Differential influence of 2'-deoxyguanosine on the induction and expression of suppressor T lymphocytes in vivo

Subcutaneous (sc) immunization of mice with allogeneic spleen cells can induce delayed-type hypersensitivity (DTH) to histocompatibility antigens. Intravenous immunization with irradiated allogeneic spleen cells, on the other hand, induces suppressor T (Ts) lymphocytes. These Ts cells are capable of suppressing the host-versus-graft (HvG) DTH reactivity which normally arises after sc immunization. Moreover they can suppress the development of antihost DTH effector T cells during graft-versus-host (GvH) reactions. These models for HvG and GvH DTH reactivity were used to study the influence of 2'-deoxyguanosine (dGuo) on the induction, further development, and expression of Ts cells in vivo. It was found that administration of dGuo inhibits the proliferation-dependent induction and further development of Ts cells, but not the suppression mediated by already activated Ts cells.     

Differential sensitivity to 2'-deoxyguanosine of antigen-specific and nonspecific suppressor T cells in delayed hypersensitivity

The cell wall components involved in the adherence of Bacillus globigii to lymphocytes were studied. Lipoteichoic acid inhibited B. globigii-lymphocyte interaction. The interaction was not affected by Escherichia coli outer membranes or lipopolysaccharides.     

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.     

In vitro and in vivo induction of effector T cells mediating DTH responses to a protein and a synthetic polypeptide antigen.

We have developed an in vitro system for the activation of T cells in order to get a better insight into the genetic and molecular mechanisms involved in the generation of delayed-type hypersensitivity (DTH) effector T cells. Low doses of fowl γ-globulin (FγG) as well as the synthetic polypeptide (T,G)-A-L were bound to splenic adherent cells and served as immunogens for the in vitro sensitization of lymphocytes. In parallel, (T,G)-A-L-specific T cells were activated in vivo in irradiated recipient mice. The ability of the in vitro- and in vivo-activated cells to mediate DTH responses was determined in naive recipient mice by the radioisotopic ear assay. Twenty to thirty × 10⁶ “educated” cells were sufficient to elicit significant DTH responses. Irradiation of the spleen cells prior to their transfer resulted in higher responses. The DTH reactivity was transferable by nylon wool-enriched T cells but not by a Thy 1.2-depleted population indicating the T-cell dependency of the response. The in vitro and in vivo antigen-activated T-cell population exhibited also helper-cell activity as determined by their cooperation with B cells in adoptive transfer experiments.     

Induction of suppressor T cells of antibody formation under conditions that preferentially stimulate DTH

These studies describe the conditions under which antibody-forming cells and TDTH cells are selectively induced in vitro. TDTH cells are preferentially stimulated when high doses of antigen are included in the culture. Antibody-forming cells, on the other hand, are optimally stimulated with a 100 to 1000-fold less concentration of antigen. The conditions that optimally stimulate TDTH cells also induce a population of suppressor T cells that inhibit the antibody response. However, although their inductive requirements are similar, the suppressor T cells of antibody formation are a distinct subpopulation of cells from the TDTH cells. Whereas the suppressor T cells are LY-1-, 2+, 4-, 6+, and Ia+; the TDTH cells are Ly-1+, 2+/-, 4-, 6+, and Ia-. Furthermore, the DTH cells are sensitive to high doses of irradiation, whereas the suppressor cells are resistant. Based on the Ly phenotype and the kinetics of suppression, the suppressor T cells are not the "feedback suppressors" that have been identified in other systems. The system described in this paper provides a means whereby the cells that regulate humoral and CMI can be studied in vitro.     

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.     

Involvement of antigen and I-J determinants in the induction of effector T suppressor cells by immune B cells

Immune B cells induce effector T suppressor cells in vitro. The B cells act as antigen-presenting cells, and express both I-A and I-J determinants. Antigen and I-J determinants are required for the induction of suppressor T cells by immune B cells, but I-A determinants are not. These findings indicate that precursors of suppressor T cells appear to recognize antigen in the context of I-J determinants on the surface of immune B cells.     

