Porcine-Stimulated Human Tr1 Cells Showed Enhanced Suppression in Xenoantigen Stimulation Response.

Type 1 regulatory T (Tr1) cells play a fundamental role in maintaining and inducing immune tolerance. Our preliminary study demonstrated that an interleukin- (IL-) 10-mediated pathway is a possible regulatory mechanism underlying the xenoantigen-specific human Treg enhanced suppressive capacity. Here, we developed a feasible protocol for expanding IL-10-induced xenoantigen-specific human Tr1 cells in vitro which would be more efficient in transplantation immunotherapy efficiency. In this study, xenoantigen-specific Tr1 cells are generated from human naive CD4+ T cells expanded for two subsequent xenoantigen-stimulation cycles with recombinant human IL-10. The phenotype and suppressive capacity of xenoantigen-stimulated Tr1 cells are assessed, and the mechanism of their suppression is studied. Tr1 cells can be induced by porcine xenoantigen stimulation combined with IL-10, IL-2, and IL-15, displaying an increased expression of CD49b, CTLA-4, and LAG-3 without expressing Foxp3 which also showed an effector memory Treg phenotype and expressed high levels of CD39. After xenoantigen stimulation, the IL-10 and IL-5 gene expression in Tr1 cells increased, secreting more IL-10, and xenoantigen-stimulated Tr1 cells changed their T cell receptor (TCR) Vß repertoire, increasing the expression of TCR Vß2, TCR Vß9, and TCR Vß13. In a pig to human mixed lymphocyte reaction (MLR), xenoantigen-stimulated Tr1 cells displayed enhanced suppressive capacity via CD39 in a dose-dependent manner. Moreover, IL-5 could affect the proliferation of xenoantigen-specific Tr1 cells, but not their phenotypes' expression. This study provides a theory and feasible method for immune tolerance induction in clinical xenotransplantation.

Development of a Recombinant Xenogeneic Tumor Necrosis Factor Alpha Protein Vaccine To Protect Mice from Experimental Colitis.

Previous studies have highlighted the efficacy of tumor necrosis factor alpha (TNF-α) inhibitors, including monoclonal antibodies and soluble receptors, in the treatment and management of intestinal bowel disease (IBD). However, because of the immunogenicity of xenogeneic TNF-α inhibitors, antidrug antibodies (ADAs) can be triggered after repeated administration. An alternative way to target TNF-α is active immunization to elicit the production of high titers of neutralizing antibodies. In this study, we prepared a xenogeneic TNF-α protein vaccine and studied the protective effects in experimental colitis models. The xenogeneic TNF-α protein vaccine could overcome self-tolerance and induce TNF-α-specific neutralizing antibody. Moreover, the xenogeneic TNF-α protein vaccine could protect mice from acute and chronic colitis induced by dextran sodium sulfate (DSS). One possible explanation for this protective effect is the production of TNF-α-specific neutralizing antibody, which absorbed the biological activity of mouse TNF-α (mTNF-α) and failed to induce T lymphocyte apoptosis. In summary, use of the xenogeneic TNF-α protein vaccine may be a potent therapeutic strategy for IBD.

Xenogeneic therapeutic cancer vaccines as breakers of immune tolerance for clinical application: to use or not to use?

Accumulation of firm evidence that clinically apparent cancer develops only when malignant cells manage to escape immunosurveillance led to the introduction of tumor immunotherapy strategies aiming to reprogramm the cancer-dysbalanced antitumor immunity and restore its capacity to control tumor growth. There are several immunotherapeutical strategies, among which specific active immunotherapy or therapeutic cancer vaccination is one of the most promising. It targets dendritic cells (DCs) which have a unique ability of inducing naive and central memory T cell-mediated immune response in the most efficient manner. DCs can be therapeutically targeted either in vivo/in situ or by ex vivo manipulations followed by their re-injection back into the same patient. The majority of current DC targeting strategies are based on autologous or allogeneic tumor-associated antigens (TAAs) which possess various degrees of inherent tolerogenic potential. Therefore still limited efficacy of various tumor immunotherapy approaches may be attributed, among various other mechanisms, to the insufficient immunogenicity of self-protein-derived TAAs. Based on such an idea, the use of homologous xenogeneic antigens, derived from different species was suggested to overcome the natural immune tolerance to self TAAs. Xenoantigens are supposed to differ sufficiently from self antigens to a degree that renders them immunogenic, but at the same time preserves an optimal homology range with self proteins still allowing xenoantigens to induce cross-reactive T cells. Here we discuss the concept of xenogeneic vaccination, describe the cons and pros of autologous/allogeneic versus xenogeneic therapeutic cancer vaccines, present the results of various pre-clinical and several clinical studies and highlight the future perspectives of integrating xenovaccination into rapidly developing tumor immunotherapy regimens.

