model by which TDH-Ts cell interactions could be analyzed. To this end, Ts cells were induced in vitro by preculture of chronic or acute infection spleen cells with soluble egg Ag (SEA) for 48 h. The induced cells suppressed the SEA-specific proliferation of acute infection spleen cells by 80 to 95%. The induced suppressor cells were Ag specific in both induction and elicitation of function, and were not cytotoxic to the acute infection splenic target cells. Suppression by the induced cells was manifested within the first 24 h of the SEA-induced response as IL-2 produced by acute infection spleen cells was suppressed 62%. Phenotypic analysis by flow cytometry of the induced suppressor cells showed that CD8+ cells from acute infection spleens and CD4+ and CD8+ cells from chronic infection spleens were effector Ts cells. Taken together, CD4+ and CD8+ SEA-specific Ts cells can be induced in vitro to effectively suppress the SEA-specific lymphoproliferation and IL-2 production of acute infection spleen cells. Establishment of this in vitro model will allow us to further analyze the mechanisms of Ts cell-mediated suppression of TDH cells.     

Immunomodulation by low-dose methotrexate. I. Methotrexate selectively inhibits Lyt-2+ cells in murine acute graft-versus-host reactions

We have studied the effect of methotrexate in murine acute graft vs host (GvH) disease at concentrations analogous to those used in human rheumatoid arthritis. The GvH reaction was induced by i.v. injection of parental spleen cells into a normal F1 recipient. The acute suppression of T cell function in GvH mice was prevented by methotrexate given orally for 10 days at 1.0 or 0.5 mg/kg but not at 0.25 mg/kg. T cell mitogen response and IL-2 secretion that were inhibited in GvH mice were restored by methotrexate. Protection from immunosuppression in drug-treated GvH mice lasted at least 3 wk after drug dosing was stopped. The mechanism of the protective effect appears to be a preferential inhibition of donor and host Lyt-2+ Ts cell proliferation. In mixing experiments we found that methotrexate inhibited Ts function in GvH mice. By dual fluorescence labeling we showed that the engraftment of donor Lyt-2+ cells was prevented by drug treatment. This was not true of donor L3T4+ cells which were clearly present in the spleens of GvH mice after methotrexate treatment. These donor L3T4 cells were functional in that they induced the production of anti-DNA autoantibodies in the methotrexate-treated GvH mice.     

Identification of suppressor T cells in virgin non-responder spleen cells responsible for primary unresponsiveness to L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT)

T cell subsets that regulate antibody responses to L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT) in mice that are Ir gene non-responders have been further characterized. We previously defined several T cell subsets in GAT-primed non-responder mice. The Lyt-2+ suppressor-effector T cells suppress responses to GAT and GAT complexed to methylated BSA (GAT-MBSA). The Lyt-1+ cell population is complex and can be separated into I-J- Th cells, which support responses to GAT and GAT-MBSA. After priming, the Lyt-1+, I-J+ cell population contains suppressor-inducer cells that activate precursors of suppressor-effector cells to suppress responses to GAT and GAT-MBSA as well as Ts cells that directly inhibit responses to GAT but not GAT-MBSA. By contrast, the Lyt-1+ cells from virgin mice contain only cells that directly suppress responses to GAT but not GAT-MBSA. The major question addressed in the present studies was whether the Lyt-1+, I-J+ Ts cells in virgin and primed mice and the suppressor-inducer cells in GAT-primed mice were functionally and serologically distinct subsets. The studies used mAb and panning procedures to separate cell populations and inhibition of PFC cell responses to functionally define the activity of the cell populations. We used the following two mAb that were raised by immunizing rats with GAT-specific suppressor factors: 1248A4.10 (known to react with suppressor-inducer cells) and 1248A4.3, another reagent from the same fusion. Lyt-1+ cells from virgin spleens contained Ts cells that were A4.10-, A4.3+ and no suppressor-inducer T cells, whereas Lyt-1+ cells from GAT-primed spleens contained Ts cells that were A4.10-, A4.3+ as well as A4.10+, A4.3- suppressor-inducer cells. Thus, the Lyt1+, I-J+ cell subset can be divided into two functionally and serologically distinct subsets, direct Ts cells (1248A4.3+), which suppress responses to GAT but not GAT-MBSA, and GAT-primed suppressor-inducer T cells (1248A4.10+).     

