Hamid Bassiri

Indefinite survival of MHC class I-deficient murine pancreatic islet allografts.

To examine the immune response to class I-deficient allogeneic tissue, we used beta 2-microglobulin-deficient mice as graft donors. These mice lack cell surface class I major histocompatibility complex antigen expression. Pancreatic islet allografts from class I-deficient donors survived indefinitely in a majority of fully allogeneic BALB/c recipients. In contrast, host recognition of graft class I antigen was unnecessary for prompt destruction of skin allografts of for autoimmune damage of transplanted pancreatic islet grafts in nonobese diabetic mice. These studies provide evidence that intentional genetic elimination of immunologically relevant donor antigens may prove an effective strategy for preventing allograft rejection.

A Requirement for IL-2/IL-2 Receptor Signaling in Intrathymic Negative Selection

The nature of the signals that influence thymocyte selection and determine the fate of CD4+8+ (double positive) thymocytes remains unclear. Cytokines produced locally in the thymus may modulate signals delivered by TCR-MHC/peptide interactions and thereby influence the fate of double-positive thymocytes. Because the IL-2/IL-2R signaling pathway has been implicated in thymocyte and peripheral T cell survival, we investigated the possibility that IL-2/IL-2R interactions contribute to the deletion of self-reactive, Ag-specific thymocytes. By using nontransgenic and transgenic IL-2-sufficient and -deficient animal model systems, we have shown that during TCR-mediated thymocyte apoptosis, IL-2 protein is expressed in situ in the thymus, and apoptotic thymocytes up-regulate expression of IL-2Rs. IL-2R+ double-positive and CD4 single-positive thymocytes undergoing activation-induced cell death bind and internalize IL-2. IL-2-deficient thymocytes are resistant to TCR/CD3-mediated apoptotic death, which is overcome by providing exogenous IL-2 to IL-2−/− mice. Furthermore, disruption or blockade of IL-2/IL-2R interactions in vivo during Ag-mediated selection rescues some MHC class II-restricted thymocytes from apoptosis. Collectively, these findings provide evidence for the direct involvement of the IL-2/IL-2R signaling pathway in the deletion of Ag-specific thymocyte populations and suggest that CD4 T cell hyperplasia and autoimmunity in IL-2−/− mice is a consequence of ineffective deletion of self-reactive T cells.

Differential requirement for the SAP-Fyn interaction during NK T cell development and function.

The adaptor molecule SAP (signaling lymphocytic activation molecule-associated protein) plays a critical role during NK T (NKT) cell development in humans and mice. In CD4+ T cells, SAP interacts with the tyrosine kinase Fyn to deliver signals required for TCR-induced Th2-type cytokine production. To determine whether the SAP-dependent signals controlling NKT cell ontogeny rely on its binding to Fyn, we used the OP9-DL1 system to initiate structure function studies of SAP in murine NKT cell development. In cultures containing wild-type (WT) hematopoietic progenitors, we noted the transient emergence of cells that reacted with the NKT cell-specific agonist α-galactosyl ceramide and its analog PBS57. Sap−/− cells failed to give rise to NKT cells in vitro; however, their development could be rescued by re-expression of WT SAP. Emergence of NKT cells was also restored by a mutant version of SAP (SAP R78A) that cannot bind to Fyn, but with less efficiency than WT SAP. This finding was accentuated in vivo in SapR78A knock-in mice as well as SapR78A competitive bone marrow chimeras, which retained NKT cells but at significantly reduced numbers compared with controls. Unlike SapR78A CD4+ T cells, which produce reduced levels of IL-4 following TCR ligation, α-galactosyl ceramide-stimulated NKT cells from the livers and spleens of SapR78A mice produced Th2 cytokines and activated NK cells in a manner mimicking WT cells. Thus, SAP appears to use differential signaling mechanisms in NKT cells, with optimal ontogeny requiring Fyn binding, while functional responses occur independently of this interaction.

