In utero exposure to alloantigens primes alloimmunization to platelet transfusion in mice.

BACKGROUND: Platelet transfusions remain a mainstay of treatment for many patients with thrombocytopenia, but can lead to alloantibodies to Human Leukocyte Antigens (anti-HLA) resulting in inadequate responses to subsequent platelet transfusions (refractoriness), as well as complicate transplantation. Despite substantial decreases in alloimmunization with the implementation of leukoreduction, a significant percentage of patients still become alloimmunized following platelet transfusions. It remains unclear why some patients make anti-HLA antibodies, but others do not make anti-HLA antibodies even with chronic transfusion. Antecedent pregnancy correlates with risk of alloimmunization due to platelet transfusion in humans - however, isolation of pregnancy as a single variable is not possible in human populations. STUDY DESIGN AND METHODS: A tractable murine model of pregnancy and transfusion was engineered by breeding C57BL/6 (H-2b ) dames with BALB/c (H-2d ) sires. After pregnancy, female mice were transfused with leukoreduced platelets from F1 (H-2b/d ) donors that expressed the same paternal major histocompatibility complex (MHC) H-2d alloantigens as the sires. Control groups allowed isolation of pregnancy or transfusion alone as independent variables. Alloimmunization was determined by testing serum for antibodies to H-2d MHC alloantigens. RESULTS: No alloantibodies were detected after pregnancy alone, or in response to transfusion of platelets alone; however, significant levels of alloantibodies were detected when pregnancy was followed by transfusion. CONCLUSIONS: These findings isolate antecedent pregnancy as a causal contribution to increased frequencies of alloimmunization by subsequent platelet transfusion in mice and provide a platform for ongoing mechanistic investigation.

Alloimmunogenicity of an isolated MHC allele is affected by the context of MHC mismatch in a murine model.

BACKGROUND: Humoral alloimmunization to human leukocyte antigen (HLA) can represent a barrier to solid-organ transplantation, can lead to a refractory state in patients requiring platelet transfusion, and can also contribute to transfusion-related acute lung injury (TRALI). While exposure to HLA-mismatched cells/tissues are generally required for HLA alloimmunization, the effect of the extent of major histocompatibility complex (MHC) mismatch between donor and recipient is poorly understood. STUDY DESIGN AND METHODS: A novel mouse was generated that allows the expression of a single MHC Class I alloantigen, Kd . Alloimmune responses to Kd were studied in C57BL/6 mice transfused with splenocytes from different donor mice, allowing the analysis of responses to Kd as an isolated alloantigen, or in the context of additional mismatched MHC molecules. Advanced tools were utilized to study responses to Kd , including T-cell receptor transgenic mice that recognize the immunodominant Kd peptide presented by C57BL/6 mice to CD4+ T cells. RESULTS: A single MHC Class I alloantigen mismatch is less immunogenic than when the same alloantigen is encountered in the context of additional mismatched MHC alloantigens. This difference is due, at least in part, to induction of CD4+ helper T cells, as the effect is overcome by increasing either mature CD4+ T-cell help through immunization or by increasing the precursor frequency of naïve CD4+ T cells by adoptive transfer from T-cell receptor transgenic donors. CONCLUSION: These findings indicate that the immunogenicity of a single alloantigen can be affected by the context in which it is encountered, demonstrating the potential for cooperative effects between different mismatched MHC alloantigens.

Transfusion-induced alloimmunization and platelet refractoriness in a mouse model: mechanisms and interventions.

BACKGROUND: Platelet (PLT) transfusions can be an essential therapy for patients with thrombocytopenia to maintain hemostasis. However, some patients become alloimmunized to antigens on PLTs (typically HLA), which can prevent efficacy of PLT transfusion due to antibody-mediated clearance. In extreme cases, patients with alloimmunization to multiple HLAs can become "refractory" to PLT transfusion, such that insufficient compatible PLT units can be found to meet transfusion needs. MATERIALS AND METHODS: An in vivo murine model of PLT-induced alloimmunization was refined so as to include both transfusion with allogeneic leukoreduced PLTs and studies of posttransfusion PLT recoveries, allowing assessment of alloimmunization and refractoriness. Basic mechanisms of antibody-mediated PLT clearance were investigated using recipients missing either the C3 complement gene or the common gamma chain for Fc receptors. In addition, the efficacy of using costimulatory blockade as a therapeutic intervention was assessed by testing CTLA4-Ig administration before PLT transfusion. RESULTS: Fcγ receptors (but not complement C3) are required for alloantibody-mediated PLT refractoriness. In addition, levels of anti-MHC predict the extent of refractoriness in a given animal. Finally, costimulatory blockade as a therapeutic modality prevents transfusion-induced PLT refractoriness. CONCLUSIONS: Together these findings introduce new experimental methods, basic mechanistic understanding, and a potential therapeutic intervention for alloimmunization to MHC-based antigens on transfused PLTs.

