γδ T Cells Activated in Different Inflammatory Environments Are Functionally Distinct.

γδ T cells are important immunoregulatory cells in experimental autoimmune uveitis (EAU), and the activation status of γδ T cells determines their disease-enhancing or inhibitory effects. Because γδ T cells can be activated via various pathways, we questioned whether the nature of their activation might impact their function. In this study, we show that γδ T cells activated under different inflammatory conditions differ greatly in their functions. Whereas anti-CD3 treatment activated both IFN-γ+ and IL-17+ γδ T cells, cytokines preferentially activated IL-17+ γδ T cells. γδ T cells continued to express high levels of surface CD73 after exposure to inflammatory cytokines, but they downregulated surface CD73 after exposure to dendritic cells. Although both CD73high and CD73low cells have a disease-enhancing effect, the CD73low γδ T cells are less inhibitory. We also show that polarized activation not only applies to αß T cells and myeloid cells, but also to γδ T cells. After activation under Th17-polarizing conditions, γδ T cells predominantly expressed IL-17 (gdT17), but after activation under Th1 polarizing conditions (gdT1) they mainly expressed IFN-γ. The pro-Th17 activity of γδ T cells was associated with gdT17, but not gdT1. Our results demonstrate that the functional activity of γδ T cells is strikingly modulated by their activation level, as well as the pathway through which they were activated.

Timing Effect of Adenosine-Directed Immunomodulation on Mouse Experimental Autoimmune Uveitis.

Adenosine is an important regulatory molecule of the immune response. We have previously reported that treatment of experimental autoimmune uveitis (EAU)-prone mice with an adenosine-degrading enzyme (adenosine deaminase) prohibited EAU development by inhibiting Th17 pathogenic T cell responses. To further validate that the targeting of adenosine or adenosine receptors effectively modulates Th17 responses, we investigated the effect of adenosine receptor antagonists. In this study, we show that the A2AR antagonist SCH 58261 (SCH) effectively modulates aberrant Th17 responses in induced EAU. However, timing of the treatment is important. Whereas SCH inhibits EAU when administered during the active disease stage, it did not do so if administered during quiescent disease stages, thus implying that the existing immune status influences the therapeutic effect. Mechanistic studies showed that inhibition of γδ T cell activation is crucially involved in adenosine-based treatment. Adenosine is an important costimulator of γδ T cell activation, which is essential for promoting Th17 responses. During ongoing disease stages, adenosine synergizes with existing high levels of cytokines, leading to augmented γδ T cell activation and Th17 responses, but in quiescent disease stages, when existing cytokine levels are low, adenosine does not enhance γδ T cell activation. Our results demonstrated that blockade of the synergistic effect between adenosine and inflammatory cytokines at active disease stages can ameliorate high-degree γδ T cell activation and, thus, suppress Th17 pathogenic T cell responses.

Rod photoreceptor clearance due to misfolded rhodopsin is linked to a DAMP-immune checkpoint switch.

Chronic endoplasmic reticulum stress resulting from misfolding of the visual pigment rhodopsin (RHO) can lead to loss of rod photoreceptors, which initiates retinitis pigmentosa, characterized initially by diminished nighttime and peripheral vision. Cone photoreceptors depend on rods for glucose transport, which the neurons use for assembly of visual pigment-rich structures; as such, loss of rods also leads to a secondary loss of cone function, diminishing high-resolution color vision utilized for tasks including reading, driving, and facial recognition. If dysfunctional rods could be maintained to continue to serve this secondary cone preservation function, it might benefit patients with retinitis pigmentosa, but the mechanisms by which rods are removed are not fully established. Using pigs expressing mutant RHO, we find that induction of a danger-associated molecular pattern (DAMP) "eat me" signal on the surface of mutant rods is correlated with targeting the live cells for (PrCR) by retinal myeloid cells. Glucocorticoid therapy leads to replacement of this DAMP with a "don't eat me" immune checkpoint on the rod surface and inhibition of PrCR. Surviving rods then continue to promote glucose transport to cones, maintaining their viability.

Short chain fatty acids inhibit endotoxin-induced uveitis and inflammatory responses of retinal astrocytes.