Deletion of donor-reactive T lymphocytes in adult mice after intrathymic inoculation with lymphoid cells

Clonal deletion of self antigen-reactive T lymphocytes is known to be a dominant mechanism of tolerance induction in the normal immune system. This report considers whether deletion of antigen-reactive T cells is also the immunologic basis for the recently described model of transplantation tolerance that follows intrathymic inoculation with allogeneic lymphoid cells. We found that the outcome of injecting Mlsa- hosts with lymphocytes from Mlsa+ donors was depletion of V beta 6+ T cells (which are known to be reactive with the Mlsa superantigen). The process was found to be specific in that a similar reduction was not seen in an irrelevant T cell population (V beta 8+) in IT injected hosts. Deletion was observed in this model only if immunosuppression with ALS or anti-CD4 accompanied intrathymic injection. When the inoculum of allogeneic lymphocytes was administered intravenously instead of intrathymically only minimal deletion was observed. The induction of transplantation tolerance by intrathymic injection of donor lymphoid cells may prove especially efficacious since it relies on deletion of only those T cells specifically reactive to donor antigens, a process analogous to tolerance induction to self antigens.

X-linked lymphoproliferative disease (XLP): a model of impaired anti-viral, anti-tumor and humoral immune responses

A major focus of our research is to understand the molecular and cellular basis of X-linked lymphoproliferative disease (XLP), a rare and often fatal immunodeficiency caused by mutations in the SH2D1A gene, which encodes the adaptor molecule SAP. Recently, we observed that SAP is essential for the development of natural killer T (NKT) cells, a lymphocyte population that participates in protection against certain tumors, infections, and autoimmune states. In this review, we describe the approaches that we are taking to understand the role of SAP in immune cells, including NKT cells. By using SAP as the focal point of our studies, we hope to identify novel signaling pathways that could be targeted to improve the treatment for patients with XLP as well as more common disorders, such as autoimmunity and cancer.

iNKT cell cytotoxic responses control T-lymphoma growth in vitro and in vivo .

Bassiri and colleagues demonstrate that the cytotoxic responses of invariant natural killer T (iNKT) cells are sufficient to limit the growth of T lymphomas, highlighting the potential utility of iNKT cells in the immunotherapy of CD1d+ cancers. Invariant natural killer T (iNKT) cells comprise a lineage of CD1d-restricted glycolipid-reactive T lymphocytes with important roles in host immunity to cancer. iNKT cells indirectly participate in antitumor responses by inducing dendritic cell maturation and producing cytokines that promote tumor clearance by CD8+ T and NK cells. Although iNKT cells thereby act as potent cellular adjuvants, it is less clear whether they directly control the growth of tumors. To gain insights into the direct contribution of iNKT cells to tumor immune surveillance, we developed in vitro and in vivo systems to selectively examine the antitumor activity of iNKT cells in the absence of other cytolytic effectors. Using the EL4 T-lymphoma cell line as a model, we found that iNKT cells exert robust and specific lysis of tumor cells in vitro in a manner that is differentially induced by iNKT cell agonists of varying T-cell receptor (TCR) affinities, such as OCH, α-galactosyl ceramide, and PBS44. In vitro blockade of CD1d-mediated lipid antigen presentation, disruption of TCR signaling, or loss of perforin expression significantly reduce iNKT cell killing. Consistent with these findings, iNKT cell reconstitution of T, B, and NK cell–deficient mice slows EL4 growth in vivo via TCR-CD1d and perforin-dependent mechanisms. Together, these observations establish that iNKT cells are sufficient to control the growth of T lymphoma in vitro and in vivo. They also suggest that the induction of iNKT cell cytotoxic responses in situ might serve as a more effective strategy to prevent and/or treat CD1d+ cancers, such as T lymphoma. Cancer Immunol Res; 2(1); 59–69. ©2013 AACR.

ISLET ALLOGRAFT, ISLET XENOGRAFT, AND SKIN ALLOGRAFT SURVIVAL IN CD8

Despite extensive study, the immunologic mechanisms mediating allograft rejection have not been completely defined. In the current study, we evaluated the T cell subsets important in islet allograft, skin allograft, and islet xenograft rejection using a genetically engineered line of mice deficient in beta 2-microglobulin expression. Because these mice lack cell surface MHC class I expression, they are deficient in T cells of the CD8 subset (class I-restricted cytotoxic T cells). Pancreatic islet allografts transplanted to CD8+ T cell-deficient recipients showed prolonged survival compared with controls. No prolongation was observed in the survival of pancreatic islet xenografts or in the survival of skin allografts transplanted to the CD8+ T cell--deficient hosts. We conclude that CD8+ T cells play a prominent role in islet allograft, but not islet xenograft or skin allograft, rejection in mice.