Metabolites in stored platelets associated with platelet recoveries and survivals.

BACKGROUND: Transfusion of platelets (PLTs) is a common therapy in a number of clinical settings. However, it is well understood that there is substantial donor-to-donor variation in how well PLTs store and thus the quality of the products that are transfused. The basis of such variation is poorly understood, and there are limited metrics by which units of PLTs can be assessed for their posttransfusion performance. It has repeatedly been demonstrated that myriad biologic changes take place during PLT storage; however, which of the changes correlate with quality of the stored PLTs and/or are mechanistically involved in PLT function remains undetermined. STUDY DESIGN AND METHODS: The current study tested stored PLTs from 21 normal subjects, combining high-resolution metabolomics of stored PLTs with in vivo PLT recoveries and survivals. Both individual analytes and metabolic pathways that correlate with posttransfusion PLT viability were identified. RESULTS: Caffeine metabolites were associated with poor PLT recovery; caffeine metabolism was not ongoing in the PLT bag and remained at prestorage levels. Acylcarnitines, particular fatty acid metabolites, and oxidized fatty acids were associated with poor PLT survivals. Of the myriad metabolic changes during PLT storage, these are the first reported metabolic findings to begin distinguishing which changes are of functional importance regarding posttransfusion PLT performance. CONCLUSIONS: Together, these findings provide novel mechanistic insights into the functional biology of the PLT storage lesion as well as identifying potential targets for modifying donor environment (e.g., caffeine consumption) and also metrics of quality assessment for stored human PLTs.

Innate B-1 B Cells Are Not Enriched in Red Blood Cell Autoimmune Mice: Importance of B Cell Receptor Transgenic Selection.

Autoimmune hemolytic anemia (AIHA) results from breakdown of humoral tolerance to RBC antigens. Past analyses of B-cell receptor transgenic (BCR-Tg) mice that recognize RBC autoantigens led to a paradigm in which autoreactive conventional B-2 B cells are deleted whereas extramedullary B-1 B cells escape deletion due to lack of exposure to RBCs. However, BCR-Tg mice utilized to shape the current paradigm were unable to undergo receptor editing or class-switching. Given the importance of receptor editing as mechanism to tolerize autoreactive B cells during central tolerance, we hypothesized that expansion of autoreactive B-1 B cells is a consequence of the inability of the autoreactive BCR to receptor edit. To test this hypothesis, we crossed two separate strains of BCR-Tg mice with transgenic mice expressing the BCR target on RBCs. Both BCR-Tg mice express the same immunoglobulin and, thus, secrete antibodies with identical specificity, but one strain (SwHEL) has normal receptor editing, whereas the other (IgHEL) does not. Similar to other AIHA models, the autoreactive IgHEL strain showed decreased B-2 B cells, an enrichment of B-1 B cells, and detectable anti-RBC autoantibodies and decreased RBC hematocrit and hemoglobin values. However, autoreactive SwHEL mice had induction of tolerance in both B-2 and B-1 B cells with anti-RBC autoantibody production without anemia. These data generate new understanding and challenge the existing paradigm of B cell tolerance to RBC autoantigens. Furthermore, these findings demonstrate that immune responses vary when BCR-Tg do not retain BCR editing and class-switching functions.

Antibodies to Senescent Antigen and C3 Are Not Required for Normal Red Blood Cell Lifespan in a Murine Model.

