Xincheng Zheng

CD24 Controls Expansion and Persistence of Autoreactive T Cells in the Central Nervous System during Experimental Autoimmune Encephalomyelitis

In the development of experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis (MS), autoreactive T cells must be activated and clonally expand in the lymphoid organs, and then migrate into the central nervous system (CNS) where they undergo further activation. It is unclear whether the autoreactive T cells further expand in the CNS and if so, what interactions are required for this process. We have demonstrated previously that expression by the host cells of the heat-stable antigen (CD24), which was recently identified as a genetic modifier for MS, is essential for their susceptibility to EAE. Here we show that CD24 is essential for local clonal expansion and persistence of T cells after their migration into the CNS, and that expression of CD24 on either hematopoietic cells or nonhematopoietic antigen-presenting cells in the recipient is sufficient to confer susceptibility to EAE.

Amelioration of sepsis by inhibiting sialidase-mediated disruption of the CD24-SiglecG interaction

Suppression of inflammation is critical for effective therapy of many infectious diseases. However, the high rates of mortality caused by sepsis attest to the need to better understand the basis of the inflammatory sequelae of sepsis and to develop new options for its treatment. In mice, inflammatory responses to host danger-associated molecular patterns (DAMPs), but not to microbial pathogen-associated molecular patterns (PAMPs), are repressed by the interaction of CD24 and SiglecG (SIGLEC10 in human). Here we use an intestinal perforation model of sepsis to show that microbial sialidases target the sialic acid–based recognition of CD24 by SiglecG/10 to exacerbate inflammation. Sialidase inhibitors protect mice against sepsis by a mechanism involving both CD24 and Siglecg, whereas mutation of either gene exacerbates sepsis. Analysis of sialidase-deficient bacterial mutants confirms the key contribution of disrupting sialic acid–based pattern recognition to microbial virulence and supports the clinical potential of sialidase inhibition for dampening inflammation caused by infection.

Perinatal blockade of b7-1 and b7-2 inhibits clonal deletion of highly pathogenic autoreactive T cells.

A number of in vitro studies have suggested that costimulatory molecules B7-1 and B7-2 and their receptor CD28 can promote clonal deletion, and limited in vivo studies have indicated that CD28 is involved in the clonal deletion of some T cells. However, the significance of B7-mediated clonal deletion in preventing autoimmune diseases has not been studied systematically. Here we report that the perinatal blockade of B7-1 and B7-2 substantially inhibits the clonal deletion of T cells in the thymus and leads to an accumulation of T cells capable of inducing fatal multiorgan inflammation. These results reveal a critical role for costimulatory molecules B7-1 and B7-2 in deleting pathogenic autoreactive T cells in the thymus. The critical role of B7-1 and B7-2 in T cell clonal deletion may explain, at least in part, the paradoxical increase of autoimmune disease in mice deficient for this family of costimulatory molecules, such as cytotoxic T lymphocyte associated molecule 4, CD28, and B7-2. The strong pathogenicity of the self-reactive T cells supports a central hypothesis in immunology, which is that clonal deletion plays an important role in preventing autoimmune diseases.

A Dinucleotide Deletion in CD24 Confers Protection against Autoimmune Diseases

It is generally believed that susceptibility to both organ-specific and systemic autoimmune diseases is under polygenic control. Although multiple genes have been implicated in each type of autoimmune disease, few are known to have a significant impact on both. Here, we investigated the significance of polymorphisms in the human gene CD24 and the susceptibility to multiple sclerosis (MS) and systemic lupus erythematosus (SLE). We used cases/control studies to determine the association between CD24 polymorphism and the risk of MS and SLE. In addition, we also considered transmission disequilibrium tests using family data from two cohorts consisting of a total of 150 pedigrees of MS families and 187 pedigrees of SLE families. Our analyses revealed that a dinucleotide deletion at position 1527∼1528 (P1527del) from the CD24 mRNA translation start site is associated with a significantly reduced risk (odds ratio = 0.54 with 95% confidence interval = 0.34–0.82) and delayed progression (p = 0.0188) of MS. Among the SLE cohort, we found a similar reduction of risk with the same polymorphism (odds ratio = 0.38, confidence interval = 0.22–0.62). More importantly, using 150 pedigrees of MS families from two independent cohorts and the TRANSMIT software, we found that the P1527del allele was preferentially transmitted to unaffected individuals (p = 0.002). Likewise, an analysis of 187 SLE families revealed the dinucleotide-deleted allele was preferentially transmitted to unaffected individuals (p = 0.002). The mRNA levels for the dinucleotide-deletion allele were 2.5-fold less than that of the wild-type allele. The dinucleotide deletion significantly reduced the stability of CD24 mRNA. Our results demonstrate that a destabilizing dinucleotide deletion in the 3′ UTR of CD24 mRNA conveys significant protection against both MS and SLE.

