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IL-17 plays critical roles in host defenses, combating bacterial and fungal infections, as well as the pathogenesis of autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE). The signaling adaptor SAP is essential for normal immune homeostasis and mutations within SH2D1A, the locus encoding this protein, result in serious and sometimes fatal syndromes, including X-linked lymphoproliferative disease and severe cases of common variable immunodeficiency. However, the precise cellular basis of how SAP deficiency contributes to immune dysfunction remains incompletely understood. In this study, we found that CD4 and CD8 T cells lacking SAP had a diminished capacity to differentiate into IL-17–producing Th17 and T cytotoxic (Tc17) cells relative to wild-type lymphocytes. The use of costimulating SLAM Abs was found to augment the differentiation of IL-17–secreting effectors in wild-type but not Sh2d1a−/− splenic T cells under IL-17–polarizing conditions. In addition, SAP’s regulation of IL-17–secreting T cells was shown to be a T cell–intrinsic role, as purified naive Sh2d1a−/− CD4 and CD8 T cells were inherently defective at converting into Th17 and Tc17 cells in vitro and in vivo. Furthermore, Sh2d1a−/− mice were protected from EAE and exhibited greatly decreased numbers of CNS-infiltrating Th17 and Tc17 effector T cells and reduced disease severity. Collectively, these results suggest that SLAM–SAP signaling drives the differentiation and function of Th17 and Tc17 cells in vitro and in vivo and contributes to the pathogenesis of autoimmunity in EAE.

B cell activation and Ab production in response to protein Ags requires presentation of peptides for recruitment of T cell help. We and others have recently demonstrated that B cells can also acquire innate help by presenting lipid Ags via CD1d to NKT cells. Given the newfound contribution of NKT cells to humoral immunity, we sought to identify the pathways that regulate CD1 molecule expression in human B cells. We show that ex vivo, activated and memory B cells expressed lower levels of CD1d compared with resting, naive, and marginal zone-like B cells. In vitro, CD1d was downregulated by all forms of B cell activation, leaving a narrow temporal window in which B cells could activate NKT cells. CD1c expression and function also decreased following activation by CD40L alone, whereas activation via the BCR significantly upregulated CD1c, particularly on marginal zone-like B cells. We found that the CD40L-induced downreglation of CD1d and CD1c correlated with diminished expression of retinoic acid receptor α (RARα) response genes, an effect that was reversed by RARα agonists. However, BCR-induced upregulation of CD1c was independent of the RAR pathway. Our findings that both CD1d and CD1c are upregulated by RARα signaling in human B cells is distinct from effects reported in dendritic cells, in which CD1c is inversely downregulated. One functional consequence of CD1d upregulation by retinoic acid was NKT cell cytotoxicity toward B cells. These results are central to our understanding of how CD1-restricted T cells may control humoral immunity.

Ag encounter by naive CD8 T cells initiates a developmental program consisting of cellular proliferation, changes in gene expression, and the formation of effector and memory T cells. The strength and duration of TCR signaling are known to be important parameters regulating the differentiation of naive CD8 T cells, although the molecular signals arbitrating these processes remain poorly defined. The Ras-guanyl nucleotide exchange factor RasGRP1 has been shown to transduce TCR-mediated signals critically required for the maturation of developing thymocytes. To elucidate the role of RasGRP1 in CD8 T cell differentiation, in vitro and in vivo experiments were performed with 2C TCR transgenic CD8 T cells lacking RasGRP1. In this study, we report that RasGRP1 regulates the threshold of T cell activation and Ag-induced expansion, at least in part, through the regulation of IL-2 production. Moreover, RasGRP1−/− 2C CD8 T cells exhibit an anergic phenotype in response to cognate Ag stimulation that is partially reversible upon the addition of exogenous IL-2. By contrast, the capacity of IL-2/IL-2R interactions to mediate Ras activation and CD8 T cell expansion and differentiation appears to be largely RasGRP1-independent. Collectively, our results demonstrate that RasGRP1 plays a selective role in T cell signaling, controlling the initiation and duration of CD8 T cell immune responses.

