IL-27 Gene Therapy Ameliorates IPEX Syndrome Caused by Germline Mutation of Foxp3 Gene: A Major Role for Induction of IL-10.

Inactivating mutations of Foxp3, the master regulator of regulatory T cell development and function, lead to immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome in mice and humans. IPEX is a fatal autoimmune disease, with allogeneic stem cell transplant being the only available therapy. In this study, we report that a single dose of adeno-associated virus (AAV)-IL-27 to young mice with naturally occurring Foxp3 mutation (Scurfy mice) substantially ameliorates clinical symptoms, including growth retardation and early fatality. Correspondingly, AAV-IL-27 gene therapy significantly prevented naive T cell activation, as manifested by downregulation of CD62L and upregulation of CD44, and immunopathology typical of IPEX. Because IL-27 is known to induce IL-10, a key effector molecule of regulatory T cells, we evaluated the contribution of IL-10 induction by crossing IL-10-null allele to Scurfy mice. Although IL-10 deficiency does not affect the survival of Scurfy mice, it largely abrogated the therapeutic effect of AAV-IL-27. Our study revealed a major role for IL-10 in AAV-IL-27 gene therapy and demonstrated that IPEX is amenable to gene therapy.

IL-27 Gene Therapy Induces Stat3-Mediated Expansion of CD11b+Gr1+ Myeloid Cells and Promotes Accumulation of M1 Macrophages in the Tumor Microenvironment.

IL-27 is a pleiotropic cytokine that exhibits stimulatory/regulatory functions on multiple lineages of immune cells and has a potential to be used as a therapeutic for cancer. We have recently demonstrated that administration of IL-27 producing adeno-associated virus (AAV-IL-27) exhibits potent inhibition of tumor growth in mouse models. In this study, we demonstrate that AAV-IL-27 treatment leads to significant expansion of CD11b+Gr1+ myeloid cells. AAV-IL-27-induced expansion of CD11b+Gr1+ cells is IL-27R-dependent and requires Stat3 signaling, but it is inhibited by Stat1 signaling. AAV-IL-27 treatment does not increase the self-renewal capacity of CD11b+Gr1+ cells but induces significant expansion of Lin-Sca1+c-Kit+ (LSK) and granulocyte-monocyte progenitor cells. Despite exhibiting significant suppression of T cells in vitro, IL-27-induced CD11b+Gr1+ cells lost the tumor-promoting activity in vivo and overall play an antitumor role. In tumors from AAV-IL-27-treated mice, CD11b+Gr1+ cells are largely F4/80+ and express high levels of MHC class I/II and M1 macrophage markers. Thus, IL-27 gene therapy induces Stat3-mediated expansion of CD11b+Gr1+ myeloid cells and promotes accumulation of M1 macrophages in the tumor microenvironment.

IL-27 Induces CCL5 Production by T Lymphocytes, Which Contributes to Antitumor Activity.

IL-27 is a pleiotropic cytokine that exhibits stimulatory/regulatory functions on multiple lineages of immune cells including T lymphocytes. In this study, we demonstrate that IL-27 directly induces CCL5 production by T lymphocytes, particularly CD8+ T cells in vitro and in vivo. IL-27-induced CCL5 production is IL-27R-dependent. In CD4+ T cells, IL-27-induced CCL5 production was primarily dependent on Stat1 activation, whereas in CD8+ T cells, Stat1 deficiency does not abrogate CCL5 induction. A chromatin immunoprecipitation assay revealed that in the CCL5 promoter region, both putative Stat3 binding sites exhibit significant binding to Stat3, whereas only one out of four Stat1 binding sites displays moderate binding to Stat1. In tumor-bearing mice, IL-27 induced dramatic production of CCL5 in tumor-infiltrating T cells. IL-27-induced CCL5 appears to contribute to an IL-27-mediated antitumor effect. This is signified by diminished tumor inhibition in anti-CCL5- and IL-27-treated mice. Additionally, intratumor delivery of CCL5 mRNA using lipid nanoparticles significantly inhibited tumor growth. Thus, IL-27 induces robust CCL5 production by T cells, which contributes to antitumor activity.

CD200-CD200R Pathway in the Regulation of Tumor Immune Microenvironment and Immunotherapy.