The role of suppressor factors in the regulation of immune responses by ultraviolet radiation-induced suppressor T lymphocytes. II. Activity of suppressor cell culture sonicates

The purpose of this study was to determine whether multiple types of suppressor factors play a role in the regulation of immune responses by ultraviolet radiation-induced suppressor T lymphocytes (UV Ts). The UV Ts were induced by applying contact allergens to the ventral, unirradiated skin of mice exposed 5 days earlier to UVB radiation. Previous studies indicated that supernatants from cultures containing UV Ts, normal lymphocytes, and hapten-modified cells suppressed contact hypersensitivity (CHS) in vivo and cytotoxic T lymphocyte (CTL) generation in vitro in a hapten-specific manner. In this report, cell-free lysates from sonically disrupted UV Ts were examined for their ability to suppress these responses. When lysates were injected into normal animals at the time of sensitization, they inhibited CHS in a hapten-nonspecific manner. In addition, the lysates suppressed not only the induction but also the elicitation of CHS, and they suppressed the generation of CTL. Lysates prepared from spleen cells obtained from non-UV-irradiated mice or UV-irradiated, unsensitized mice failed to inhibit either response. Moreover, in contrast to the lysates, the hapten-specific UV Ts culture supernatants inhibited the induction but not the elicitation of CHS. These results suggest that both hapten-specific and nonspecific inhibitory factors may participate in the regulation of immune responses by UV Ts.     

Soluble factors in tolerance and contact sensitivity to DNFB in mice. X. IL-2 is the activation signal mediating release of synthesized suppressor factor

Two signals are required for the in vitro activation of Lyt2+ T suppressor cells (Ts) from mice tolerized with 2,4-dinitrobenzene sulfonate (DNBS) to produce soluble suppressor factors (SSF) which suppress the transfer of contact sensitivity to dinitrofluorobenzene (DNFB). Recognition of DNP/class I MHC (signal one) stimulates the Ts to synthesize SSF. Release of SSF requires a soluble mediator (signal two) produced by the interaction of L3T4+ T cells from tolerant mice with I-A on metabolically functional cells in the DNP-presenting cell population. The purpose of this study was to examine the nature of this second Ts activation signal. Coculture of tolerant spleen cells and glutaraldehyde-fixed (Glu-) DNP-labeled spleen cells (DNP-SC) resulted in the synthesis but not release of SSF. Addition of either IL-1 or IL-2 to these cultures induced SSF release. Treatment of such cultured cells with the anti-murine IL-2 receptor antibody PC 61.5.3 blocked the IL-2- and IL-1-stimulated release of SSF. Release of SSF was also blocked when tolerant cells were cultured with (unfixed) DNP-SC in the presence of a monoclonal anti-IL-2 antibody. IL-2 but not IL-1 was able to stimulate the Ts to release synthesized SSF in the absence of L3T4+ TH activity. First, addition of IL-2 to cocultures of tolerant cells and DNP-presenting I-A- cells induced release of the synthesized SSF, whereas addition of IL-1 did not. Second, IL-2 also stimulated SSF release in cocultures of L3T4+ T cell-depleted tolerant cells and Glu-DNP-SC, whereas IL-1 did not. Tolerant cells pretreated with IL-2 and then washed were able to synthesize and release SSF upon culture with Glu-DNP-SC. Pretreatment of tolerant cells with IL-1 did not stimulate SSF release upon subsequent culture with Glu-DNP-SC. These results indicate that the Lyt2+ Ts from DNBS-tolerant mice express IL-2 receptors and IL-2 is the lymphokine which induces the Ts to release synthesized SSF. Thus, IL-2 provides a differentiative signal during the functional activation of these regulatory T cells.     

Genetic control of contact photosensitivity to tetrachlorosalicylanilide. II. Igh complex controls the sensitivity induced by photohapten-modified spleen cells but not epidermal cells

We studied the genetic control of murine contact photosensitivity (CPS)1 to 3,3',4',5-tetrachlorosalicylanilide (TCSA) that was induced by subcutaneous injection of TCSA-photomodified epidermal cells (photoTCSA-EC) and spleen cells (photoTCSA-SC). With regard to the H-2 locus, sensitization with both types of photohaptenated cells showed the same pattern of CPS responses: H-2k and H-2b,d haplotypes were closely associated with low and high responders, respectively. On the other hand, the Igh locus affected the CPS reaction induced by photoTCSA-SC but not -EC; the Igh-1d allotype was related to low responsiveness, while high responders possessed Igh-1a,b. Thus, the photoTCSA-SC sensitization was controlled by H-2 and Igh in a codominant manner. The photoTCSA-SC-induced responses of H-2k but not Igh-1d mice were enhanced by CY pretreatment, suggesting that the mechanisms of low responsiveness in H-2k and Igh-1d mice were different. H-2 identity between donors of photoTCSA-EC and recipients was sufficient for effective sensitization, whereas both H-2 and Igh between donors of photoTCSA-SC and recipients should be identical to obtain maximum sensitization. This further confirmed the involvement of the Igh complex in the genetic control of CPS evoked by photoTCSA-SC. B cells as well as macrophages served as an effective presentation template for the photoTCSA-SC sensitization in the high responder Igh-1a mice, whereas B cells failed in inducing the CPS reaction in the low responder Igh-1d mice. These results suggest that B cells play an essential role in the Igh control phenomenon seen in the photoTCSA-SC sensitization. The present study demonstrated that CPS induced by photohapten-modified cells are differentially regulated by the H-2 and Igh gene loci depending on the cell type used for sensitization.     