Characterization of two suppressor cells that together prevent in vivo development of cytolytic T cells to hapten-altered self

Two suppressor cell populations that interact to down-regulate in vivo development of the cytolytic T-cell (CTL) response to trinitrophenyl-modified syngeneic spleen cells (TNP-SC) have been further characterized. Suppressor cells induced by the iv injection of trinitrophenyl-modified syngeneic spleen cells possess Thy 1.2 antigen. Their precursors are insensitive to pretreatment of host animals with cyclophosphamide (CY). Suppressor cells that arise after dermal sensitization with trinitrochlorobenzene are also Thy 1.2 antigen positive but their precursors are sensitive to pretreatment with CY. These characteristics of the two suppressor T cells (Ts) are identical to those of the two Ts that are generated by similar methodologies and that together suppress contact sensitivity (CS) to picryl chloride. Neither the CS nor CTL response was suppressed when host animals possessed only one set of Ts. In contrast to suppression of CS at the efferent phase, development of CTL was suppressed only when the two Ts were present early during sensitization (afferent phase). Since the results point to several similarities between the two sets of Ts that are active in the down-regulation of the CS and CTL responses, it is suggested that the two dissimilar immune responses directed to the same hapten, namely CS and CTL, may be controlled by the same suppressor cells. Since it appears that the two sets of Ts interact to affect different phases of the CS and CTL responses, down-regulation of each must be accomplished through different mechanisms.     

Dynamics of pathogenic and suppressor T cells in autoimmune diabetes development

In the nonobese diabetic (NOD) mouse, pathogenic and suppressor CD4(+) T cells can be distinguished by the constitutive expression of CD25. In this study, we demonstrate that the progression of autoimmune diabetes in NOD mice reflects modifications in both T cell subsets. CD4(+)CD25(+) suppressor T cells from 8-, but not 16-wk-old NOD mice delayed the onset of diabetes transferred by 16-wk-old CD25-depleted spleen cells. These results were paralleled by the inhibition of alloantigen-induced proliferation of CD4(+)CD25(-) cells, indicating an age-dependent decrease in suppressive activity. In addition, CD4(+)CD25(-) pathogenic T cells became progressively less sensitive to immunoregulation by CD4(+)CD25(+) T cells during diabetes development. CD4(+)CD25(-) T cells showed a higher proliferation and produced more IFN-gamma, but less IL-4 and IL-10, whereas CD4(+)CD25(+) T suppressor cells produced significantly lower levels of IL-10 in 16- compared with 8-wk-old NOD mice. Consistent with these findings, a higher frequency of Th1 cells was observed in the pancreas of 16-wk-old compared with 8-wk-old NOD mice. An increased percentage of CD4(+)CD25(-) T cells expressing CD54 was present in 16-wk-old and in diabetic NOD, but not in BALB/c mice. Costimulation via CD54 increased the proliferation of CD4(+)CD25(-) T cells from 16-, but not 8-wk-old NOD mice, and blocking CD54 prevented their proliferation, consistent with the role of CD54 in diabetes development. Thus, the pathogenesis of autoimmune diabetes in NOD mice is correlated with both an enhanced pathogenicity of CD4(+)CD25(-) T cells and a decreased suppressive activity of CD4(+)CD25(+) T cells.     

Myeloid-derived suppressor cells accumulate in kidney allograft tolerance and specifically suppress effector T cell expansion

The immune tolerance to rat kidney allografts induced by a perioperative treatment with anti-CD28 Abs is associated with a severe unresponsiveness of peripheral blood cells to donor Ags. In this model, we identified an accumulation in the blood of CD3(-)class II(-)CD11b(+)CD80/86(+) plastic-adherent cells that additionally expressed CD172a as well as other myeloid markers. These cells were able to inhibit proliferation, but not activation, of effector T cells and to induce apoptosis in a contact-dependent manner. Their suppressive action was found to be under the control of inducible NO synthase, an enzyme also up-regulated in tolerated allografts. Based on these features, these cells can be defined as myeloid-derived suppressor cells (MDSC). Interestingly, CD4(+)CD25(high)FoxP3(+) regulatory T cells were insensitive in vitro to MDSC-mediated suppression. Although the adoptive transfer of MDSC failed to induce kidney allograft tolerance in recently transplanted recipients, the maintenance of tolerance after administration of anti-CD28 Abs was found to be dependent on the action of inducible NO synthase. These results suggest that increased numbers of MDSC can inhibit alloreactive T cell proliferation in vivo and that these cells may participate in the NO-dependent maintenance phase of tolerance.     