NK cells and CD8+ T cells bind MHC-I molecules using distinct topological interactions. Specifically, murine NK inhibitory receptors bind MHC-I molecules at both the MHC-I H chain regions and β2-microglobulin (β2m) while TCR engages MHC-I molecules at a region defined solely by the class I H chain and bound peptide. As such, alterations in β2m are not predicted to influence functional recognition of MHC-I by TCR. We have tested this hypothesis by assessing the capability of xenogeneic β2m to modify the interaction between TCR and MHC-I. Using a human β2m-transgenic C57BL/6 mouse model, we show that human β2m supports formation and expression of H-2Kb and peptide:H-2Kb complexes at levels nearly equivalent to those in wild-type mice. Despite this finding, the frequencies of CD8+ single-positive thymocytes in the thymus and mature CD8+ T cells in the periphery were significantly reduced and the TCR Vβ repertoire of peripheral CD8+ T cells was skewed in the human β2m-transgenic mice. Furthermore, the ability of mouse β2m-restricted CTL to functionally recognize human β2m+ target cells was diminished compared with their ability to recognize mouse β2m+ target cells. Finally, we provide evidence that this effect is achieved through subtle conformational changes occurring in the distal, peptide-binding region of the MHC-I molecule. Our results indicate that alterations in β2m influence the ability of TCR to engage MHC-I during normal T cell physiology.

Recently, it has been shown that human beta(2)-microglobulin (h-beta(2)m) blocks the association between the NK cell inhibitory receptor Ly49C and H-2K(b). Given this finding, we therefore sought to assess the immunobiology of NK cells derived from C57BL/6 (H-2(b)) mice expressing exclusively h-beta(2)m. Initial analysis revealed that the Ly49C expression profile of NK cells from h-beta(2)m(+) mice was modified, despite the fact that H-2K(b) expression was normal in these mice. Moreover, the NK cells were not anergic in that IL-2 treatment of h-beta(2)m(+) NK cells in vitro enabled efficient lysis of prototypic tumor cell lines as well as of syngeneic h-beta(2)m(+) lymphoblasts. This loss of self-tolerance appeared to correlate with the activation status of h-beta(2)m(+) NK cells because quiescent h-beta(2)m(+) transplant recipients maintained h-beta(2)m(+) grafts but polyinosine:polycytidylic acid-treated recipients acutely rejected h-beta(2)m(+) grafts. NK cell reactivity toward h-beta(2)m(+) targets was attributed to defective Ly49C interactions with h-beta(2)m:H-2K(b) molecules. With regard to NK cell regulatory mechanisms, we observed that h-beta(2)m:H-2K(b) complexes in the cis-configuration were inefficient at regulating Ly49C and, furthermore, that receptor-mediated uptake of h-beta(2)m:H-2K(b) by Ly49C was impaired compared with uptake of mouse beta(2)m:H-2K(b). Thus, we conclude that transgenic expression of h-beta(2)m alters self-MHC class I in such a way that it modulates the NK cell phenotype and interferes with regulatory mechanisms, which in turn causes in vitro-expanded and polyinosine:polycytidylic acid-activated NK cells to be partially self-reactive similar to what is seen with NK cells derived from MHC class I-deficient mice.

Abnormal placentation results in either inadequate (consequences: recurrent miscarriage, intrauterine growth restriction, and preeclampsia) or overzealous (consequences: placenta accreta, increta, and percreta) placentation. NK cells dominate in first trimester decidua and probably control extravillous cytotrophoblast (EVT) invasion. We examined this interaction in a novel way, using NK cells and villous explants from concordant first trimester pregnancies cocultured using a new collagen (two-dimensional) model of placentation. Decidual NK (dNK) cells exerted contact-independent inhibition of normal cytotrophoblast migration, associated with changes in the cytotrophoblast expression of metalloproteases-2 and -9, and plasminogen activator inhibitor-1. dNK cells did not affect EVT proliferation and apoptosis, and cell column formation. dNK cell effects were partially reversed by neutralizing Abs against IFN-γ. We provide ex vivo human evidence of a direct role for dNK in modulating EVT differentiation as they form columns and then migrate from anchoring villi.

Human and mouse NK cells use different families of receptors to recognize MHC class I (MHC I) on target cells. Although human NK cells express both Ig-like receptors and lectin-like receptors specific for MHC I, all the MHC I-specific receptors identified on mouse NK cells to date are lectin-like receptors, and no Ig-like receptors recognizing MHC I have been identified on mouse NK cells. In this study we report the first MHC I-specific Ig-like receptor on mouse NK cells, namely, murine CD160 (mCD160). The expression of mCD160 is restricted to a subset of NK cells, NK1.1+ T cells, and activated CD8+ T cells. The mCD160-Ig fusion protein binds to rat cell lines transfected with classical and nonclassical mouse MHC I, including CD1d. Furthermore, the level of mCD160 on NK1.1+ T cells is modulated by MHC I of the host. Overexpression of mCD160 in the mouse NK cell line KY-2 inhibits IFN-γ production induced by phorbol ester plus ionomycin, whereas it enhances IFN-γ production induced by NK1.1 cross-linking or incubation with dendritic cells. Cross-linking of mCD160 also inhibits anti-NK1.1-mediated stimulation of KY-2 cells. Anti-mCD160 mAb alone has no effect. Thus, mCD160, the first MHC I-specific Ig-like receptor on mouse NK cells, regulates NK cell activation both positively and negatively, depending on the stimulus.