Tumor-associated inflammation and immune responses are key components in the tumor microenvironment (TME) which regulate tumor growth, progression, and metastasis. Tumor-associated myeloid cells (TAMCs) are a group of cells that play multiple key roles including induction of tumor-associated inflammation/angiogenesis and regulation of tumor-specific T-cell responses. Thus, identification and characterization of key pathways that can regulate TAMCs are of critical importance for developing cancer immunotherapy. Recent studies suggest that CD200-CD200 receptor (CD200R) interaction may be important in regulating the TME via affecting TAMCs. In this chapter, we will give a brief overview of the CD200-CD200R axis, including the biology behind CD200-CD200R interaction and the role(s) it plays in tumor microenvironment and tumor growth, and activation/effector functions of T cells. We will also discuss CD200-CD200R's role as potential checkpoint molecules for cancer immunotherapy. Further investigation of the CD200-CD200R pathway will not only advance our understanding of tumor pathogenesis and immunity but also provide the rationale for CD200-CD200R-targeted immunotherapy of human cancer.

A Critical Role for CD200R Signaling in Limiting the Growth and Metastasis of CD200+ Melanoma.

CD200 is a cell surface glycoprotein that functions through engaging CD200R on cells of the myeloid lineage and inhibits their functions. Expression of CD200 was implicated in a variety of human cancer cells, including melanoma cells; however, its roles in tumor growth and immunity are not clearly understood. In this study, we used CD200R-deficient mice and the B16 tumor model to evaluate this issue. We found that CD200R-deficient mice exhibited accelerated growth of CD200(+), but not CD200(-), B16 tumors. Strikingly, CD200R-deficient mice receiving CD200(+) B16 cells i.v. exhibited massive tumor growth in multiple organs, including liver, lung, kidney, and peritoneal cavity, whereas the growth of the same tumors in wild-type mice was limited. CD200(+) tumors grown in CD200R-deficient mice contained higher numbers of CD11b(+)Ly6C(+) myeloid cells, exhibited increased expression of VEGF and HIF1α genes with increased angiogenesis, and showed significantly reduced infiltration of CD4(+) and CD8(+) T cells, presumably as the result of reduced expression of T cell chemokines, such as CXCL9 and CXCL16. The liver from CD200R-deficient mice, under metastatic growth of CD200(+) tumors, contained significantly increased numbers of CD11b(+)Gr1(-) myeloid cells and Foxp3(+) regulatory T cells and reduced numbers of NK cells. Liver T cells also had a reduced capacity to produce IFN-γ or TNF-α. Taken together, we revealed a critical role for CD200R signaling in limiting the growth and metastasis of CD200(+) tumors. Thus, targeting CD200R signaling may potentially interfere with the metastatic growth of CD200(+) tumors, like melanoma.

Interleukin-27 signalling induces stem cell antigen-1 expression in T lymphocytes in vivo.

Stem cell antigen-1 (Sca-1/Ly6A/E) is a cell surface glycoprotein that is often used as a biomarker for stem cells and cell stemness. However, it is not clear what factors can directly induce the expression of Sca-1/Ly6A/E in T lymphocytes in vivo, and if induction of Sca-1 is associated with T cell stemness. In this study, we show that interleukin-27 (IL-27), a member of the IL-12 family of cytokines, directly induces Sca-1 expression in T cells in vivo. We found that mice-deficient for IL-27 (either P28 or EBI3) or its signalling (IL-27Rα) had profound reduction of Sca-1 expression in naive (CD62L+  CD44- ), memory (CD62L+  CD44+ ) and effector (CD62L-  CD44+ ) T cells. In contrast, in vivo delivery of IL-27 using adeno-associated viral vectors strongly induced the expression of Sca-1 in naive and memory/effector T-cell populations in an IL-27 receptor- or signal transducer and activator of transcription 1-dependent manner. Interestingly, IL-27-induced Sca-1+ T cells do not express or up-regulate classic stem cell-associated genes such as Nanog, Oct4, Sox2 and Ctnnb1. However, IL-27-induced Sca-1+ T cells had increased expression of effector/memory-associated transcription factor T-bet, Eomes and Blimp1. Hence, IL-27 signalling directly induces the expression of Sca-1/Ly6A/E expression in T cells. Direct expansion of Sca-1+  CD62L+  CD44- T memory stem cells may explain why IL-27 enhances T-cell memory.