The use of nonspecific T acceptor cells to overcome the aberrant function of antigen-specific third-order T suppressor cells

Earlier studies in the phenyltrimethylamino (TMA) hapten system demonstrated that under certain conditions idiotype-specific second-order T suppressor (Ts2)-bearing mice fail to suppress TMA-specific delayed-type hypersensitivity. This was due to a functional deletion in the third-order T suppressor (Ts3) subset. In this report we have confirmed and extended these findings to show that only homologous TMA-specific Ts3 can restore suppressor function, both heterologous Ts3 and unprimed T-cell populations failed to do so. Furthermore, attempts to induce Ts3 function in the defective mice after reconstitution with normal precursor Ts3 cells also failed. In contrast, protocols which induce heterologous contact and cutaneous hypersensitivity reactions readily induced cell populations capable of restoring suppression in the Ts3-defective mice. Analysis of the lymphoid populations from the contact-sensitized defective mice revealed that these cells were not the prototypical Ts3 but were similar to the previously reported nonspecific T acceptor cell. The results further indicated that the T acceptor cell functioned as the active terminal-phase Ts subset, and this could be used as an alternative to the TMA-specific Ts3. The importance of multiple suppressor pathways at the terminal phase of immune suppression is discussed.     

Hapten-specific responses to the phenyltrimethylamino hapten. IV. Occurrence of mechanistically distinct idiotypic suppressor T cells before the appearance of anti-idiotypic suppressor T cells induced by the monovalent antigen L-tyrosine-p-azophenyltrimethylammonium

We have previously shown that a single i.p. injection of the monovalent synthetic antigen, L-tyrosine-p-azophenyltrimethylammonium [tyr(TMA)] in complete Freund's adjuvant induces an anti-idiotypic T suppressor cell (Ts2) population that can be detected 6 wk later by its ability to shut down delayed-type hypersensitivity (DTH) specific for the TMA hapten. In this paper we present evidence that 2 wk after tyr(TMA) administration, a subset of Ts, termed Ts1, appears that is both functionally and phenotypically distinct from the late appearing Ts2 population. The early occurring Ts1 act only at the induction phase of the DTH response and can also suppress this response intrinsically. This latter point is in marked contrast to our previous observation that the tyr(TMA)-induced anti-idiotypic Ts2 fail to function intrinsically and can only be detected upon adoptive transfer into naive mice. Ts1 bear idiotypic receptors and are Ly-1+,2- in contrast to the anti-idiotypic Ly-1-,2+ Ts2 population. In addition, unlike the Ts2 population, Ts1 are comparatively nylon wool-adherent. Adsorption of Ts1 on either antigen- or idiotype-coated petri dishes indicate that the suppressor activity can be transferred only by antigen-binding cells. Cellfree factors prepared from spleens containing the Ts1 population can suppress DTH only if administered at the induction phase of the response, in contrast to the factors derived from the Ts2 population that act both at induction as well as effector phases, suggesting that Ts1 and Ts2 can function via soluble mediators. Finally, we show that when Ts1-bearing mice are primed and boosted for anti-TMA antibody formation, the resulting response was overall reduced with respect to the idiotype-positive and negative plaque-forming cells that differs from the Ts2-bearing hosts wherein the idiotypic component is preferentially suppressed. The appearance of Ts1 before the detection of Ts2 in the same experimental animals is discussed with reference to a normal physiologic sequence of events involved in suppressor pathways.     

Functional analysis of cloned macrophage hybridomas. V. Induction of suppressor T cell responses

It has been suggested that macrophage-like accessory cells are involved in suppressor T cell (Ts) induction. To further analyze this issue, we obtained several cloned macrophage hybridoma cell lines by somatic cell fusion of the macrophage tumor P388D1 of DBA/2 (H-2d) origin with splenic adherent cells of CKB mice (H-2k). Several cloned lines displayed the serological and functional characteristics of macrophages. We evaluated the ability of these hybridomas to induce third order or effector Ts (Ts3) to suppress the contact sensitivity response against the hapten 4-hydroxy-3-nitrophenyl acetyl (NP). In contrast to the parental P388D1 and two other macrophage hybridomas, one macrophage hybridoma clone, termed 63, when conjugated with NP, induced Ts3, which suppressed contact sensitivity responses against NP but not DNFB, showing that the Ts3 were antigen specific. Macrophage hybridoma 63 could specifically induce Ts3 activity in either H-2k, H-2d, or H-2k/H-2d heterozygous hosts. Thus, macrophage hybridoma 63 functionally expressed major histocompatibility complex-related restricting determinants, and the fusion with cells from a H-2k macrophage donor caused a functional complementation of H-2d-related, Ts-inducing elements. The genetic restriction governing induction of Ts3 was controlled by genes that mapped to I-J region. Furthermore, NP-conjugated macrophage hybridoma 63 could serve as a target for elicitation of suppressor responses after administration of I-Jk, but not I-Jb, restricted suppressor factor. The data suggest that macrophage hybridomas represent a means to dissect heterogeneity within the macrophage population. The data also imply that the I-J determinants expressed on macrophages represent a ligand for the antigen receptor of Ts.     