X-linked lymphoproliferative disease (XLP) is a fatal immunological disorder that typically manifests following EBV infection. XLP patients exhibit a number of immune defects including abnormal T, B, and NK lymphocyte function. These defects have been attributed to mutations of Src homology 2 domain-containing gene 1A (SH2D1A), the gene encoding signaling lymphocytic activation molecule-associated protein (SAP), an intracellular adaptor molecule expressed in lymphocytes. We have observed that SAP knockout (SAPKO) mice and humans with XLP have a complete lack of CD1d-restricted NKT cells. As expected, SAPKO mice injected with the NKT cell agonist, α-galactosylceramide failed to generate NKT cell IFN-γ or IL-4. Furthermore, in contrast to wild-type littermates, SAPKO mice coinjected with OVA and α-galactosylceramide failed to mount OVA-specific CTL responses. These data suggest that an absence of NKT cells may underlie part of the immune dysregulation seen in SAPKO mice and in XLP patients.

Autoimmune (type 1) diabetes results from a loss of β cells that is mediated by self-reactive T cells. Previous studies have shown that a single injection of CFA prevents diabetes in nonobese diabetic (NOD) mice, but the mechanism(s) of protection remain unknown. We show here that NOD mice immunized with CFA have a markedly reduced incidence of diabetes and that this reduced incidence is associated with a decrease in the number of β cell-specific, autoreactive CTL. In addition, the adoptive transfer of diabetes into syngeneic NOD/SCID recipients was prevented by CFA immunization, and the protective effects of CFA were lost when cells expressing the NK cell marker, asialo GM1, were removed from both donor cells and recipient mice. Returning a population of CD3−DX5+ cells to the adoptive transfer restored the protective effects of CFA. Therefore, NK cells mediate the protective effects of CFA possibly through the down-regulation of autoreactive CTL and stimulation of NK cells represents a novel approach to the prevention of autoimmune diabetes.

Activated T cells and their naive precursors display different functional avidities for peptide/MHC, but are thought to have identical antigenic repertoires. We show that, following activation with a cognate mimotope (NRP), diabetogenic CD8+ T cells expressing a single TCR (8.3) respond vigorously to numerous peptide analogs of NRP that were unable to elicit any responses from naive 8.3-CD8+ T cells, even at high concentrations. The NRP-reactive, in vivo activated CD8+ cells arising in pancreatic islets of nonobese diabetic mice are similarly promiscuous for peptide/MHC, and paradoxically this promiscuity expands as the aviditiy of the T cell population for NRP/MHC increases with age. Thus, activation and avidity maturation of T lymphocyte populations can lead to dramatic expansions in the range of peptides that elicit functional T cell responses.

X-linked lymphoproliferative disease (XLP) is characterized by a selective immune deficiency to EBV. The molecular basis of XLP has been attributed to mutations of signaling lymphocytic activation molecule-associated protein, an intracellular molecule known to associate with the lymphocyte-activating surface receptors SLAM and 2B4. We have identified a single nucleotide mutation in SLAM-associated protein that affects the NK cell function of males carrying the mutated gene. In contrast to normal controls, both NK and lymphokine-activated killer cell cytotoxicity was significantly reduced in two XLP patients. In addition to decreased baseline cytotoxicity, ligation of 2B4 significantly augmented NK lytic function in normal controls but failed to enhance the cytotoxicity of NK cells from XLP patients. These findings suggest that association of SAP with 2B4 is necessary for optimal NK/lymphokine-activated killer cytotoxicity and imply that alterations in SAP/2B4 signaling contribute to the immune dysfunction observed in XLP.