[Therapeutic intervention alternatives in cancer, using attenuated live bacterial vectors: Salmonella enterica as a carrier of heterologous molecules]. / Alternativas de intervención terapéutica en cáncer usando vectores bacterianos vivos atenuados: Salmonella enterica como acarreador de moléculas heterólogas.

Salmonella enterica is a facultative anaerobic bacteria, whose ability to colonize antigen-presenting cells (APCs) such as dendritic cells and macrophages, has allowed its successful use as an alive, attenuated bacterial vector for vaccination. Salmonella enterica elicits efficient cellular, humoral and mucosal immune responses, against heterologous antigens including viruses, parasites, other bacterial species and tumor-associated antigens, since it is capable of delivering these antigens to cells of the immune system. The extracellular expression of heterologous antigens on the surface of Salmonella enterica via its type I, III and V secretion systems, and their delivery into infected cells is essential for its stimulation of immune responses against these antigens. Moreover, Salmonella enterica is a promising therapeutic agent against cancer, as demonstrated by reports of pre-clinical and clinical studies indicating that, after systemic administration, Salmonella enterica preferentially localizes in solid tumors and metastases as compared to normal tissues. In this review, we focus on novel prophylactic and therapeutic anti-cancer approaches using Salmonella enterica as a delivery system of heterologous molecules with the aim of inhibiting tumor growth.

Heterologous prime-boost-boost immunisation of Chinese cynomolgus macaques using DNA and recombinant poxvirus vectors expressing HIV-1 virus-like particles.

BACKGROUND: There is renewed interest in the development of poxvirus vector-based HIV vaccines due to the protective effect observed with repeated recombinant canarypox priming with gp120 boosting in the recent Thai placebo-controlled trial. This study sought to investigate whether a heterologous prime-boost-boost vaccine regimen in Chinese cynomolgus macaques with a DNA vaccine and recombinant poxviral vectors expressing HIV virus-like particles bearing envelopes derived from the most prevalent clades circulating in sub-Saharan Africa, focused the antibody response to shared neutralising epitopes. METHODS: Three Chinese cynomolgus macaques were immunised via intramuscular injections using a regimen composed of a prime with two DNA vaccines expressing clade A Env/clade B Gag followed by boosting with recombinant fowlpox virus expressing HIV-1 clade D Gag, Env and cholera toxin B subunit followed by the final boost with recombinant modified vaccinia virus Ankara expressing HIV-1 clade C Env, Gag and human complement protein C3d. We measured the macaque serum antibody responses by ELISA, enumerated T cell responses by IFN-γ ELISpot and assessed seroneutralisation of HIV-1 using the TZM-bl ß-galactosidase assay with primary isolates of HIV-1. RESULTS: This study shows that large and complex synthetic DNA sequences can be successfully cloned in a single step into two poxvirus vectors: MVA and FPV and the recombinant poxviruses could be grown to high titres. The vaccine candidates showed appropriate expression of recombinant proteins with the formation of authentic HIV virus-like particles seen on transmission electron microscopy. In addition the b12 epitope was shown to be held in common by the vaccine candidates using confocal immunofluorescent microscopy. The vaccine candidates were safely administered to Chinese cynomolgus macaques which elicited modest T cell responses at the end of the study but only one out of the three macaques elicited an HIV-specific antibody response. However, the antibodies did not neutralise primary isolates of HIV-1 or the V3-sensitive isolate SF162 using the TZM-bl ß-galactosidase assay. CONCLUSIONS: MVA and FP9 are ideal replication-deficient viral vectors for HIV-1 vaccines due to their excellent safety profile for use in humans. This study shows this novel prime-boost-boost regimen was poorly immunogenic in Chinese cynomolgus macaques.