Abrogation of SRC homology region 2 domain-containing phosphatase 1 in tumor-specific T cells improves efficacy of adoptive immunotherapy by enhancing the effector function and accumulation of short-lived effector T cells in vivo

T cell expression of inhibitory proteins can be a critical component for the regulation of immunopathology owing to self-reactivity or potentially exuberant responses to pathogens, but it may also limit T cell responses to some malignancies, particularly if the tumor Ag being targeted is a self-protein. We found that the abrogation of Src homology region 2 domain-containing phosphatase-1 (SHP-1) in tumor-reactive CD8(+) T cells improves the therapeutic outcome of adoptive immunotherapy in a mouse model of disseminated leukemia, with benefit observed in therapy employing transfer of CD8(+) T cells alone or in the context of also providing supplemental IL-2. SHP-1(-/-) and SHP-1(+/+) effector T cells were expanded in vitro for immunotherapy. Following transfer in vivo, the SHP-1(-/-) effector T cells exhibited enhanced short-term accumulation, followed by greater contraction, and they ultimately formed similar numbers of long-lived, functional memory cells. The increased therapeutic effectiveness of SHP-1(-/-) effector cells was also observed in recipients that expressed the tumor Ag as a self-antigen in the liver, without evidence of inducing autoimmune toxicity. SHP-1(-/-) effector CD8(+) T cells expressed higher levels of eomesodermin, which correlated with enhanced lysis of tumor cells. Furthermore, reduction of SHP-1 expression in tumor-reactive effector T cells by retroviral transduction with vectors that express SHP-1-specific small interfering RNA, a translatable strategy, also exhibited enhanced antitumor activity in vivo. These studies suggest that abrogating SHP-1 in effector T cells may improve the efficacy of tumor elimination by T cell therapy without affecting the ability of the effector cells to persist and provide a long-term response.     

The indispensable role of CCR5 for in vivo suppressor function of tumor-derived CD103+ effector/memory regulatory T cells

CD103 is a marker for identification of effector/memory regulatory T cells (Tregs). CD103(+) Tregs are potent suppressors of tissue inflammation in several infectious diseases, autoimmune diseases, and cancers. However, the underlying mechanisms for this potent suppression ability remain unclear. The current study was designed to clarify this issue. Unexpectedly, we found both CD103(+) and CD103(-) Tregs had similar suppression capacity in vitro. We then chose a murine tumor model for investigation of the in vivo behavior of these Tregs. The suppression ability in vivo against the anti-tumor ability of CD8(+) T cells was restricted to CD103(+) Tregs although both Tregs had equal in vitro suppression ability. In addition, CD103(+) Tregs expressed significantly higher levels of CCR5 than those of CD103(-) Tregs and accumulated more in tumors than did CD103(-) Tregs. Furthermore, blockade of CCR5 signaling, either by CCR5(-/-)CD103(+) Tregs or by CCL5 knockdown tumor, could reduce the migration of CD103(+) Tregs into tumors and impair their in vivo suppression ability. In conclusion, these results indicate that the potent in vivo suppression ability of CD103(+) Tregs is due to the tissue-migration ability through CCR5 expression.     

Characteristics of DTH suppressor cells in mice infected with Candida albicans

Inoculation of 10⁸ C. albicans intraperitoneally into Balb/c mice at given dosage was reported to induce suppression of antigen-specific delayed-type hypersensitivity. Adoptive transfer of spleen cells into normal syngeneic mice pre-treated with Cyclophosphamide confirmed the existence of suppressor cells in mice. Such cells were sensitive to treatment with anti- serum and complement, non-adherent to Sephadex G-10. A pretreatment of the mice with Cyclophosphamide eliminated DTH suppression. Treatment with antimacrophage agents via intraperitoneal abrogated suppression only if being effected before inoculation of alive 10⁸ Candida albicans.It is concluded that the spleen suppressor cell is a T-lymphocyte whose precursor is Cyclophosphamide-sensitive, requiring the macrophage to be induced.     

Delayed-type hypersensitivity (DTH) in BCG-sensitized mice I. Lack of suppressor T cell activity on DTH to sheep red blood cells.