Regulatory T cells (Tregs) control immune homeostasis by preventing inappropriate responses to self and non-harmful foreign antigens. Tregs use multiple mechanisms to control immune responses, all of which require Tregs to be near their targets of suppression, but how Treg-to-target proximity is controlled is unknown. We found that Tregs produce chemokines to attract CD4+ and CD8+ T cells close to their proximity in vitro and in vivo. Mouse and human lineage committed Tregs, as well as murine in vitro induced Tregs, produced CCL3 and CCL4 at message and protein level. Chemokine producing Tregs had an activated Treg phenotype with high expression of multiple proteins associated with suppressive function. Furthermore the human CCL3 and CCL4 promoter could be transactivated by FOXP3. In vitro and in vivo migration experiments indicated that CCR5 was the major receptor required by target cells. CCL3 and CCL4 deficient Tregs were impaired in their ability to prevent experimental autoimmune encephalomyelitis or islet allograft rejection, but suppressive in a standard in vitro assay. Moreover, Tregs from subjects with established type 1 were impaired in their ability to produce CCL3 and CCL4. These results demonstrate a previously unknown facet of Treg function and suggest that chemokine secretion by Tregs is a fundamental aspect of their in vivo function and therapeutic effect in autoimmunity and transplantation.

Mutations in SH2D1A gene that encodes SAP (SLAM-associated protein) result in X-linked lymphoproliferative disease (XLP), a rare primary immunodeficiency defined by exquisite sensitivity to the B-lymphotropic Epstein-Barr virus (EBV) and B cell lymphomas. However, the precise mechanism of how the loss of SAP function contributes to extreme vulnerability to EBV and the development of B cell lymphomas remains unclear. Here, we utilized SAP-deficient (Sh2d1a−/−) mice to investigate the hypothesis that SAP is critical for CD8 T cell immune surveillance of antigen-expressing B cells or B lymphoma cells. Sh2d1a−/− CD8 T cells exhibited greatly diminished proliferation relative to wild type when antigen-presenting-B cells or -B lymphoma cells served as the primary APC. By contrast, Sh2d1a−/− CD8 T cells responded equivalently to wild type CD8 T cells when B cell-depleted splenocytes, melanoma cells or breast carcinoma cells performed antigen presentation. Through application of SLAM family receptor blocking antibodies or SLAM family receptor-deficient CD8 T cells and APCs, we found that CD48 engagement on the B cell surface by 2B4 is crucial for initiating SAP-dependent signaling required for the antigen-driven CD8 T cell proliferation and differentiation. Altogether, a pivotal role for SAP in promoting the expansion and differentiation of B cell-primed viral-specific naïve CD8 T cells may explain the selective immune deficiency of XLP patients to EBV and B cell lymphomas.

Epstein-Barr virus (EBV) poses serious health consequences for immunocompromised individuals, such as patients undergoing transplant or chemotherapeutic regimens, and is strongly associated with an expanding list of human cancers. To gain insight into immunity against EBV, a rare congenital immunodeficiency defined by exquisite sensitivity to EBV but not other pathogens called X-linked lymphoproliferative disease, a syndrome caused by mutations in the SH2D1A gene that encodes SLAM-associated protein (SAP), has been the subject of intense investigation. The unique B cell tropism of EBV led us to speculate that SAP and SLAM family receptors are especially critical for optimal CD8 T cell immunity against antigen-expressing B cells and B lymphoma cells. Consistent with this hypothesis, Sh2d1a-/- CD8 T cells exhibited greatly diminished proliferation and effector functions relative to wild type CD8 T cells when cultured with antigen-coated B cells or B lymphoma cells in vitro. By contrast, both wild type and Sh2d1a-/- CD8 T cells responded robustly when stimulated with either antigen-coated B cell-depleted splenocytes or antigen-presenting tumor cells lacking SLAM family receptor expression. Collectively, these observations suggest that the recognition of SLAM family receptors on the surface of antigen presenting-B cells and -B lymphoma cells are essential for antigen priming and immunosurveillance by CD8 T cells.