Red blood cells (RBCs) have a well-defined lifespan, indicating a mechanism by which senescent cells of a certain age are removed from circulation. However, the specifics by which senescent cells are recognized and removed are poorly understood. There are multiple competing hypotheses for this process, perhaps the most commonly cited is that senescent RBCs expose neoantigens [or senescent antigen(s)] that are then recognized by naturally occurring antibodies, which opsonize the senescent cells and result in clearance from circulation. While there are a large volume of published data to indicate that older RBCs accumulate increased levels of antibody on their surface, to the best of our knowledge, the causal role of such antibodies in clearance has not been rigorously assessed. In the current report, we demonstrate that RBC lifespan and clearance patterns are not altered in mice deficient in antibodies, in C3 protein, or missing both. These data demonstrate that neither antibody nor C3 is required for clearance of senescent RBCs, and questions if they are even involved, in a murine model of RBC lifespan.

Regulatory T Cells Are Dispensable for Tolerance to RBC Antigens.

Autoimmune hemolytic anemia (AIHA) occurs when pathogenic autoantibodies against red blood cell (RBC) antigens are generated. While the basic disease pathology of AIHA is well studied, the underlying mechanism(s) behind the failure in tolerance to RBC autoantigens are poorly understood. Thus, to investigate the tolerance mechanisms required for the establishment and maintenance of tolerance to RBC antigens, we developed a novel murine model. With this model, we evaluated the role of regulatory T cells (Tregs) in tolerance to RBC-specific antigens. Herein, we show that neither sustained depletion of Tregs nor immunization with RBC-specific proteins in conjunction with Treg depletion led to RBC-specific autoantibody generation. Thus, these studies demonstrate that Tregs are not required to prevent autoantibodies to RBCs and suggest that other tolerance mechanisms are likely involved.

Regulation of CD8+ cytolytic T lymphocyte differentiation by a cholinergic pathway.

In this report, we provide evidence that muscarinic receptors play a role in the generation of CD8+ cytolytic T lymphocytes. Analysis of mice with targeted deletions of each of the known muscarinic receptors (M1-M5) showed that CD8+ T cells from M1 receptor-deficient mice had a defect in the ability to differentiate into cytolytic T lymphocytes. Additional pharmacological experiments support the role of muscarinic receptors in wild type mice and suggest that acetylcholine may be involved. Together, these findings suggest that the M1 muscarinic receptor is involved in CTL development, thus providing novel insights into CD8+ T cell biology and the potential role of cholinergic signaling in immune regulation.

Inhibition of cellular immune responses to encapsulated porcine islet xenografts by simultaneous blockade of two different costimulatory pathways.

BACKGROUND: Transplantation of human islets has been successful clinically. Since human islets are scarce, we are studying microencapsulated porcine islet xenografts in nonobese diabetic (NOD) mice. We have evaluated the cellular immune response in NOD mice with and without dual costimulatory blockade. METHODS: Alginate-poly-L-lysine-encapsulated adult porcine islets were transplanted i.p. in untreated diabetic NODs and NODs treated with CTLA4-Ig to block CD28/B7 and with anti-CD154 mAb to inhibit CD40/CD40-ligand interactions. Groups of mice were sacrificed on subsequent days; microcapsules were evaluated by histology; peritoneal cells were analyzed by FACS; and peritoneal cytokines were quantified by ELISA. Controls included immunoincompetent NOD-Scids and diabetic NODs given sham surgery or empty microcapsules. RESULTS: Within 20 days, encapsulated porcine islets induced accumulation of large numbers of macrophages, eosinophils, and significant numbers of CD4 and CD8 T cells at the graft site, and all grafts were rejected. During rejection, IFNgamma, IL-12 and IL-5 were significantly elevated over sham-operated controls, whereas IL-2, TNFalpha, IL-4, IL-6, IL-10, IL-1beta and TGFbeta were unchanged. Treatment with CTLA4-Ig and anti-CD154 prevented graft destruction in all animals during the 26 days of the experiment, dramatically inhibited recruitment of host inflammatory cells, and inhibited peritoneal IFNgamma and IL-5 concentrations while delaying IL-12 production. CONCLUSIONS: When two different pathways of T cell costimulation were blocked, T cell-dependent inflammatory responses were inhibited, and survival of encapsulated islet xenografts was significantly prolonged. These findings suggest synergy between encapsulation of donor islets and simultaneous blockade of two host costimulatory pathways in prolonging xenoislet transplant survival.