The Adaptor Molecule Signaling Lymphocytic Activation Molecule-Associated Protein (SAP) Regulates IFN-γ and IL-4 Production in Vα14 Transgenic NKT Cells via Effects on GATA-3 and T-bet Expression

NKT cells comprise a rare regulatory T cell population of limited TCR diversity, with most cells using a Vα14Jα18 TCR. These cells exhibit a critical dependence on the signaling adapter molecule, signaling lymphocytic activation molecule-associated protein (SAP), for their ontogeny, an aspect not seen in conventional αβ T cells. Prior studies demonstrate that SAP enhances TCR-induced activation of NF-κB in CD4+ T cells. Because NF-κB is required for NKT cell development, SAP might promote the ontogeny of this lineage by signaling to NF-κB. In this study, we demonstrate that forced expression of the NF-κB target gene, Bcl-xL, or inhibitory NF-κB kinase β, a catalytic subunit of the IκB kinase complex essential for NF-κB activation, fails to restore NKT cell development in sap−/− mice, suggesting that SAP mediates NKT cell development independently of NF-κB. To examine the role of SAP in NKT cell function, we generated NKT cells in sap−/− mice by expressing a transgene encoding the Vα14Jα18 component of the invariant TCR. These cells bound α-galactosylceramide-loaded CD1d tetramers, but exhibited a very immature CD24+NK1.1− phenotype. Although sap−/− tetramer-reactive cells proliferated in response to TCR activation, they did not produce appreciable levels of IL-4 or IFN-γ. The reduction in cytokine production correlated with the near absence of GATA-3 and T-bet, key transcription factors regulating cytokine expression and maturation of NKT cells. Ectopic expression of GATA-3 partially restored IL-4 production by the NKT cells. Collectively, these data suggest that by promoting GATA-3 and T-bet expression, SAP exerts control over NKT cell development and mature NKT cell cytokine production.

Murine natural killer immunoreceptors use distinct proximal signaling complexes to direct cell function

Signaling pathways leading to natural killer (NK)-cell effector function are complex and incompletely understood. Here, we investigated the proximal signaling pathways downstream of the immunotyrosine-based activation motif (ITAM) bearing activating receptors. We found that the adaptor molecule SH2 domain-containing leukocyte protein of 76 kD (SLP-76) is recruited to microclusters at the plasma membrane in activated NK cells and that this is required for initiation of downstream signaling and multiple NK-cell effector functions in vitro and in vivo. Surprisingly, we found that 2 types of proximal signaling complexes involving SLP-76 were formed. In addition to the canonical membrane complex formed between SLP-76 and linker for activation of T cells (LAT) family members, a novel LAT family-independent SLP-76-dependent signaling pathway was identified. The LAT family-independent pathway involved the SH2 domain of SLP-76 and adhesion and degranulation-promoting adaptor protein (ADAP). Both the LAT family-dependent and ADAP-dependent pathway contributed to interferon-gamma production and cytotoxicity; however, they were not essential for other SLP-76-dependent events, including phosphorylation of AKT and extracellular signal-related kinase and cellular proliferation. These results demonstrate that NK cells possess an unexpected bifurcation of proximal ITAM-mediated signaling, each involving SLP-76 and contributing to optimal NK-cell function.

Thymic Stromal-Cell Abnormalities and Dysregulated T-Cell Development in IL-2-Deficient Mice

The role that interleukin-2 (IL-2) plays in T-cell development is not known. To address this issue, we have investigated the nature of the abnormal thymic development and autoimmune disorders that occurs in IL-2-deficient (IL-2–/–) mice. After 4 to 5 weeks of birth, IL-2–/– mice progressively develop a thymic disorder resulting in the disruption of thymocyte maturation. This disorder is characterized by a dramatic reduction in cellularity, the selective loss of immature CD4-8- (double negative; DN) and CD4+8+ (double positive; DP) thymocytes and defects in the thymic stromal-cell compartment. Immunohistochemical staining of sections of thymuses from specific pathogen-free and germ-free IL-2–/– mice of various ages showed a progressive ,loss of cortical epithelial cells, MHC class II-expressing cells, monocytes, and macrophages. Reduced numbers of macrophages were apparent as early as week after birth. Since IL-2–/– thymocyte progenitor populations could mature normally on transfer into a normal thymus, the thymic defect in IL-2–/– mice appears to be due to abnormalities among thymic stromal cells. These results underscore the role of IL-2 in maintaining functional microenvironments that are necessary to support thymocyte growth, development, and selection.