Clonal Deletion of Simian Virus 40 Large T Antigen-Specific T Cells in the Transgenic Adenocarcinoma of Mouse Prostate Mice: An Important Role for Clonal Deletion in Shaping the Repertoire of T Cells Specific for Antigens Overexpressed in Solid Tumors

In addition to their overexpression in cancer cells, most of the tumor-associated Ags are expressed at low but detectable levels in normal tissues. It is not clear whether the repertoire of T cells specific for unmutated tumor Ags is shaped by negative selection during T cell development. The transgenic adenocarcinoma of mouse prostate (TRAMP) model is transgenic for the SV40 large T Ag (Tag) under the control of the rat probasin regulatory elements. Although it has been established that T lymphocytes from TRAMP mice are tolerant to SV40 Tag, the mechanism of the tolerance is largely unknown. To examine whether the T cell clonal deletion is responsible for the tolerance, we crossed the TRAMP mice with mice transgenic for a rearranged TCR specific for SV40 Tag presented by the H-2Kk. Double transgenic TRAMP/TCR mice showed profound thymic deletion of SV40 Tag-reactive T cells, including a 6- to 10-fold reduction in the total thymocyte numbers and a >50-fold reduction in phenotypically mature T cells. Consistent with this finding, we observed that the SV40 Tag and endogenous mouse probasin genes are expressed at low levels in the thymus. These results demonstrate that clonal deletion is a major mechanism for tolerance to Ags previously regarded as prostate-specific, and provide direct evidence that the T cell repertoire specific for an unmutated tumor Ag can be shaped by clonal deletion in the thymus.

Expression of tissue‐specific autoantigens in the hematopoietic cells leads to activation‐induced cell death of autoreactive T cells in the secondary lymphoid organs

Many tissue‐specific antigens are expressed in specialized cells called peripheral antigen‐expressing cells (PAE) in the thymus and can induce central tolerance. While thymic medullary epithelial cells are the prototypic PAE that express peripheral antigens via an aire‐dependent mechanism, some studies also describe bone marrow (BM)‐derived dendritic cells (DC) and macrophages as PAE in both the thymus and secondary lymphoid organs. However, the role of these cells in development of tolerance to tissue‐specific antigens has not been elucidated. Here we use BM radiation chimeric mice to study the existence of hematopoietic PAE and their contribution to tolerance to tissue‐specific antigens. Our results reveal that BM‐derived PAE exist in both central and secondary lymphoid organs and that the expression of peripheral antigens in the BM‐derived cells does not correlate with aire expression. Using double‐transgenic mice expressing TCR specific for a model antigen expressed under the control of a prostate‐specific promoter, we show that expression of self antigen in PAE of non‐hematopoietic origin is both necessary and sufficient to induce clonal deletion. Surprisingly, while BM‐derived PAE fail to induce clonal deletion, they do cause the activation‐induced cell death of autoreactive cells in the secondary lymphoid organs. Thus, BM‐derived PAE have a distinct function in the maintenance of tolerance to tissue‐specific antigens.