Short chain fatty acids (SCFAs) are produced by gut microbiota as fermentation products of digestion-resistant oligosaccharides and fibers. Their primary roles are functioning as major energy sources for colon cells and assisting in gut homeostasis by immunomodulation. Recent evidence suggests that they affect various organs both at cellular and molecular levels, and regulate functions in distance sites including gene expression, cell proliferation, cell differentiation, apoptosis and inflammation. In this study, we examined whether SCFAs are present in the mouse eye and whether SCFAs affect inflammatory responses of the eye and retinal astrocytes (RACs). We observed that intra-peritoneal injected SCFAs were detected in the eye and reduced intraocular inflammation induced by lipopolysaccharide (LPS). Moreover, SCFAs displayed two disparate effects on LPS-stimulated RACs - namely, cytokine and chemokine production was reduced, but the ability to activate T cells was enhanced. Our results support the existence of gut-eye cross talk and suggest that SCFAs can cross the blood-eye-barrier via the systemic circulation. If applied at high concentrations, SCFAs may reduce inflammation and impact cellular functions in the intraocular milieu.

Yap1 is required for maintenance of adult RPE differentiation.

Nuclear YAP1 plays a critical role in regulation of stem cell proliferation, tissue regeneration, and organ size in many types of epithelia. Due to rapid turnover of most epithelial cell types, the cytoplasmic function of YAP1 in epithelial cells has not been well studied. The retinal pigment epithelium (RPE) is a highly polarized epithelial cell type maintained at a senescence state, and offers an ideal cell model to study the active role of YAP1 in maintenance of the adult epithelial phenotype. Here, we show that the cytoplasmic function of YAP1 is essential to maintain adult RPE differentiation. Knockout of Yap1 in the adult mouse RPE caused cell depolarization and tight junction breakdown, and led to inhibition of RPE65 expression, diminishment of RPE pigments, and retraction of microvilli and basal infoldings. These changes in RPE further prompted the loss of adjacent photoreceptor outer segments and photoreceptor death, which eventually led to decline of visual function in older mice between 6 and 12 months of age. Furthermore, nuclear ß-catenin and its activity were significantly increased in mutant RPE. These results suggest that YAP1 plays an important role in active inhibition of Wnt/ß-catenin signaling, and is essential for downregulation of ß-catenin nuclear activity and prevention of dedifferentiation of adult RPE.

Sustained dasatinib treatment prevents early fibrotic changes following ocular trauma.

PURPOSE: Posterior ocular trauma and the subsequent fibrotic retinal complication termed proliferative vitreoretinopathy (PVR) are leading causes of blindness in children and young adults. A previous study suggested that changes occurring within the first month post-trauma can lead to development of PVR later. The aim of this study was to examine the effect of dasatinib, a tyrosine kinase inhibitor clinically used to treat chronic myeloid leukemia, on fibrotic changes occurring within the first month following ocular trauma. METHODS: A previously established swine ocular trauma model that mimics both contusion and penetrating injuries was used. Dasatinib was administered on days 4 and 18 post-trauma via intravitreal injection of either bolus solution or suspension of a sustained release system incorporated in biodegradable poly (lactic-co-glycolic acid) (PLGA) nanoparticles. Animals were followed up to day 32, and the development of traction full-thickness fold in the posterior retina was assessed. RESULTS: A full-thickness retinal fold extending from the wound site developed in 3 out of 4 control eyes injected with PLGA nanoparticles alone at 1 month. Administration of dasatinib solution had little preventative effect with 6 out of 7 eyes developing a fold. In contrast, dasatinib-incorporated PLGA nanoparticle injection significantly reduced the incidence of fold to 1 out of 10 eyes. CONCLUSIONS: Injection of dasatinib-incorporated PLGA significantly reduced early fibrotic retinal changes which eventually lead to PVR following posterior ocular trauma. Thus, our sustained dasatinib release system can potentially be used to both prevent and/or broaden the surgical treatment window for PVR.

Vaccination with circulating exosomes in autoimmune uveitis prevents recurrent intraocular inflammation.