In GWA studies, CLEC16A has been identified as a risk gene for several major autoimmune diseases, including multiple sclerosis (MS). We show that CLEC16A is increased in MS and functionally links to HLA-II, and uncovered the molecular mechanism of CLEC16A as key regulator of HLA-II antigen presentation. We used the human melanoma cell line MelJuSo and primary moDC as APC models to silence CLEC16A with different siRNA and lentiviral shRNA. CLEC16A knockdown in MelJuSo cells and moDC resulted in cytoplasmic dispersion and strong accumulation of HLA-II+ late endosomes, suggesting impaired formation of multivesicular late endosomal compartments, i.e. MIIC. Indeed, in CLEC16A shRNA transductants, MIIC contained no or a few abnormally enlarged internal vesicles and showed an increased number of HLA-II molecules as compared to scrambled shRNA transductants of MelJuSo cells. CLEC16A associated with Rab7-interacting lysosomal protein (RILP) and the homotypic fusion and protein sorting (HOPS) complex, two members of the dynein motor complex regulating late endosomal trafficking and maturation. In addition, CLEC16A silencing disrupted RILP-mediated recruitment of HLA-II+ late endosomes to perinuclear regions. We reveal direct involvement of CLEC16A in the machinery regulating late endosomal processing of HLA-II. This novel C-type lectin function supports a pathogenic role of CLEC16A in autoimmune disease by promoting HLA-II antigen presentation via the formation of MIIC.

Mutations in the SH2D1A gene, that encodes SLAM-associated protein (SAP), cause X-linked lymphoproliferative disease (XLP), a rare congenital immunodeficiency defined by exquisite sensitivity to Epstein-Barr virus (EBV). The inability of boys with XLP to control EBV infection results in fulminant, often fatal, infectious mononucleosis, massive expansions of EBV-infected B cells and malignant B cell lymphomas. However, the precise mechanism of how SAP loss contributes to susceptibility to EBV remains unclear. Given the unique B cell tropism of EBV and the purported role of SAP in regulating lymphocyte/lymphocyte interactions, we hypothesized that SAP expression within CD8 T cells is critical for T cells to mount responses towards antigen-expressing B cells. Wild type and Sh2d1a-/- CD8 T cells responded strongly when stimulated in vitro with cognate antigen-coated whole splenocytes. However, Sh2d1a-/- CD8 T cells exhibited greatly diminished proliferation and effector functions relative to wild type CD8 T cells when cultured with antigen-coated B cells in vitro or challenged with antigen-expressing B cell lymphoma in vivo. These results demonstrate that SAP is required for the priming of CD8 T cells when B cells act as the primary APC. Together, our findings suggest that the susceptibility of XLP patients to EBV infection may at least in part be a consequence of an inability of their naïve CD8 T cells to differentiate into effectors upon encountering virally-infected B cells.

FOXP3+ T regulatory cells (Tregs) function to restrain immune responses. Accumulating evidence has shown that Tregs mediate their suppression by migrating to tissues based on their distinct expression of chemokine receptors. Yet how Tregs ensure they are close to their targets is unknown. Using ELISA and intracellular staining we discovered that both human and mouse Tregs produce CCL3 and CCL4, ligands for CCR5. Since chemokines recruit immune cells and the broad suppressive activities of Tregs require intimate cell proximity, we hypothesized that the secretion of the pro-inflammatory chemokines may bring immune cells closer to Tregs to be suppressed. To determine whether Tregs can recruit CD4+ and CD8+ cells via a CCR5-dependent mechanism, we performed migration assays and showed CCL3- and CCL4- containing-supernatants from Tregs attracted both subsets of T cells as well Tregs. Additionally, CCR5-/- T cells had a significantly reduced capacity to migrate towards Tregs in vitro and in vivo, supporting that chemokine signalling through CCR5 is critical. Furthermore, decreased proportion of FOXP3+CCL3+ Tregs was observed in patients with type 1 diabetes compared to healthy controls, implying that the altered chemokine secretion could contribute to the inadequate Treg function. These results suggest that active recruitment of their targets of suppression may be a novel mechanism of action by Tregs and that this pathway could be a potential therapeutic target in autoimmunity.

Type 1 diabetes (T1D) results from the destruction of beta cells by autoreactive lymphocytes. Regulatory T cells (Tregs) maintain self-tolerance in part by suppressing the effector functions of self-reactive lymphocytes. Tregs require the cytokine interleukin-2 (IL-2) for survival and maintenance of their suppressive function. IL-2 binds to the high-affinity IL-2 receptor (IL-2R), leading to phosphorylation of signal transducer and activator of transcription 5 (STAT5) and downstream transcription of effector proteins.The IL-2 receptor gene has single nucleotide polymorphisms (SNPs), which are associated with T1D incidence. We hypothesized that T1D subjects with SNPs in the IL-2R gene possess Tregs that respond aberrantly to IL-2 and do not optimally induce signaling in the STAT5 pathway. PBMC from T1D subjects with a homozygous susceptible (GG), heterozygous (AG) and homozygous wild-type (AA) genotype for a SNP located in the rs3118470 locus of the IL-2R gene were stimulated with IL-2. Our results indicate that Tregs from individuals with the homozygous susceptible GG genotype have reduced levels of pSTAT5 compared to those with the AA genotype (p=0.0133). This provides evidence that Tregs from T1D subjects with SNPs in the rs3118470 locus of the IL-2R gene have diminished STAT5 phosphorylation in response to IL-2. This is a potential mechanism of defective Treg function in a genetically identifiable subset of children with T1D.