Immune suppression produced by intrathymic inoculation with xenogeneic antigen and whole-body γ-irradiation in a pig-to-monkey heart transplantation model.

OBJECTIVE: To validate the use of pig-to-monkey transplantation as an experimental animal model for study of the induction of immune suppression of cardiac xenografts via intrathymic inoculation with xenogeneic spleen cells and whole-body γ-irradiation. MATERIALS AND METHODS: Donors (Meishan pigs) and recipients (Rhesus monkeys) were randomized into 3 groups. Group 2 was the control group; group 2 received whole-body irradiation, and group 3 received whole-body irradiation and intrathymic inoculation. In each group, survival time of heterotopic heart xenografts in monkeys were observed. A mixed lymphocyte reaction assay with (3)H thymidine was used to examine changes in lymphocyte function in all groups on the day of transplantation. In each group, recipient serum was harvested for analysis of IL-2 and IL-10 concentrations. RESULTS: Mean (SD) survival time of donor hearts in group 3 was significantly longer (91.1 [22.8] hours) than in group 1 (36.6 [5.8] hours) (P < .01). The results of mixed lymphocyte reaction assay demonstrated a significant reduction in the stimulation index in group 3 compared with groups 1 and 2 (P < .01) when recipient splenocytes responded to the donor stimulator. The IL-2 concentration in recipients in all groups was much higher during rejection of xenografts than before transplantation (P < .01). CONCLUSION: These results suggest that pretreatment with intrathymic inoculation or whole-body irradiation induces T-cell immunosuppression. The IL-2 concentration is closely correlated with xenograft rejection.

Immunologic response to xenogeneic gp100 DNA in melanoma patients: comparison of particle-mediated epidermal delivery with intramuscular injection.

PURPOSE: Prior studies show that i.m. injection of xenogeneic orthologues of melanosomal antigens (tyrosinase, gp100) induces CD8(+) T-cell responses to the syngeneic protein. To further define the optimal vaccination strategy, we conducted a pilot clinical trial comparing i.m. injection with particle-mediated epidermal delivery (PMED). EXPERIMENTAL DESIGN: Human leukocyte antigen (HLA)-A*0201(+) disease-free melanoma patients were randomized to the PMED or i.m. arm, receiving eight vaccinations over 4 months. Patients received 4 microg or 2,000 microg per injection, respectively, of mouse gp100 DNA. Peripheral blood mononuclear cells were collected, cultured with gp100 peptides, and analyzed by tetramer and intracellular cytokine staining for responses to HLA-A*0201-restricted gp100 epitopes [gp100(209-217) (ITDQVPFSV) and gp100(280-288) (YLEPGPVTA)]. RESULTS: Twenty-seven patients with stage IIB-IV melanoma were analyzable for immune response. The only common toxicity was grade 1 injection site reaction in nine patients with no intergroup difference, and one dose-limiting toxicity of acute hypersensitivity occurred in a PMED patient with undiagnosed gold allergy. Four of 27 patients produced gp100 tetramer(+)CD8(+) T cells, all carrying the CCR7(lo)CD45RA(lo) effector-memory phenotype. Five of 27 patients generated IFN-gamma(+)CD8(+) T cells, one who was also tetramer-positive. Overall, vaccination induced a response in 30% of patients, which was not significantly associated with study arm or clinical outcome. However, the PMED group showed a trend toward increased IFN-gamma(+)CD8(+) T-cell generation (P = 0.07). CONCLUSION: A comparable efficacy and safety profile was shown between the i.m. and PMED arms, despite a significantly decreased dose of DNA used for PMED injection.

Xenogeneic immunization with human tyrosine hydroxylase DNA vaccines suppresses growth of established neuroblastoma.