BACKGROUND: Exosomes participate in intercellular communication and act as important molecular vehicles in the regulation of numerous physiological and pathological processes, including autoimmune development. The role of circulating exosomes in the development of autoimmune uveitis is unknown. In this study, using the rat model of experimental autoimmune uveitis, which has clinical and histological features of pan uveitis in man, we evaluated the immunoregulatory function of circulating exosomes. METHODS: Experimental autoimmune uveitis was induced in Lewis rats either immunised with interphotoreceptor retinoid-binding protein R16 peptides or injected with activated R16-specific T cells. The disease incidence and severity were examined by indirect fundoscopy and flow cytometry. Circulating exosomes were isolated from peripheral blood of naïve and Day 14 R16 immunised Lewis rats. The effect of exosomes on specific T cells was evaluated by R16-specific T cell proliferation, cytokine production and recurrent uveitis induction. RESULTS: Circulating exosomes derived from active immunised uveitis rats selectively inhibited immune responses of R16-specific T cells in vitro. Vaccination of naïve rats with these exosomes reduced the incidence of recurrent uveitis in an antigen-specific manner. Antigen-specific uveitogenic T cells reduced IFN-γ production and increased IL-10 after vaccination. CONCLUSIONS: Circulating exosomes in autoimmune uveitis have the potential to be a novel treatment for recurrent autoimmune uveitis.

Adenosine receptor activation in the Th17 autoimmune responses of experimental autoimmune uveitis.

Th17-type autoreactive T cells contribute to pathogenicity in autoimmune diseases, including autoimmune uveitis. However, the mechanisms of regulation of Th17 cell activities remain unsolved and are likely to be tissue- and disease specific. In this review, we have summarized our studies from the murine model of experimental autoimmune uveitis (EAU). The resultsdemonstrate that γδ T cells have a regulatory effect on Th17 response. The regulatory effects of γδ T cells depend on their action state. Activated γδ T cells express significantly high levels of adenosine receptor A2 (A2AR) but low CD73. Both molecules are crucially involved in adenosine generation, thus modifying T cell responses. While the increased expression of A2AR-allows activated γδ T cells to bind adenosine more effectively than other immune cells, the decreased CD73 restricts their ability to convert AMP to adenosine. Adenosine affects Th1 and Th17 autoimmune responses differently. Its activation of γδ T cells shifts the Th1/Th17 balance towards the Th17 autoreactivity.

Connection between γδ T-cell- and Adenosine- Mediated Immune Regulation in the Pathogenesis of Experimental Autoimmune Uveitis.

Regulatory effects of γδ T-cells on immune responses have been studied for years. We have investigated the regulatory effect of γδ T-cells on Th1 and Th17 autoimmune responses, and have studied molecular and cellular mechanisms by which γδ T-cells enhance or inhibit immune responses, exploiting a well-characterized murine model of experimental autoimmune uveitis (EAU). Our results show that (1) aberrant γδ T-cell activation is an important pathogenic event in EAU; (2) γδ T-cells have a unique regulatory effect on Th17 autoimmune responses, which is shaped by the activation status of γδ T-cells; and (3) γδ-mediated immunoregulation is closely linked with the extracellular adenosine metabolism. Reciprocal interactions between γδ T-cells and extracellular adenosine partially determine the development of EAU.

Functional Conversion and Dominance of γδ T Subset in Mouse Experimental Autoimmune Uveitis.

We have previously shown that activated γδ T cells have a much stronger proinflammatory effect in the development of experimental autoimmune uveitis than their nonactivated counterparts. Our present study explored γδ T cell subsets are functionally distinct in autoimmune pathogenesis and determined the pathogenic contribution of biased Vγ4+ γδ T cell activation in this disease. By systematically comparing two major peripheral γδ T cell subsets, the Vγ1+ and the Vγ4+ cells, we found that the Vγ4+ cells were readily activated in B6 mice during experimental autoimmune uveitis development, whereas Vγ1+ cells remained nonactivated. Cytokines that were abundantly found in the serum of immunized mice activated Vγ4+, but did not activate Vγ1+, cells. The Vγ4+ cells had a strong proinflammatory activity, whereas the Vγ1+ cells remained nonactivated when tested immediately after isolation from immunized mice. However, when the Vγ1+ cells were activated in vitro, they promoted inflammation. Our results demonstrated that activation is a major factor in switching the enhancing and inhibiting effects of both Vγ1+ and Vγ4+ γδ T cell subsets, and that γδ T cell subsets differ greatly in their activation requirements. Whether the enhancing or inhibiting function of γδ T cells is dominant is mainly determined by the proportion of the γδ T cells that are activated versus the proportion not activated.

Sphere-Induced Rejuvenation of Swine and Human Müller Glia Is Primarily Caused by Telomere Elongation.