Tumor-derived IL-35 promotes tumor growth by enhancing myeloid cell accumulation and angiogenesis.

IL-35 is a member of the IL-12 family of cytokines that is comprised of an IL-12 p35 subunit and an IL-12 p40-related protein subunit, EBV-induced gene 3 (EBI3). IL-35 functions through IL-35R and has a potent immune-suppressive activity. Although IL-35 was demonstrated to be produced by regulatory T cells, gene-expression analysis revealed that it is likely to have a wider distribution, including expression in cancer cells. In this study, we demonstrated that IL-35 is produced in human cancer tissues, such as large B cell lymphoma, nasopharyngeal carcinoma, and melanoma. To determine the roles of tumor-derived IL-35 in tumorigenesis and tumor immunity, we generated IL-35-producing plasmacytoma J558 and B16 melanoma cells and observed that the expression of IL-35 in cancer cells does not affect their growth and survival in vitro, but it stimulates tumorigenesis in both immune-competent and Rag1/2-deficient mice. Tumor-derived IL-35 increases CD11b(+)Gr1(+) myeloid cell accumulation in the tumor microenvironment and, thereby, promotes tumor angiogenesis. In immune-competent mice, spontaneous CTL responses to tumors are diminished. IL-35 does not directly inhibit tumor Ag-specific CD8(+) T cell activation, differentiation, and effector functions. However, IL-35-treated cancer cells had increased expression of gp130 and reduced sensitivity to CTL destruction. Thus, our study indicates novel functions for IL-35 in promoting tumor growth via the enhancement of myeloid cell accumulation, tumor angiogenesis, and suppression of tumor immunity.

IL-27 enhances the survival of tumor antigen-specific CD8+ T cells and programs them into IL-10-producing, memory precursor-like effector cells.

IL-27 is a member of the IL-12 family of cytokines that is comprised of an IL-12 p40-related protein subunit, EBV-induced gene 3, and a p35-related subunit, p28. IL-27 functions through IL-27R and has been shown to have potent antitumor activity via activation of a variety of cellular components, including antitumor CD8(+) T-cell responses. However, the exact mechanisms of how IL-27 enhances antitumor CD8(+) T-cell responses remain unclear. Here we show that IL-27 significantly enhances the survival of activated tumor antigen-specific CD8(+) T cells in vitro and in vivo, and programs tumor antigen-specific CD8(+) T cells into memory precursor-like effector cells, characterized by upregulation of Bcl-6, SOCS3, Sca-1, and IL-10. While STAT3 activation and the CTL survival-enhancing effects can be independent of CTL IL-10 production, we show here that IL-27-induced CTL IL-10 production contributes to memory precursor cell phenotype induction, CTL memory, and tumor rejection. Thus, IL-27 enhances antitumor CTL responses via programming tumor antigen-specific CD8(+) T cells into a unique memory precursor type of effector cells characterized by a greater survival advantage. Our results have important implications for designing immunotherapy against human cancer.

Increased Th17 and regulatory T cell responses in EBV-induced gene 3-deficient mice lead to marginally enhanced development of autoimmune encephalomyelitis.

EBV-induced gene 3 (EBI3)-encoded protein can form heterodimers with IL-27P28 and IL-12P35 to form IL-27 and IL-35. IL-27 and IL-35 may influence autoimmunity by inhibiting Th17 differentiation and facilitating the inhibitory roles of Foxp3(+) regulatory T (Treg) cells, respectively. In this study, we have evaluated the development of experimental autoimmune encephalomyelitis (EAE) in EBI3-deficient mice that lack both IL-27 and IL-35. We found that myelin oligodendrocyte glycoprotein peptide immunization resulted in marginally enhanced EAE development in EBI3-deficient C57BL6 and 2D2 TCR-transgenic mice. EBI3 deficiency resulted in significantly increased Th17 and Th1 responses in the CNS and increased T cell production of IL-2 and IL-17 in the peripheral lymphoid organs. EBI3-deficient and -sufficient 2D2 T cells had equal ability in inducing EAE in Rag1(-/-) mice; however, more severe disease was induced in EBI3(-/-)Rag1(-/-) mice than in Rag1(-/-) mice by 2D2 T cells. EBI3-deficient mice had increased numbers of CD4(+)Foxp3(+) Treg cells in peripheral lymphoid organs. More strikingly, EBI3-deficient Treg cells had more potent suppressive functions in vitro and in vivo. Thus, our data support an inhibitory role for EBI3 in Th17, Th1, IL-2, and Treg responses. Although these observations are consistent with the known functions of IL-27, the IL-35 contribution to the suppressive functions of Treg cells is not evident in this model. Increased Treg responses in EBI3(-/-) mice may explain why the EAE development is only modestly enhanced compared with wild-type mice.