Neuroblastoma (NB) is a challenging malignancy of the sympathetic nervous tissue characterized by a very poor prognosis. One important marker for NB is the expression of tyrosine hydroxylase (TH), the first-step enzyme of catecholamine biosynthesis. We could show stable and high TH gene expression in 67 NB samples independent of the clinical stage. Based on this observation, we addressed the question of whether xenogeneic TH DNA vaccination is effective in inducing an anti-NB immune response. For this purpose, we generated three DNA vaccines based on pCMV-F3Ub and pBUD-CE4.1 plasmids encoding for human (h)THcDNA (A), hTH minigene (B), and hTHcDNA in combination with the proinflammatory cytokine interleukin 12 (C), and tested prophylactic and therapeutic efficacy to suppress primary tumor growth and spontaneous metastasis. Here we report that xenogeneic TH DNA vaccination was effective in eradicating established primary tumors and inhibiting metastasis. Interestingly, this effect could not be enhanced by adding the Th1 cytokine interleukin 12. However, increased IFN-gamma production and NB cytotoxicity of effector cells harvested from vaccinated mice suggested the participation of tumor-specific CTLs in the immune response. The depletion of CD8(+)T cells completely abrogated the hTH vaccine-mediated anti-NB immune response. Furthermore, rechallenging of surviving mice resulted in reduced primary tumor growth, indicating the induction of a memory immune response. In conclusion, xenogeneic immunization with TH-derived DNA vaccines is effective against NB, and may open a new venue for a novel and effective immunotherapeutic strategy against this challenging childhood tumor.

A new strategy to induce effective antitumour response in vitro and in vivo.

To induce Her2-specific cell immune response, we used xenogeneic antigen rat neu L2-S2 domains as the vaccine antigen. The antigenic protein was engineered as a chimeric protein with human IgG1 Fc region (neu-Fc). Neu-Fc could stimulate the cell proliferation in mixed lymphocyte reaction effectively. Simultaneous neu-Fc and IFN-gamma stimulation dramatically elevated IL-12 secretion and reduced IL-10 production in PBMC. To further augment the activating effects on Th1-type response, Bacille Calmette-Guerin (BCG) was utilized as a non-specific stimulus. Neu-Fc, IFN-gamma and BCG costimulation exhibited the most conspicuous effects on the reversal of the Th1-type inhibitory effects by MCF-7 cell supernatant compared with neu-Fc alone or IFN-gamma and BCG costimulation. The lytic activity of effector cells to Her2 overexpressing cells was greatly promoted by neu-Fc, IFN-gamma and BCG stimulation simultaneously. Neu-Fc led to considerable retardation in EMT6/Her2 tumour growth in Balb/c mice. IFN-gamma and BCG efficiently enhanced the antitumour activity. A large amount of inflammatory cells were found to be accumulated in the tumour tissues or surrounded tumours in mice treated with neu-Fc, IFN-gamma and BCG but no inflammatory cell infiltration was observed in control tumours, indicating that the strategy is potent enough to support the initiation and propagation of tumour-specific immune response in an established tumour and generate a proinflammatory environment.

Double-negative T cells, activated by xenoantigen, lyse autologous B and T cells using a perforin/granzyme-dependent, Fas-Fas ligand-independent pathway.

The ability to control the response of B cells is of particular interest in xenotransplantation as Ab-mediated hyperacute and acute xenograft rejection are major obstacles in achieving long-term graft survival. Regulatory T cells have been proven to play a very important role in the regulation of immune responses to self or non-self Ags. Previous studies have shown that TCRalphabeta+CD3+CD4-CD8- (double-negative (DN)) T cells possess an immune regulatory function, capable of controlling antidonor T cell responses in allo- and xenotransplantation through Fas-Fas ligand interaction. In this study, we investigated the possibility that xenoreactive DNT cells suppress B cells. We found that DNT cells generated from wild-type C57BL/6 mice expressed B220 and CD25 after rat Ag stimulation. These xenoreactive B220+CD25+ DNT cells lysed activated, but not naive, B and T cells. This killing, which took place through cell-cell contact, required participation of adhesion molecules. Our results indicate that Fas ligand, TGF-beta, TNF-alpha, and TCR-MHC recognition was not involved in DNT cell-mediated syngenic cell killing, but instead this killing was mediated by perforin and granzymes. The xenoreactive DNT cells expressed high levels of granzymes in comparison to allo- or xenoreactive CD8+ T cells. Adoptive transfer of DNT cells in combination with early immune suppression by immunosuppressive analog of 15-deoxyspergualin, LF15-0195, significantly prolonged rat heart graft survival to 62.1 +/- 13.9 days in mice recipients. In conclusion, this study suggests that xenoreactive DNT cells can control B and T cell responses in perforin/granzyme-dependent mechanisms. DNT cells may be valuable in controlling B and T cell responses in xenotransplantation.