Müller cells are the major supportive and protective glial cells in the retina with important functions in histogenesis and synaptogenesis during development, and in maintenance of mature neurons as they show to secrete various cytokines and manifest potentials of self-renewal and transdifferentiation into retinal neurons following injury in the vertebrate retinas. The swine retina has a visual streak structure similar to the human macular where cone photoreceptors are highly concentrated, thereby can serve as a better model for studying retinal diseases and for formulating cell-based therapeutics than the rodent retinas. Like most differentiated somatic mammalian cells, the isolated swine and human Müller glia become senescent over passages in culture, which restricts their potential application in basic and clinic researches. Here, we demonstrate that the senescence of swine and human Müller cells is caused by telomere attrition upon multiplications in vitro; and the senescent cells can be rejuvenated by sphere suspension culture. We also provide evidence that sphere-induced extension of telomeres in swine and human Müller glia is achieved by alternative lengthening of telomeres or/and by telomerase activation. Stem Cells 2017;35:1579-1591.

Regulation of Adenosine Deaminase on Induced Mouse Experimental Autoimmune Uveitis.

Adenosine is an important regulator of the immune response, and adenosine deaminase (ADA) inhibits this regulatory effect by converting adenosine into functionally inactive molecules. Studies showed that adenosine receptor agonists can be anti- or proinflammatory. Clarification of the mechanisms that cause these opposing effects should provide a better guide for therapeutic intervention. In this study, we investigated the effect of ADA on the development of experimental autoimmune uveitis (EAU) induced by immunizing EAU-prone mice with a known uveitogenic peptide, IRBP1-20. Our results showed that the effective time to administer a single dose of ADA to suppress induction of EAU was 8-14 d postimmunization, shortly before EAU expression; however, ADA treatment at other time points exacerbated disease. ADA preferentially inhibited Th17 responses, and this effect was γδ T cell dependent. Our results demonstrated that the existing immune status strongly influences the anti- or proinflammatory effects of ADA. Our observations should help to improve the design of ADA- and adenosine receptor-targeted therapies.

Focal adhesion kinase family is involved in matrix contraction by transdifferentiated Müller cells.

Transdifferentiated Müller cells that adopt a fibroblastic/myofibroblastic phenotype have been identified in epiretinal membranes (ERMs) in several ocular disorders, and have been implicated to play a role in the formation and/or the contraction of ERMs. We have previously demonstrated that dasatinib, a dual inhibitor of Src-family kinases and Abl kinase, can prevent matrix contraction by transdifferentiated Müller cells. In this study, we examined molecules involved in matrix contraction downstream of primary dasatinib targets. Tyrosine phosphorylation of focal adhesion kinase (FAK) family members FAK and PYK2 was significantly reduced by dasatinib, and select inhibitors for these kinases PF431396, which inhibits both FAK and PYK2, and PF573228, which only inhibits FAK and not PYK2, significantly reduced matrix contraction by transdifferentiated Müller cells. Dasatinib and PF431396 significantly reduced phosphorylation of Hic-5, a protein implicated to play a role in focal adhesions and cell signaling. Our data shows that FAK family members are involved in matrix contraction by transdifferentiated Müller cells, and also implicates that Hic-5 is situated downstream of the FAK family within the signaling pathway.

Role of epithelial-mesenchymal transition in proliferative vitreoretinopathy.

Proliferative vitreoretinopathy (PVR) is a potentially blinding fibrotic complication. It is caused by the formation and contraction of epiretinal membranes (ERMs) that ultimately lead to retinal folds and traction retinal detachments. While multiple cell types have been identified in ERMs, retinal pigment epithelial (RPE) cells have long been implicated as a key player in the pathophysiology of PVR. Clinical and experimental evidence has shown that RPE cells undergo epithelial-mesenchymal transition (EMT) to adopt a fibroblastic phenotype. Cell-cell adhesions maintained by adherens and tight junctions are important for the maintenance of RPE phenotype, and disruption of these junctional complexes results in EMT via activation of signaling pathways such as ß-catenin/Wnt and Hippo signaling, as well as transcription factors involving Zeb1, Snail, and ZONAB. Upon EMT, RPE cells can further differentiate into myofibroblasts in the presence of TGF-ß with cytoskeletal tension mediated by RhoGTPase. These fibroblasts and myofibroblasts derived from RPE cells can contribute to ERM formation by cell migration, proliferation and matrix modification, and play a key role in ERM contraction. It is not solely the proliferation of these cells that results in PVR but rather the contraction of these cells in the ERM.