CD24 on thymic APCs regulates negative selection of myelin antigen-specific T lymphocytes.

Negative selection plays a key role in the clonal deletion of autoreactive T cells in the thymus. However, negative selection is incomplete; as high numbers of autoreactive T cells can be detected in normal individuals, mechanisms that regulate negative selection must exist. In this regard, we previously reported that CD24, a GPI-anchored glycoprotein, is required for thymic generation of autoreactive T lymphocytes. The CD24-deficient 2D2 TCR transgenic mice (2D2(+) CD24(-/-) ), whose TCR recognizes myelin oligodendrocyte glycoprotein (MOG), fail to generate functional 2D2 T cells. However, it was unclear if CD24 regulated negative selection, and if so, what cellular mechanisms were involved. Here, we show that elimination of MOG or Aire gene expression in 2D2(+) CD24(-/-) mice - through the creation of 2D2(+) CD24(-/-) MOG(-/-) or 2D2(+) CD24(/) ∼Aire(-/-) mice - completely restores thymic cellularity and function of 2D2 T cells. Restoration of CD24 expression on DCs, but not on thymocytes also partially restores 2D2 T-cell generation in 2D2(+) CD24(-/-) mice. Taken together, we propose that CD24 expression on thymic antigen-presenting cells (mTECs, DCs) down-regulates autoantigen-mediated clonal deletion of autoreactive thymocytes.

Dendritic cell expression of CD24 contributes to optimal priming of T lymphocytes in lymph nodes.

CD24 is a GPI anchored cell surface glycoprotein whose function as a co-stimulatory molecule has been implicated. However, the function of CD24 on antigen presenting cells during T cell responses is not well understood. Here we show that in the CD24-deficient host, adoptively transferred CD4+ T cells undergo inefficient expansion and have accelerated cell death in lymph nodes, which results in insufficient priming of T cells. Insufficient expansion of T cells in the CD24-deficient host was not due to host anti-CD24 response by NK, T and B lymphocytes. Transgenic expression of CD24 on DC in CD24-/- mice restored T cell accumulation and survival in draining lymph nodes. Consistent with these findings, MHC II tetramer staining also revealed that an antigen-specific polyclonal T cell response was reduced in lymph nodes of CD24-/- mice. Taken together, we have revealed a novel role of CD24 on DC in optimal T cell priming in lymph nodes. These data suggest that CD24 blockade should lower unwanted T cell responses such as those in autoimmune diseases.

Tenofovir amibufenamide vs tenofovir alafenamide for treating chronic hepatitis B: A real-world study.

BACKGROUND: The efficacy and safety profile of tenofovir amibufenamide (TMF) in chronic hepatitis B (CHB) patients is not well-established. AIM: To compare the efficacy and safety of TMF and tenofovir alafenamide (TAF) over a 48-wk period in patients with CHB. METHODS: A total of 215 subjects meeting the inclusion criteria were enrolled and divided into two groups: TMF group (n = 106) and the TAF group (n = 109). The study included a comparison of virological response (VR): Undetectable hepatitis B virus DNA levels, alanine transaminase (ALT) normalization rates, renal function parameters, and blood lipid profiles. RESULTS: At 24 and 48 wk, VR rates for the TMF group were 53.57% and 78.57%, respectively, compared with 48.31% and 78.65% for the TAF group (P > 0.05). The VR rates were also similar in both groups among patients with low-level viremia, both hepatitis B e antigen (HBeAg)-positive and HBeAg-negative subgroups. The TMF cohort showed ALT normalization and renal safety profiles similar to the TAF group. There was a notable increase in total cholesterol levels in the TAF group (P = 0.045), which was not observed in the TMF group (P > 0.05). In patients with liver cirrhosis, both groups exhibited comparable VR and ALT normalization rates and renal safety profiles. However, the fibrosis 4 score at 48 wk showed a significant reduction in the TAF group as compared to the TMF group within the liver cirrhosis subgroup. CONCLUSION: Our study found TMF is as effective as TAF in treating CHB and has a comparable safety profile. However, TAF may be associated with worsening lipid profiles.