Mice pretreated with an intravenous (i.v.) injection of BCG (BCG-sensitized mice) and then immunized intravenously with a high dose (10(8)--10(9)) of sheep red blood cells (SRBC) 2 weeks later developed strong delayed-type hypersensitivity (DTH) to SRBC, as in mice pretreated with cyclophosphamide (CY) (CY-treated mice) and then immunized with SRBC 2 days later; normal mice given the same dose of SRBC did not show such DTH. The mechanism of this strong DTH to SRBC which developed in BCG-sensitized mice was studied, by comparing it with that in CY-treated mice. The transfer of either whole spleen cells or thymus cells, but not serum, obtained from mice immunized with i.v. injections of 10(9) SRBC 4 days previously (hyperimmune mice) did not suppress either the induction or the expression of DTH to SRBC in BCG-sensitized mice, but suppressed those in CY-treated mice. The suppressor cells were SRBC-specific T cells. Adoptive transfer of DTH to SRBC by spleen cells from either BCG-sensitized mice of CY-treated mice to hyperimmune recipients failed. The adoptive transfer of DTH from BCG-sensitized mice to normal recipients also failed if the spleen cells from hyperimmune mice were cotransferred. Whole body irradiation (600 rad) of mice 2 hr before or after the time of immunization with SRBC reduced significantly DTH to SRBC in both BCG-sensitized and CY-treated mice. It was noticed that the total number of spleen cells in BCG-sensitized mice was 3--4 times larger than that in CY-treated mice. From these results, we conclude that the entity of effector T cells of DTH to SRBC induced in BCG-sensitized mice and in CY-treated mice was not different in terms of susceptibility to suppressor T cells and irradiation, but that the total numbers of effector T cells generated in these mice differed remarkably, resulting in the above-described different responsiveness to suppressor T cells transferred passively.     

A role for L3T4+ T cells and their lymphokines in the generation of suppressor effector (TS3) cells

The cellular requirements for the in vitro induction of antigen-specific suppressor T cells were examined. Previous reports indicated that Ia-bearing macrophages and anti-idiotypic B cells are required as accessory cells to facilitate the generation of suppressor effector (TS3) cells which regulate the response to the 4-hydroxy-3-nitrophenyl acetyl (NP) hapten. The present study describes two distinct T cell populations which interact to generate antigen-specific TS3. Fractionation of the T cell populations with monoclonal antibody to the L3T4 determinant led to the identification of an NP-specific L3T4- TS3 progenitor population and an L3T4+ helper/inducer subset. In the presence of NP-coupled antigen, the L3T4+ subset could induce progenitor TS3 to differentiate into mature TS3 cells. The activity of the L3T4+ inducer population could be replaced with specifically activated cloned helper cells which were not NP-reactive since an I-Ab-restricted, insulin-reactive, L3T4+ clone was capable of supporting the generation of NP-specific TS3. Inducer activity appeared to be confined to the Th1 but not the Th2 subset. In addition, 18-hr supernatants from antigen-activated clones were capable of substituting for L3T4+ cells or T cell clones in TS3 induction cultures. The TS maturation/differentiation factor(s) active in these supernatants does not appear to be IL-1, IL-2, IL-3, or interferon-gamma alone since purified sources of these lymphokines failed to induce TS3 activity.     

Tumor-induced L-selectinhigh suppressor T cells mediate potent effector T cell blockade and cause failure of otherwise curative adoptive immunotherapy

Tumor-specific effector T cells (T(E)) are naturally sensitized within the L-selectin(low) (CD62L(low)) fraction of tumor-draining lymph nodes (TDLN). Whether isolated from day 9 (D9) or day 12 (D12) TDLN, 5 million L-selectin(low) T(E) could be culture activated and adoptively transferred to achieve complete rejection of established intradermal, pulmonary, and brain tumors. Surprisingly, although 25 million unfractionated T cells from D9 TDLN were equally effective, even 100 million unfractionated T cells from D12 TDLN seldom prevented lethal intradermal tumor progression, despite a pronounced therapeutic excess of T(E). This highly reproducible treatment failure was due to cotransfer of tumor-induced, L-selectin(high) suppressor T cells (T(S)) which were also present in D12 TDLN. In contrast, D9 TDLN and normal spleens lacked L-selectin(high) T(S). Only those L-selectin(high) D12 TDLN T cells that down-regulated L-selectin during culture activation were suppressive in vivo and in vitro, and, like L-selectin(low) T(E), trafficked promptly into tumors following i.v. administration. This is the first demonstration that adoptive immunotherapy can fail as a direct result of passenger T(S) that share certain phenotypic and trafficking features of T(E), even when otherwise curative doses of T(E) have been administered. Furthermore, in contrast to recently described CD4(+)CD25(+) T(S) and plasmacytoid dendritic cell-activated T(S), tumor-induced L-selectin(high) T(S) prevent tumor rejection via blockade of sensitized, activated T(E) rather than via afferent blockade.     