Combination of low-dose cisplatin and recombinant xenogeneic endoglin as a vaccine induces synergistic antitumor activities.

Angiogenesis is critical to the growth and metastasis of solid tumors, and acquired drug resistance is one of the major hindrances to chemotherapy. Thus, we sought a rational strategy using the combination of antiangiogenic biotherapy and chemotherapy for cancer therapy. We explored the efficacy of a strategy combining low-dose cisplatin and a recombinant xenogeneic endoglin as a protein vaccine, which we previously demonstrated to have effective antiangiogenic effects in several mouse models. We found that both low-dosage cisplatin and xenogeneic endoglin vaccine individually resulted in effective suppression of tumor growth in 2 tumor models via inhibition of tumor angiogenesis. Remarkably, the combination therapy resulted in not only significant antiangiogenic effects but also additional promotion of tumor cell apoptosis and inhibition of tumor cell proliferation, without any ensuing increase in host toxicity during the course of treatment, which lasted for 6 months. In addition, the combination demonstrated a synergistic relationship, which was shown in all of the synergistic indexes, i.e., tumor volume, angiogenesis, apoptosis and proliferation. Both antibodies and antibody-producing B cells against mouse self-endoglin were observed in all mice immunized by the xenogeneic endoglin vaccine (alone and combination), which suggested that low-dose cisplatin did not suppress the host immune response but potentiated the antitumor activity of the xenogeneic endoglin vaccine. These observations may provide the basis for an effective alternative strategy for cancer therapy in the near future.

Direct antigen presentation by a xenograft induces immunity independently of secondary lymphoid organs.

The location of immune activation is controversial during acute allograft rejection and unknown in xenotransplantation. To determine where immune activation to a xenograft occurs, we examined whether splenectomized alymphoplastic mice that possess no secondary lymphoid organs can reject porcine skin xenografts. Our results show that these mice rejected their xenografts, in a T cell-dependent fashion, at the same tempo as wild-type recipients, demonstrating that xenograft rejection is not critically dependent on secondary lymphoid organs. Furthermore, we provide evidence that immune activation in the bone marrow did not take place during xenograft rejection. Importantly, immunity to xenoantigens was only induced after xenotransplantation and not by immunization with porcine spleen cells, as xenografted mutant mice developed an effector response, whereas mutant mice immunized by porcine spleen cells via i.p. injection failed to do so. Moreover, we provide evidence that antixenograft immunity occurred via direct and indirect Ag presentation, as recipient T cells could be stimulated by either donor spleen cells or recipient APCs. Thus, our data provide evidence that direct and indirect Ag presentation by a xenograft induces immunity in the absence of secondary lymphoid organs. These results have important implications for developing relevant xenotransplantation protocols.

Generation and regulation of human Th1-biased immune responses in vivo: a critical role for IL-4 and IL-10.

Tissue damage in many human autoimmune diseases is mediated by activated autoantigen-specific Th1 cells. Delineation of the regulatory mechanisms controlling a Th1-biased human immune reaction and its pathologic potential is, therefore, a critical step in the understanding of autoimmune diseases. In this study, we introduce a novel means to investigate human Th1-biased immune responses in vivo. Intraperitoneal injection of human mononuclear cells into immunodeficient mice generates a xenogeneic Th1-biased human immune response characterized by systemic inflammation and leukocytic infiltrates with a granuloma-like architecture in the liver, and the perigastrointestinal and perirenal fatty tissue. Th1 cell activation was dependent on the presence of APCs and could be blocked by cyclosporine. Importantly, neutralization of endogenously produced IL-4 and IL-10 markedly exaggerated the immune response, whereas exogenous IL-4 and IL-10 inhibited systemic Th1 immunity. Thus, the model described in this paper presents a useful means to analyze the regulation of human immune reactions in an in vivo situation. The results suggest that both IL-4 and IL-10 contribute to controlling the development of a human Th1-biased immune reaction.