CD200R signaling contributes to unfavorable tumor microenvironment through regulating production of chemokines by tumor-associated myeloid cells.

CD200 is overexpressed in many solid tumors and considered as an immune checkpoint molecule dampening cancer immunity. In this study, we found that CD200R-/- mice were significantly more potent in rejecting these CD200+ tumors. scRNA sequencing demonstrated that tumors from CD200R-/- mice had more infiltration of CD4+ and CD8+ T cells, and NK cells but less infiltration of neutrophils. Antibody depletion experiments revealed that immune effector cells are crucial in inhibiting tumor growth in CD200R-/- mice. Mechanistically, we found that CD200R signaling regulates the expression of chemokines in tumor-associated myeloid cells (TAMCs). In the absence of CD200R, TAMCs increased expression of CCL24 and resulted in increased infiltration of eosinophils, which contributes to anti-tumor activity. Overall, we conclude that CD200R signaling contributes to unfavorable TME through chemokine-dependent recruitment of immune suppressive neutrophils and exclusion of anti-cancer immune effectors. Our study has implications in developing CD200-CD200R targeted immunotherapy of solid tumors.

Targeting activation-induced cytidine deaminase overcomes tumor evasion of immunotherapy by CTLs.

Activation-induced cytidine deaminase (AID) is an enzyme essential for the generation of Ab diversity in B cells and is considered to be a general gene mutator. In addition, AID expression was also implicated in the pathogenesis of human B cell malignancies and associated with poor prognosis. In this study, we report that small interfering RNA silencing of AID in plasmacytoma dramatically increased its susceptibility to immunotherapy by CTLs. AID silencing did not decrease the mutation frequencies of tumor Ag gene P1A. Gene-array analysis showed dramatically altered expression of a number of genes in AID-silenced plasmacytoma cells, and upregulation of CD200 was shown to be in favor of tumor eradication by CTLs. Taken together, we demonstrate a novel function of AID in tumor evasion of CTL therapy and that targeting AID should be beneficial in the immunotherapy of AID-positive tumors.

CD24 is expressed on FoxP3+ regulatory T cells and regulates their function.

CD24 is a glycosyl-phosphatidylinositol (GPI) anchored cell surface glycoprotein with a variety of immunomodulatory functions such as inhibition of thymic generation of autoreactive T cells, regulation of antigen presenting cell functions, and mediation of autoimmunity. Given the autoimmune nature of FoxP3+ regulatory T cells and their importance in autoimmune diseases, we hypothesize that CD24 regulates the generation and functions of Treg cells. Through the analysis of the Treg repertoire in two strains of CD24-deficient mice, we found that CD24 does not globally affect the thymic generation of Treg cells. However, CD24 is abundantly expressed on Treg cells, and CD24 antibody treatment of Treg cells enhances their suppressive functions. Concurrently, we observed CD24-deficient Treg cells exhibit increased suppressive functions and produce more IL-10 compared to their wild type counterparts. In addition, CD24-deficient Treg cells exhibited more potent suppressive capacity in inhibiting the development of experimental autoimmune encephalomyelitis (EAE) in mice. Thus, CD24 on Treg cells regulates their suppressive functions. Our findings can partially explain the resistance of EAE development in CD24-deficient mice and CD24 polymorphism-associated susceptibility of human autoimmune diseases. Further investigations regarding mechanisms of CD24 regulation of Treg function may lead to a new approach for the immunotherapy of human autoimmune diseases.

Intratumoral delivery of IL-12 and IL-27 mRNA using lipid nanoparticles for cancer immunotherapy.