Autoreactive T Cells Escape Clonal Deletion in the Thymus by a CD24-Dependent Pathway

Despite negative selection in the thymus, significant numbers of autoreactive T cells still escape to the periphery and cause autoimmune diseases when immune regulation goes awry. It is largely unknown how these T cells escape clonal deletion. In this study, we report that CD24 deficiency caused deletion of autoreactive T cells that normally escape negative selection. Restoration of CD24 expression on T cells alone did not prevent autoreactive T cells from deletion; bone marrow chimera experiments suggest that CD24 on radio-resistant stromal cells is necessary for preventing deletion of autoreactive T cells. CD24 deficiency abrogated the development of experimental autoimmune encephalomyelitis in transgenic mice with a TCR specific for a pathogenic autoantigen. The role of CD24 in negative selection provides a novel explanation for its control of genetic susceptibility to autoimmune diseases in mice and humans.

B7-CD28 Interaction Promotes Proliferation and Survival but Suppresses Differentiation of CD4−CD8− T Cells in the Thymus

Costimulatory molecules play critical roles in the induction and effector function of T cells. More recent studies reveal that costimulatory molecules enhance clonal deletion of autoreactive T cells as well as generation and homeostasis of the CD25+CD4+ regulatory T cells. However, it is unclear whether the costimulatory molecules play any role in the proliferation and differentiation of T cells before they acquire MHC-restricted TCR. In this study, we report that targeted mutations of B7-1 and B7-2 substantially reduce the proliferation and survival of CD4−CD8− (double-negative (DN)) T cells in the thymus. Perhaps as a result of reduced proliferation, the accumulation of RAG-2 protein in the DN thymocytes is increased in B7-deficient mice, which may explain the increased expression of TCR gene and accelerated transition of CD25+CD44− (DN3) to CD25−CD44− (DN4) stage. Qualitatively similar, but quantitatively less striking effects were observed in mice with a targeted mutation of CD28, but not CTLA4. Taken together, our results demonstrate that the development of DN in the thymus is subject to modulation by the B7-CD28 costimulatory pathway.

Intracellular CD24 disrupts the ARF–NPM interaction and enables mutational and viral oncogene-mediated p53 inactivation

CD24 is overexpressed in nearly 70% human cancers, whereas TP53 is the most frequently mutated tumour-suppressor gene that functions in a context-dependent manner. Here we show that both targeted mutation and short hairpin RNA (shRNA) silencing of CD24 retard the growth, progression and metastasis of prostate cancer. CD24 competitively inhibits ARF binding to NPM, resulting in decreased ARF, increase MDM2 and decrease levels of p53 and the p53 target p21/CDKN1A. CD24 silencing prevents functional inactivation of p53 by both somatic mutation and viral oncogenes, including the SV40 large T antigen and human papilloma virus 16 E6-antigen. In support of the functional interaction between CD24 and p53, in silico analyses reveal that TP53 mutates at a higher rate among glioma and prostate cancer samples with higher CD24 mRNA levels. These data provide a general mechanism for functional inactivation of ARF and reveal an important cellular context for genetic and viral inactivation of TP53.

B7 Blockade Alters the Balance between Regulatory T cells and Tumor-reactive T Cells for Immunotherapy of Cancer

Purpose: In prostate cancer-bearing host, regulatory T (Treg) cells restrain activity of tumor antigen-specific T cells. Because B7:CD28 interactions are needed for both function of CD4+CD25+ Treg cells and CD8+ effective T cells, targeting this pathway may help to overcome the immunotherapy barriers. Experimental Design: The anti-B7-1/B7-2 monoclonal antibodies were administered to a transgenic mouse model of prostate cancer (TRAMP) ectopically expressing SV40 large T antigen in different tumor development stages for prevention and therapy of prostate cancer. The treatment was also tested in treating transplanted MC38 colon adenocarcinoma in mice. Results: Here, we showed that short-term administration of anti-B7-1/B7-2 monoclonal antibodies in TRAMP mice leads to significant inhibited primary tumor growth and the size of metastatic lesions. The treatment is effective to inhibit MC38 colon cancer growth. Correspondingly, this treatment results in a transient reduction of Treg in both thymus and the periphery. In vivo cytotoxicity assay revealed T antigen-specific CTL effectors in anti-B7-treated but not control IgG-treated TRAMP mice. Conclusions: Transient blockade of B7-1/B7-2 alters the balance between Treg and cancer-reactive T cells to enhance cancer immunotherapy.