Anti-inflammatory or proinflammatory effect of an adenosine receptor agonist on the Th17 autoimmune response is inflammatory environment-dependent.

Adenosine is a key endogenous signaling molecule that regulates a wide range of physiological functions, including immune system function and inflammation. Studies have shown that adenosine receptor (AR) agonists can be either anti-inflammatory or proinflammatory in immune responses and in inflammation, and the clarification of the mechanisms causing these opposing effects should provide a better guide for therapeutic intervention. Whereas previous studies mostly examined the effects of AR agonists on Th1-type immune responses, in this study, we compared their effect on Th17 and Th1 autoimmune responses in experimental autoimmune uveitis, a mouse model of human uveitis induced by immunization with the human interphotoreceptor retinoid-binding protein peptides 1-20. We showed that injection of mice with a nonselective AR agonist, 5'-N-ethylcarboxamidoadenosine (NECA), at an early stage after immunization had an inhibitory effect on both Th1 and Th17 responses, whereas injection of the same amount of NECA at a late stage inhibited the Th1 response but had an enhancing effect on the Th17 response. We also showed that the effects of NECA on Th1 and Th17 responses were completely dissociated, that the enhancing effect of NECA on Th17 responses was modulated by γδ T cells, and that the response of γδ T cells to NECA was determined by their activation status. We conclude that the inflammatory environment has a strong impact on converting the effect of AR agonist on the Th17 autoimmune response from anti-inflammatory to proinflammatory. Our observation should help in the designing of better AR-targeted therapies.

HMGB1 release triggered by the interaction of live retinal cells and uveitogenic T cells is Fas/FasL activation-dependent.

BACKGROUND: It is not clear how invading autoreactive T cells initiate the pathogenic process inside the diseased organ in T cell-mediated organ-specific autoimmune disease. In experimental autoimmune uveitis (EAU) induced by adoptive transfer of interphotoreceptor retinoid-binding protein (IRBP)-specific T cells in mice, we have previously reported that intraocular inflammation was initiated by infiltrating IRBP-specific T cells that directly interacted with retinal cells and resulted in the active release of high mobility group box 1 (HMGB1), an important member of damage associate molecular patterns (DAMPs). Furthermore, blockade of HMGB1 in our murine model reduced intraocular inflammation via suppression of IRBP-specific T cell functions. These results have demonstrated that HMGB1 is an early and critical mediator of induction of intraocular inflammation. The present study identified the cell surface molecule that triggers HMGB1 secretion. METHODS: Retinal explants from Fas-deficient (Fas(lpr)) and wild-type (Wt) C57BL/6 (B6) mice were cultured with activated IRBP 1-20 peptide-specific T cells or with a Fas-activating antibody (Jo2), and then the level of HMGB1 in culture supernatants were detected by ELISA. In addition, released HMGB1 was examined in the eye of Fas(lpr) and Wt mice after IRBP-specific T cell transfer. Uveitis was evaluated in the IRBP-specific T cell transferred Fas(lpr) mice after recombinant HMGB1 was restored within the eye and in the IRBP-specific T cell transferred Wt mice after they were treated with a Fas antagonist (Met12). RESULTS: In contrast to retinal explants from Wt mice, those from Fas(lpr) mice did not release HMGB1 after exposure to IRBP-specific T cells or to Jo2. The release of HMGB1 by Wt retinal explants was suppressed by Met 12. Moreover, after IRBP-specific T cell injection, Fas(lpr) mice did not release HMGB1 in the eye or develop EAU, but intravitreous injection of HMGB1 resulted in intraocular inflammation. Finally, tEAU in Wt mice was attenuated by local treatment with Met 12. Unlike HMGB1, Fas-induced IL-1 and IL-18 were not essential for tEAU induction. CONCLUSION: Our results show that interaction of retinal cells with infiltrating uveitogenic T cells leads to rapid release of HMGB1 via the Fas/FasL inflammatory signaling pathway.

Role of retinal pigment epithelial cell ß-catenin signaling in experimental proliferative vitreoretinopathy.