Cytokines are important immunotherapeutics with approved drugs for the treatment of human cancers. However, systemic administration of cytokines often fails to achieve adequate concentrations to immune cells in tumors due to dose-limiting toxicity. Thus, developing localized therapy that directly delivers immune-stimulatory cytokines to tumors may improve the therapeutic efficacy. In this study, we generated novel lipid nanoparticles (LNPs) encapsulated with mRNAs encoding cytokines including IL-12, IL-27 and GM-CSF, and tested their anti-tumor activity. We first synthesized ionizable lipid materials containing di-amino groups with various head groups (DALs). The novel DAL4-LNP effectively delivered different mRNAs in vitro to tumor cells and in vivo to tumors. Intratumoral injection of DAL4-LNP loaded with IL-12 mRNA was most potent in inhibiting B16F10 melanoma tumor growth compared to IL-27 or GM-CSF mRNAs in monotherapy. Furthermore, intratumoral injection of dual DAL4-LNP-IL-12 mRNA and IL-27 mRNA showed a synergistic effect in suppressing tumor growth without causing systematic toxicity. Most importantly, intratumoral delivery of IL-12 and IL-27 mRNAs induced robust infiltration of immune effector cells, including IFN-γ and TNF-α producing NK and CD8+ T cells into tumors. Thus, intratumoral administration of DAL-LNP loaded with IL-12 and IL-27 mRNA provides a new treatment strategy for cancer.

Tumor expression of CD200 inhibits IL-10 production by tumor-associated myeloid cells and prevents tumor immune evasion of CTL therapy.

CD200 is a cell-surface glycoprotein that functions through interaction with the CD200 receptor on myeloid lineage cells to regulate myeloid cell functions. Expression of CD200 has been implicated in multiple types of human cancer; however, the impact of tumor expression of CD200 on tumor immunity remains poorly understood. To evaluate this issue, we generated CD200-positive mouse plasmacytoma J558 and mastocytoma P815 cells. We found that established CD200-positive tumors were often completely rejected by adoptively transferred CTL without tumor recurrence; in contrast, CD200-negative tumors were initially rejected by adoptively transferred CTL but the majority of tumors recurred. Tumor expression of CD200 significantly inhibited suppressive activity and IL-10 production by tumor-associated myeloid cells (TAMC), and as a result, more CTL accumulated in the tumor and exhibited a greater capacity to produce IFN-gamma in CD200-positive tumors than in CD200-negative tumors. Neutralization of IL-10 significantly inhibited the suppressor activity of TAMC, and IL-10-deficiency allowed TAMC to kill cancer cells and their antigenic variants, which prevented tumor recurrence during CTL therapy. Thus, tumor expression of CD200 prevents tumor recurrence via inhibiting IL-10 production by TAMC.

CD200 Blockade Modulates Tumor Immune Microenvironment but Fails to Show Efficacy in Inhibiting Tumor Growth in a Murine Model of Melanoma.

CD200-CD200R pathway regulates immune responses and has been implicated in the pathogenesis of a number of cancer types. CD200 blockade is considered a strategy for immunotherapy of CD200-positive cancers such as melanoma. Thus, it is critical to understand the potential impacts of CD200 blockade in a more human relevant tumor model. In this study, we evaluated these issues using the CD200+ Yumm1.7 mouse melanoma model. Yumm1.7 cells bear Braf/Pten mutations resembling human melanoma. We found that Yumm1.7 tumors grow significantly faster in CD200R-/- mice compared to wild type mice. Analysis of tumor immune microenvironment (TIME) revealed that tumors from CD200R-/- or anti-CD200 treated mice had downregulated immune cell contents and reduced TCR clonality compared to tumors from untreated wild type mice. T cells also showed impaired effector functions, as reflected by reduced numbers of IFN-γ+ and TNF-α+ T cells. Mechanistically, we found upregulation of the CCL8 gene in CD200R-/- tumors. In vitro co-culture experiments using Yumm1.7 tumor cells with bone marrow derived macrophages (BMDM) from WT and CD200R-/- mice confirmed upregulation of macrophage CCL8 in the absence of CD200-CD200R interaction. Finally, we found that anti-CD200 therapy failed to show efficacy either alone or in combination with checkpoint inhibitors such as anti-PD-1 or anti-CTLA4 in inhibiting Yumm1.7 tumor growth. Given that CD200R-deficiency or anti-CD200 treatment leads to reduced T cell responses in TME, using blockade of CD200 as an immunotherapy for cancers such as melanoma should be practiced with caution.

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 approximately 1528 (P1527(del)) 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 P1527(del) 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.