A single heteroclitic epitope determines cancer immunity after xenogeneic DNA immunization against a tumor differentiation antigen.

Successful active immunization against cancer requires induction of immunity against self or mutated self Ags. However, immunization against self Ags is difficult. Xenogeneic immunization with orthologous Ags induces cancer immunity. The present study evaluated the basis for immunity induced by active immunization against a melanoma differentiation Ag, gp100. Tumor rejection of melanoma was assessed after immunization with human gp100 (hgp100) DNA compared with mouse gp100 (mgp100). C57BL/6 mice immunized with xenogeneic full-length hgp100 DNA were protected against syngeneic melanoma challenge. In contrast, mice immunized with hgp100 DNA and given i.p. tolerizing doses of the hgp100 D(b)-restricted peptide, hgp100(25-33), were incapable of rejecting tumors. Furthermore, mice immunized with DNA constructs of hgp100 in which the hgp100(25-27) epitope was substituted with the weaker D(b)-binding epitope from mgp100 (mgp100(25-27)) or a mutated epitope unable to bind D(b) did not reject B16 melanoma. Mice immunized with a minigene construct of hgp100(25-33) rejected B16 melanoma, whereas mice immunized with the mgp100(25-33) minigene did not develop protective tumor immunity. In this model of xenogeneic DNA immunization, the presence of an hgp100 heteroclitic epitope with a higher affinity for MHC created by three amino acid (25 to 27) substitutions at predicted minor anchor residues was necessary and sufficient to induce protective tumor immunity in H-2(b) mice with melanoma.

Human serum induces apoptosis of xenogenic cardiomyocytes in vivo and in vitro.

Discordant xenotransplantation is complicated by delayed xenograft rejection (DXR). Previous studies have demonstrated that anti-apoptotic genes are protective against DXR. This study examines the hypothesis that apoptosis plays a role in human anti-xenograft responses. C57BL/6 mice and NOD SCID mice were given a single intravenous injection of either a lethal dose (LD, survival < 30 min) or a sublethal dose (SLD) of human serum, and isolated pig and mouse rod-shaped cardiomyocytes were exposed to human serum in vitro. In situ detection of apoptotic cells in mouse hearts was assessed using a terminal deoxynucleotidyl transferase-mediated dUTP nicked-end labeling assay. Mice transfused with human serum had approximately a 10-fold increased percentage of apoptotic cells after SLD 18 h post-injection compared with animals given saline, and a fourfold increase over LD. Administration of cobra venom factor (CVF) decomplemented SLD 18 h did not significantly (P > 0.05) alter the percentage apoptosis. The addition of 20 mM Gal-alpha-1,3-Gal to SLD 18 h significantly (P < 0.05) reduced percentage apoptosis to levels comparable to saline treated control animals. In vitro using mouse and pig cardiomyocytes demonstrated parallel results as in vivo experiments. Human serum induces apoptosis of cardiomyocytes in immunocompetent and immunoincompetent mice in vivo, as well as mouse and pig cardiomyocytes in vitro. Further, this apoptotic response can be inhibited by the addition of Gal-alpha-1,3-Gal without affecting the capacity of the serum to cause HAR. These results demonstrate that a putative human serum factor induces a delayed apoptotic injury of xenograft tissues, and supports the hypothesis that apoptosis may be an important mediator of DXR.

Intrathymic inoculation of donor antigen: an ineffective strategy for prolonging xenograft survival.