Individuals with X-linked lymphoproliferative disease (XLP) lack invariant natural killer T (iNKT) cells and are uniquely susceptible to Epstein-Barr virus (EBV). To determine whether iNKT cells recognize or regulate EBV replication, human B cells were infected with GFP+ EBV 95.8 in the presence or absence of iNKT cells. We observed that iNKT cells significantly reduced early EBV titres and GFP+ B cell expansion but that within 72 hours of infection, B cells transformed into lymphoblastoid cell lines (LCL) by EBV had downregulated the iNKT antigen receptor CD1d, completely abrogating iNKT recognition of the lipid agonist α-galactosylceramide (αGalCer). To determine whether ectopic CD1d expression could restore iNKT recognition, LCL were treated with a synthetic retinoic acid receptor-α agonist AM580 known to modulate CD1d expression by regulating the nuclear protein lymphoid enhancer-binding factor-1 (LEF-1). AM580 treatment significantly reduced LEF-1 at the CD1d promoter, induced surface CD1d expression and enabled iNKT recognition of LCL loaded with αGalCer. Unexpectedly, AM580-treated LCL also activated iNKT function even in the absence of αGalCer suggesting that EBV infection and transformation triggers endogenous iNKT antigen expression. These observations indicate that iNKT cells may be important for the early recognition and control of EBV-infected B cells prior to CD1d downregulation and the loss of CD1d may allow EBV to circumvent iNKT-mediated immunosurveillance.

Mutations in SAP (SLAM-associated protein) underlie the majority of cases of X-linked lymphoproliferative disease (XLP), a rare congenital disorder that often leads to fatal complications upon infection with Epstein-Barr virus. The small SH2-containing adaptor SAP transmits signaling via SLAM (signaling lymphocytic activation molecule) family receptors and has been shown to be critical for development and function of multiple immune cell types. Here, we have investigated the role of SLAM and SAP signaling in regulating the differentiation of naïve CD4 T cells into IL-17 secreting effectors (TH17 cells), a pro-inflammatory lineage implicated in host defense as well as autoimmune diseases. T cell receptor activation along with co-stimulating SLAM antibodies was found to augment TH17 cell differentiation in wild type but not SAP-deficient splenic T cells under IL-17 polarizing conditions. Furthermore, SAP-/- mice were protected from experimental autoimmune encephalomyelitis (EAE), exhibiting greatly decreased numbers of CNS-infiltrating TH17 cells and dramatically reduced disease severity. Collectively, these results suggest that SLAM-SAP interactions promote the differentiation of IL-17 secreting effector CD4 T cells both in vitro and in vivo.

CD4+CD25+ T regulatory (Treg) cells prevent spontaneous development of autoimmune diabetes and may delay islet allograft rejection. Tumour cells attract Treg cells to foster immune privilege by producing the chemokine CCL22, which promotes trafficking of Treg cells to the tumour. Therefore, manipulating Treg migration with CCL22 may provide a means of preventing islet transplant rejection in autoimmune diabetes. To drive islet-specific expression of CCL22 we created an adenovirus (Ad-CCL22) in which a mouse CCL22 cDNA is expressed downstream of the CMV promoter. Isolated islets transduced with Ad-CCL22 (10 MOI) produced and secreted high levels of CCL22 in vitro whereas CCL22 production by non-transduced islets was undetectable. To determine whether CCL22 expression prevents islet allograft rejection, donor Balb/c islets were isolated and transduced overnight with Ad-CCL22 (n = 6) or Ad-lacZ (n = 5). Islets (300 per recipient) were then transplanted into the left renal subcapsular space of age-matched female C57/BL6 diabetic recipients. By 5 weeks post-transplant, only 1 of 7 CCL22-expressing islet grafts had been rejected whereas 4 of 5 Ad-LacZ-transduced grafts were rejected. Induced expression of CCL22 by transplanted islets thus delays islet allograft rejection. Manipulation of Tregs cells by CCL-22 in transplanted islets may be a novel therapeutic strategy for diabetes.