Gammadelta T cells play an essential role in several forms of tolerance.

Gammadelta T cells were discovered in the mid-1980s, but the antigens they recognize and the biological functions they mediate are poorly understood. Although gammadelta T cells have the capacity to augment immunity to certain infections and kill certain tumor cells, they are generally not required for development of antibody responses, for graft rejection, or for development of autoimmune diseases. Nevertheless, gammadelta T cells accumulate at sites of inflammation induced by infection, tumor growth, and autoimmune lesions, where they have been shown to reduce the inflammatory reaction and tissue damage. In this review, we summarize the evidence that gammadelta T cells play an important role in the induction of various forms of tolerance.

Antigen, in the presence of TGF-beta, induces up-regulation of FoxP3gfp+ in CD4+ TCR transgenic T cells that mediate linked suppression of CD8+ T cell responses.

CD4(+)CD25(+) regulatory T cells (Tregs) inhibit immune responses to a variety of Ags, but their specificity and mechanism of suppression are controversial. This controversy is largely because many studies focused on natural Tregs with undefined specificities and suppression has frequently been measured on polyclonal T cell responses. To address the issue of specificity further, we have bred K(d)-specific, CD4(+) TCR (TCR75) transgenic mice to Foxp3(gfp) knockin reporter mice to permit sorting of Tregs with a known specificity. Foxp3(gfp).TCR75 mice did not express significant numbers of natural FoxP3(+) Tregs expressing the TCR75 transgenes, but FoxP3 expression was induced by stimulating with K(d) plus TGF-beta. The resulting GFP(+) TCR75 cells were anergic, whereas the GFP(-) TCR75 cells proliferated upon restimulation with K(d) peptide. Yet both exhibited severely reduced expression of intracellular IFN-gamma and TNF-alpha upon restimulation. GFP(+), but not GFP(-), TCR75 T cells suppressed responses by naive TCR75 T cells and by nontransgenic spleen cells stimulated with anti-CD3. GFP(+) TCR75 cells also inhibited polyclonal C57BL/6 anti-K(d) CTL responses if the APC expressed K(d) and both MHC class I and class II, and responses by OT1 T cells to B6.K(d).OVA but not B6.K(d) plus OVA expressing APC, demonstrating linked-suppression of CD8 responses. Thus, Tregs exhibit a greater degree of specificity in vitro than previously appreciated. The observation that Tregs and responder T cells must recognize the same APC provides a mechanistic explanation for the observation that Tregs must be in direct contact with effector T cells to suppress their responses.

TCR transgenic CD8+ T cells activated in the presence of TGFbeta express FoxP3 and mediate linked suppression of primary immune responses and cardiac allograft rejection.

Although CD4+CD25+FoxP3+ regulatory T cells play a role in allograft tolerance, the role of CD8+ cells with immunosuppressive function is less clear. To address this issue, spleen cells from Rag-1-deficient TCR transgenic (Tg) mice expressing a receptor for ovalbumin (OVA) in the context of MHC class I (OT1) were activated with OVA expressing antigen-presenting cell (APC) in the presence or absence of exogenous transforming growth factor beta (TGFbeta). TGFbeta inhibited the expression of IFN-gamma, granzyme B and the lytic activity of the OT1 T cells while inducing FoxP3 expression in 5-15% of the cells. By contrast, FoxP3 expression was not detected in naive OT-1 T cells or OT-1 T cells activated without exogenous TGFbeta. TGFbeta-activated OT1 cells inhibited the activation of Kd-specific CD8+ CTL responses by normal B6 T cells and the proliferation by Kd-specific CD4+ TCR Tg T cells, but only if the OVA epitope was co-expressed by Kd+ APC. This antigen-specific inhibitory activity, referred to as linked suppression, was neither mediated by residual lytic activity within the activated OT1 T cells nor did it depend upon IL-10 or TGFbeta. Suppression correlated with inhibition of CD86 expression on CD11c+ APC. TGFbeta-activated OT1 T cells also delayed the rejection of heterotopic, vascularized cardiac allografts mediated by anti-Kd-specific CD4+ TCR Tg T cells, but only if the cardiac allograft expressed both OVA and Kd as transgenes. Prolonged survival of allografts was associated with rapid migration of the FoxP3+ OT1 T cells into the donor heart raising the possibility that suppression may be mediated within the allograft. These data show that TGFbeta-activated CD8+ T cells mediate antigen-specific, APC-focused patterns of suppression in vitro and in vivo.