Characterization of suppressor T cell clones derived from a mouse tolerized with conjugates of ovalbumin and monomethoxypolyethylene glycol.

The induction of antigen-specific tolerance in mice by conjugates of ovalbumin (OVA) and monomethoxypolyethylene glycol (mPEG) previously had been shown to be associated with the generation of antigen-specific suppressor T (Ts) cells. For the elucidation of the nature of these Ts cells, five nonhybridized OVA-specific Ts cell clones were generated from the spleen cells of a BDF1 mouse which had been immunosuppressed by the tolerogenic conjugate, OVA(mPEG)12. The cloned Ts cells were maintained in vitro by periodic stimulation with OVA and feeder cells and were able to suppress the in vitro antibody production in an OVA-specific and MHC class I (H-2Kd or H-2Dd)-restricted manner. All these Ts cell clones were shown to be Thy1.2+, CD4-, CD5-, CD8+, and to express CD3 and the alpha beta heterodimer of the T cell receptor. The cell-free extracts of these cells contained soluble suppressor factors which could mimic in vitro the suppressive activity of the intact cells. In contrast to cytotoxic T lymphocytes (CTL), none of the cloned Ts cells were endowed with cytolytic activity as revealed in the perforin-mediated microhemolysis and in the 18-hr51Cr release assays. These results demonstrate that (i) OVA-specific Ts cell clones can be generated from mice pretreated with OVA(mPEG)12 by employing conventional T cell culture techniques, and (ii) these Ts cells are functionally different from conventional CD8+ CTL.     

Characterization of L-glutamic acid60-L-alanine30-L-tyrosine10-specific suppressor T cells in responder mice restricted by Igh-C-linked genes

A Ts cell subset has been identified in the spleens of responder mice 3 to 6 wk after immunization with an optimally immunogenic dose of L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT). These Ts were positively selected by panning procedures by using a mAb (1248 A4.10) produced by immunization of rats with semipurified mouse GAT-specific, single polypeptide chain suppressor factor. These Ts cells inhibited the activity of virgin Th cells but not memory Th cells and this activity was genetically restricted by genes which are linked to the Ig H chain (Igh) locus on chromosome 12. Use of the Igh recombination strain, BAB.14, which has a crossover near the VHCH region junction, demonstrated that the genes regulating the Igh restriction map telomeric to the VH genes. The Igh-linked restriction regulated the interaction of A4.10+ Ts cells with virgin T cells and not B cells. However, A4.10+ Ts did not act directly on Lyt-2-Th cells, but required the presence of Lyt-2+ cells for suppression. Suppression by GAT-primed A4.10+-Ts cells also required syngenicity at Igh-linked genes by both Lyt-2- and Lyt-2+ T cells. These results indicated that A4.10+-Ts cells were inducer Ts cells which activated Lyt-2+ effector Ts cells which prevented primary GAT specific Th cell activity. The interaction between A4.10+-Ts inducer and effector Ts cells and/or the interaction of the effector Ts and its target cell were restricted by genes linked to the Igh constant region.     

Requirements for activation of contrasuppressor T cells by type III pneumococcal polysaccharide

Either S3-coupled spleen cells (S3-SC) or soluble S3 activates two populations of regulatory T cells, T suppressor cells (Ts) and contrasuppressor T cells (Tcs). The latter cells function to mask the activity of Ts in unfractionated T cell populations, so that Ts can be detected only after removal of Tcs. Activation of Tcs by S3 may be required for induction of an antibody response to S3. This is suggested by the findings that Tcs are activated only by immunogenic doses of S3, that Tcs are not detectable in the spleens of mice tolerant to S3, and that (CBA/N X BALB/c)F1 male (xid) mice, which are genetically unresponsive to S3, do not develop Tcs after immunization with S3. Moreover, the kinetics of activation of Tcs by S3 closely parallels the kinetics of the antibody response to S3. Tcs have no detectable activity in the absence of Ts, indicating that these cells do not function as amplifier or helper T cells.