The aim of this study was to determine whether intrathymic inoculation of the recipient with donor antigen and short-term depletion of peripheral lymphocytes would lead to donor-specific unresponsiveness to rat pancreatic islet xenografts. The results were compared directly with an allograft model to determine whether there were substantial differences in the mechanisms of graft prolongation between allografts and xenografts using an identical conditioning regimen. Streptozotocin-induced diabetic C57B6 mice were injected with up to 0.3 ml of rat anti-mouse lymphocyte serum (ALS) 1 day before intrathymic injection of donor splenocytes. DA rat islet xenografts or Balb/c mouse islet allografts were transplanted 3 days later. ALS depleted CD3+ and CD4+ peripheral blood T lymphocytes to less than 5% of values in control mice by 24 h. Median islet xenograft survival (MGS) was 9 days in untreated mice, 28 days in mice receiving 0.2 ml of ALS and 32 days in mice receiving 0.3 ml of ALS alone. Intrathymic injection of 5 x 10(6) DA splenocytes plus 0.2 ml of ALS did not improve islet xenograft survival beyond 28 days. Increasing the intrathymic inoculum to 10(7) DA splenocytes with or without a higher dose of ALS (0.3 ml) did not increase MGS beyond 26 days, although 2 out of 18 animals survived beyond 100 days. These long-term surviving mice rejected a second DA rat islet graft in less than 22 days, indicating that tolerance was not achieved. To confirm the efficacy of this treatment regimen in allotransplantation, diabetic C57B6 mice received 10(7) Balb/c splenocytes intrathymically and 0.3 ml of ALS. A Balb/c islet allograft was performed 3 days later. Allograft survival was similar to that of rat islet xenografts with 40%, (4 out of 10) of grafts surviving beyond 100 days. In contrast to the xenograft recipients, a second Balb/c islet allograft survived indefinitely, indicating that tolerance was achieved. Histology of the long-surviving allografts showed intact islets with a sparse cellular infiltrate, whereas the long-surviving xenografts (> 100 days) showed a large cellular infiltrate and significant islet destruction. To investigate further the role of the thymus, adult thymectomized C57B6 mice were treated with 0.3 ml of ALS and received a DA rat islet xenograft. The median graft survival was 52 days and no graft survived beyond 80 days, suggesting that peripheral xenoreactive T cells remained after ALS treatment and greater T-cell depletion was necessary to obtain permanent engraftment. These results show that peripheral xenoreactive T cells which remain after profound T-cell depletion are capable of rejecting an islet xenograft despite intrathymic inoculation of donor antigen. The T-cell-mediated xenograft response appears to be stronger and more difficult to suppress than the allograft response using this strategy.

Isotype-specific immunoregulation. Evidence for a distinct subset of T contrasuppressor cells for IgA responses in murine Peyer's patches.

The ability of murine Peyer's patch (PP) T contrasuppressor cells (Tcs) to reverse oral tolerance to the T cell-dependent (TD) antigen SRBC was studied both in vivo and in vitro. C3H/HeJ mice given SRBC orally for 4 wk are not rendered tolerant to this antigen and were used as a source of PP Tcs cells for adoptive transfer to identically treated, orally tolerized C3H/HeN mice. Transfer of 10(4) or 5 X 10(4) V. villosa-adherent PP T cells resulted in splenic IgM, IgG, and mainly IgA responses in C3H/HeN mice challenged systemically with SRBC. The T cell responsible was Lyt-1+, 2-, L3T4-, I-JK+ and V. villosa lectin-adherent, all characteristics of mature effector Tcs cells. This C3H/HeJ PP Tcs cell subset was also effective when added to in vitro cultures of tolerized spleen cells derived from SRBC-fed, C3H/HeN mice. Interestingly, C3H/HeJ PP Tcs cells restored mainly IgA responses when transferred in vivo or when added to suppressed C3H/HeN splenic cultures. Comparison of the functional activity of Tcs cells derived from spleen or PP of orally immunized C3H/HeJ mice revealed that splenic Tcs cells supported responses of all 3 isotypes; however, PP Tcs cells yielded three-fourfold higher IgA responses, when compared with IgM or IgG anti-SRBC responses. Adherence of C3H/HeJ PP Tcs to an Fc alpha R+ T cell line derived from IgA-specific Th cells resulted in a nonadherent cell fraction that potentiated only IgM and IgG responses, while bound Tcs cells preferentially supported IgA responses. These results suggest that murine PP contain IgA-specific Tcs cells that allow IgA response induction in the presence of Ts cells that mediate oral tolerance.