Inhibition of tumor rejection by gammadelta T cells and IL-10.

Although many tumors express tumor-specific antigens, most fail to stimulate effective immune responses. Tumors generally lack co-stimulatory molecules, which can lead to tolerance of tumor-specific T cells and progressive tumor growth. Here, we demonstrate that the ovalbumin (OVA) transfected EL4 tumor, E.G7-OVA, grows progressively in syngeneic mice even though the tumor can be rejected if the mice are immunized with OVA in adjuvant. E.G7-OVA grew more rapidly in RAG-1 deficient than sufficient mice suggesting that normal mice make an abortive immune response to this tumor. Depletion of gammadelta T cells or IL-10 augmented the ability of B6 mice to reject E.G7-OVA. Spleen cells from normal, but not IL-10 knockout, mice reconstituted rapid tumor growth in gammadelta T cell-deficient mice. Thus, gammadelta T cells play an important role in preventing immune elimination of this tumor by a mechanism that directly or indirectly involves IL-10.

gammadelta T-cell clones from intestinal intraepithelial lymphocytes inhibit development of CTL responses ex vivo.

Oral administration of antigen induces a state of tolerance that is associated with activation of CD8+ T cells that can transfer unresponsiveness to naïve syngeneic hosts. These T cells are not lytic, but they inhibit development of antibody, CD4+ T helper cell, and CD8+ cytotoxic T lymphocyte (CTL) responses upon adoptive transfer into naïve, syngeneic mice. In addition, we have shown that depletion of gammadelta T cells by injection of the anti-delta chain antibody (GL3) down modulates the expression of gammadelta T-cell receptor (TCR) and inhibits the induction of oral tolerance to ovalbumin. Oral administration of antigen also fails to induce tolerance in TCR delta-chain knockout mice suggesting that gammadelta T cells play a critical, active role in tolerance induced by orally administered antigen. To further study the contribution of gammadelta T cells to tolerance, murine gammadelta T cells were isolated from intraepithelial lymphocytes (IEL) of the small intestine by stimulation with splenic filler cells, concanavalin A and growth factors. gammadelta IEL lines demonstrated lytic activity in a redirected lysis assay. gammadelta T-cell clones express different gammadelta TCR genes and secrete large amounts of interleukin (IL)-10, but little or no IL-2, IL-4, or interferon-gamma. gammadelta IEL clones expressed transforming growth factor-beta1 and macrophage migration inhibitory factor, as well as IL-10, mRNA. Moreover, gammadelta T-cell clones potently inhibited the generation of CTL responses by secreted molecules rather than by direct cell-to-cell contact.

CD75s is a marker of murine CD8(+) suppressor T cells.

We have previously described a monoclonal antibody, 984, which specifically recognizes murine CD8(+) suppressor T cells (Ts) but not CD8(+) cytolytic T lymphocytes (CTLs). Removal of 984(+) cells abrogates the suppressive effect of CD8(+) Ts generated either in vivo or in vitro while having no effect upon CTL. In this report, the molecules recognized by 984 are identified as 2-6 sialylated neolacto series gangliosides, which are members of the newly defined CD75s cluster. We proceed to demonstrate that like 984, a separate anti-CD75s antibody (CRIS-4), recognizes primary CD8(+) Ts cells. In addition, the 2,6 sialyltransferase responsible for the synthesis of the 984 epitope is identified, allowing the manipulation and study of the regulation of this epitope. This is the first report of CD75s on murine cells and the first report that delineates lymphocyte function based upon CD75s expression. In addition to contributing to the growing body of evidence that lineage dependent gangliosides are expressed by T lymphocytes, these findings suggest that CD8(+) CD75s(+) T lymphocytes represent a functionally distinct subset of CD8(+) T cells with negative regulatory function.