The role of B cells in the development of CD4 effector T cells during a polarized Th2 immune response

Previous studies have suggested that B cells promote Th2 cell development by inhibiting Th1 cell differentiation. To examine whether B cells are directly required for the development of IL-4-producing T cells in the lymph node during a highly polarized Th2 response, B cell-deficient and wild-type mice were inoculated with the nematode parasite, Nippostrongylus brasiliensis. On day 7, in the absence of increased IFN-gamma, IL-4 protein and gene expression from CD4 T cells in the draining lymph nodes were markedly reduced in B cell-deficient mice and could not be restored by multiple immunizations. Using a DO11.10 T cell adoptive transfer system, OVA-specific T cell IL-4 production and cell cycle progression, but not cell surface expression of early activation markers, were impaired in B cell-deficient recipient mice following immunization with N. brasiliensis plus OVA. Laser capture microdissection and immunofluorescent staining showed that pronounced IL-4 mRNA and protein secretion by donor DO11.10 T cells first occurred in the T cell:B cell zone of the lymph node shortly after inoculation of IL-4-/- recipients, suggesting that this microenvironment is critical for initial Th2 cell development. Reconstitution of B cell-deficient mice with wild-type naive B cells, or IL-4-/- B cells, substantially restored Ag-specific T cell IL-4 production. However, reconstitution with B7-1/B7-2-deficient B cells failed to rescue the IL-4-producing DO11.10 T cells. These results suggest that B cells, expressing B7 costimulatory molecules, are required in the absence of an underlying IFN-gamma-mediated response for the development of a polarized primary Ag-specific Th2 response in vivo.     

In vivo generation of hapten-specific killer T cells without elimination of suppressor cells.

We have provided evidence to demonstrate that hapten-specific killer cells can be generated in vivo toward hapten-conjugated syngeneic splenic cells. The critical aspect is to provide an auxillary cellular antigenic stimulus in addition to the hapten-conjugated syngeneic cells. In our experiments this stimulus was CBA/J splenic cells that possess MIs disparate but H-2 compatible antigens with C3H/HeN hosts. The Mls antigen has been shown by others to activate helper T cells in vitro to synthesize KAF, a signal that prekiller cells require besides target antigen. Killer cells (shown to be T cells) as well as helper T cells were found to be derived from the C3H host. Induction in the host animal of partial tolerance to the auxiliary cells possessing Mls antigen abrogated the response. This system, besides providing a better understanding of the control mechanisms involved in the development of T killer cells in vivo, points to a way in which the latter may be generated-ng suppressor cells. The latter principle may prove highly useful in certain clinical situations.     

Limited in vivo reactivity of polyclonal effector cytotoxic T cells towards altered peptide ligands

T cell responses are regulated by the affinity/avidity of the T cell receptor for the MHC/peptide complex, available costimulation and duration of antigenic stimulation. Altered peptide ligands (APLs) are usually recognized with a reduced affinity/avidity by the T cell receptor and are often able to only partially activate T cells in vitro or may even function as antagonists. Here we assessed the ability of APLs derived from peptide p33 of lymphocytic choriomeningitis virus (LCMV) to mediate lysis of target cells in vivo, confer anti-viral protection and cause auto-immune disease. In general, in vitro cross-reactivity between APLs was rather limited, and even strongly cross-reactive cytotoxic T lymphocytes were only able to mediate moderate anti-viral protection. Partial protection was observed for infection with LCMV or low doses of recombinant vaccinia virus, while no reduced viral titers could be seen upon infection with high dose of vaccinia virus. In a transgenic mouse model expressing LCMV glycoprotein in the islets of the pancreas, APLs induced a transient insulitis but failed to induce autoimmune diabetes. Thus, effector functions induced by even highly homologous APLs are rather limited in vivo.