Linda M. Kapp

γδ 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 γδ T cells by injection of the anti‐δ chain antibody (GL3) down modulates the expression of γδ T‐cell receptor (TCR) and inhibits the induction of oral tolerance to ovalbumin. Oral administration of antigen also fails to induce tolerance in TCR δ‐chain knockout mice suggesting that γδ T cells play a critical, active role in tolerance induced by orally administered antigen. To further study the contribution of γδ T cells to tolerance, murine γδ T cells were isolated from intraepithelial lymphocytes (IEL) of the small intestine by stimulation with splenic filler cells, concanavalin A and growth factors. γδ IEL lines demonstrated lytic activity in a redirected lysis assay. γδ T‐cell clones express different γδ TCR genes and secrete large amounts of interleukin (IL)‐10, but little or no IL‐2, IL‐4, or interferon‐γ. γδ IEL clones expressed transforming growth factor‐β1 and macrophage migration inhibitory factor, as well as IL‐10, mRNA. Moreover, γδ T‐cell clones potently inhibited the generation of CTL responses by secreted molecules rather than by direct cell‐to‐cell contact.

γδ T cells play an essential role in several forms of tolerance

Abstractγδ T cells were discovered in the mid-1980s, but the antigens they recognize and the biological functions they mediate are poorly understood. Although γδ 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, γδ 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 γδ T cells play an important role in the induction of various forms of tolerance.

Inhibition of tumor rejection by γδ 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 γδ 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 γδ T cell-deficient mice. Thus, γδ T cells play an important role in preventing immune elimination of this tumor by a mechanism that directly or indirectly involves IL-10.

Antigen, in the Presence of TGF-β, 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 Kd-specific, CD4+ TCR (TCR75) transgenic mice to Foxp3gfp knockin reporter mice to permit sorting of Tregs with a known specificity. Foxp3gfp.TCR75 mice did not express significant numbers of natural FoxP3+ Tregs expressing the TCR75 transgenes, but FoxP3 expression was induced by stimulating with Kd plus TGF-β. The resulting GFP+ TCR75 cells were anergic, whereas the GFP− TCR75 cells proliferated upon restimulation with Kd peptide. Yet both exhibited severely reduced expression of intracellular IFN-γ and TNF-α 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-Kd CTL responses if the APC expressed Kd and both MHC class I and class II, and responses by OT1 T cells to B6.Kd.OVA but not B6.Kd 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.

Metabolites in stored platelets associated with platelet recoveries and survivals

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.

Mechanisms of alloimmunization and subsequent bone marrow transplantation rejection induced by platelet transfusion in a murine model.

For many nonmalignant hematological disorders, HLA‐matched bone marrow transplantation (BMT) is curative. However, due to lack of neoplasia, the toxicity of stringent conditioning regimens is difficult to justify, and reduced intensity conditioning is used. Unfortunately, current reduced intensity regimens have high rates of BMT rejection. We have recently reported in a murine model that mHAs on transfused platelet products induce subsequent BMT rejection. Most nonmalignant hematological disorders require transfusion support prior to BMT and the rate of BMT rejection in humans correlates with the number of transfusions given. Herein, we perform a mechanistic analysis of platelet transfusion‐induced BMT rejection and report that unlike exposure to alloantigens during transplantation, platelet transfusion primes alloimmunity but does not stimulate full effector function. Subsequent BMT is itself an additional and distinct immunizing event, which does not induce rejection without antecedent priming from transfusion. Both CD4+ and CD8+ T cells are required for priming during platelet transfusion, but only CD8+ T cells are required for BMT rejection. In neither case are antibodies required for rejection to occur.

Serological blind spots for variants of human IgG3 and IgG4 by a commonly used anti-immunoglobulin reagent.

Human immunoglobulin G (IgG) includes four different subtypes (IgG1, IgG2, IgG3, and IgG4), and it is also now appreciated that there are genetic variations within IgG subtypes (called isoallotypes). Twenty‐nine different isoallotypes have been described, with 7, 4, 15, and 3 isoallotypes described for IgG1, IgG2, IgG3, and IgG4, respectively. The reactivity of anti‐IgG with different isoallotypes has not been characterized.

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.

Common murine immunoglobulin detection reagents have diminished reactivity with IgG3 – A vulnerability to misinterpretation

Methods designed to monitor humoral immune responses, in a variety of settings, typically use a broadly reactive detection reagent (e.g. polyclonal anti-Ig (immunoglobulin)) in order to characterize antibody responses. In the context of murine models of immunity, which are widely used, this would typically be antisera to mouse Ig or mouse IgG. However, there are 4 different subtypes of mouse IgG; thus, the validity of the above approach, as a general screen for humoral immune responses, depends upon the assumption that the antisera recognize all IgG subtypes. This seems like a reasonable assumption, since polyclonal antisera recognize multiple epitopes; however, herein we report that two commercial sources of goat anti-mouse Ig are hyporeactive with IgG3. Given that relative IgG3 levels are different in distinct types of immune response, these findings demonstrate a potential for misinterpretation, and suggest a need to modify immunological methods in this context.

Identification of IgG3-specific epitope that remedies problem in diagnostic IgG subclass determination due to human genetic variation

There are four subtypes of human IgG with different effector functions. Quantifying the relative amount of each IgG subtype is important for laboratory diagnosis in multiple settings. However, in an evolving landscape of the appreciation of human variability and the need for precision/personalised laboratory diagnosis, it has also been shown that there are numerous natural variants of IgG subtypes, with at least 29 having been described. It has recently been reported that commercially available polyclonal antisera to IgG3 cross react with variants of other IgG subtypes, while available monoclonal anti-IgG3 have a blind-spot for the IgG3-04 variant. Herein, we report that IgG3-04 contains an epitope in common with all known IgG3 variants and absent in other subtypes. A novel monoclonal anti-IgG3 is described that is specific to IgG3 but without any blind-spots for known IgG3 variants, providing a remedy to the problem of genetic variability of IgG3.