Proliferative vitreoretinopathy is caused by the contraction of fibrotic membranes on the epiretinal surface of the neurosensory retina, resulting in a traction retinal detachment and loss of visual acuity. Retinal pigment epithelial (RPE) cells play an important role in formation of such fibrotic, contractile membranes. We investigated the role of Wnt/ß-catenin signaling, a pathway implicated in several fibrotic diseases, in RPE cells in proliferative vitreoretinopathy. In vitro culture of swine RPE sheets resulted in nuclear translocation of ß-catenin in dedifferentiated RPE cells. FH535, a specific inhibitor of ß-catenin signaling, reduced the outgrowth of cultured RPE sheets and prevented dedifferentiated RPE cell proliferation and migration. It also inhibited formation of contractile membranes by dedifferentiated RPE cells on collagen I matrices. Expression and function of the ß-catenin signaling target connexin-43 were down-regulated by FH535, and functional blockade of connexins with carbenoxolone also prevented the in vitro formation of fibrotic, contractile membranes. Intravitreal injection of FH535 in swine also inhibited formation of dense, contractile membranes on the epiretinal surface and prevented development of traction retinal detachment. These findings demonstrate that ß-catenin signaling is involved in formation of contractile membranes by dedifferentiated RPE cells and suggest that adjunctive treatment targeting this pathway could be useful in preventing proliferative vitreoretinopathy.

The role of Th17-associated cytokines in the pathogenesis of experimental autoimmune uveitis (EAU).

The proinflammatory and pathogenic function of Th17 cells in autoimmune diseases have been established but the mechanism by which such cells cause disease remains to be determined. Inflammatory cytokines produced by Th17 cells may either promote or inhibit disease development. The major cytokines produced by the uveitogenic T cells, such as IL-17 and IL-22, are not always pathogenic, and the disease-inducing ability of pathogenic T cells is not immediately correlated to the amount of cytokine they produce. Future studies identifying factors causing increased Th17 responses and determining the types of cells that regulating Th17 autoreactive T cells should facilitate our effort of understanding Th17-mediated disease pathogenesis.

Dasatinib affects focal adhesion and myosin regulation to inhibit matrix contraction by Müller cells.

Epiretinal membrane (ERM) contraction is associated with a variety of ocular diseases that cause macular dysfunction. Trans-differentiated Müller cells have been identified in ERMs, and have been implicated to be involved in the contractile process. In this study, we tested the effect of dasatinib, an FDA-approved tyrosine kinase inhibitor, on matrix contraction caused by Müller cells, and examined molecular mechanism of action. Type I collagen matrix contraction assays were used to examine the effect of drugs on matrix contraction by trans-differentiated Müller cells. Fluophore-conjugated phalloidin was used for the detection of actin cytoskeleton, and Western-blot analyses were carried out to examine protein expression and phosphorylation status. Dasatinib inhibited collagen matrix contraction by trans-differentiated Müller cells that was associated with decreased cell spreading and reduction of actomyosin stress fibers. Concomitantly, dasatinib-treated Müller cells had reduced phosphorylation of Src family kinase, paxillin, as well as myosin II light chain. Specific inhibitors of Rho/ROCK and myosin II confirmed the critical role played by this pathway in Müller cell contraction. Our data demonstrate that dasatinib significantly reduced matrix contraction by Müller cells via inhibition of focal adhesion, as well as actomyosin contraction.

IL-23 receptor expression on γδ T cells correlates with their enhancing or suppressive effects on autoreactive T cells in experimental autoimmune uveitis.

We have previously reported that, depending on their activation status, mouse γδ T cells can either enhance or inhibit the activity of IL-17(+) autoreactive T cells in experimental autoimmune uveitis. In this study, we showed that γδ T cells in naive C57BL/6 (B6) mouse do not express the IL-23R, whereas in immunized mice, it is expressed on >50% of γδ T cells. In vitro studies showed that IL-23R expression on γδ T cells is modulated by their state of activation, as weakly activated γδ T cells expressed the IL-23R, but highly activated γδ T cells did not. Functional studies showed that IL-23R(+) γδ T cells had the strongest suppressive effect on IL-17(+) autoreactive T cells, and that this effect was inhibited when the IL-23R was blocked by anti-IL-23R Ab or in the presence of excessive amounts of exogenous IL-23. We conclude that the balance between the enhancing and inhibitory effects of γδ T cells is regulated by their level of IL-23R expression. The expression of variable IL-23R levels allows γδ T cells to have different regulatory effects on adaptive immune responses, conceivably as a result of αß and γδ T cells competing for IL-23.