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The interaction between follicular T helper cells (TFH) and B cells in the lymph nodes and spleen has a major impact on the development of antigen-specific B cell responses during infection or vaccination. Recent studies described a functional equivalent of these cells among circulating CD4 T cells, referred to as peripheral TFH cells. Here, we characterize the phenotype and in vitro B cell helper activity of peripheral TFH populations, as well as the effect of HIV infection on these populations. In co-culture experiments we confirmed CXCR5+ cells from HIV-uninfected donors provide help to B cells and more specifically, we identified a CCR7(high)CXCR5(high)CCR6(high)PD-1(high) CD4 T cell population that secretes IL-21 and enhances isotype-switched immunoglobulin production. This population is significantly decreased in treatment-naïve, HIV-infected individuals and can be recovered after anti-retroviral therapy. We found impaired immunoglobulin production in co-cultures from HIV-infected individuals and found no correlation between the frequency of peripheral TFH cells and memory B cells, or with neutralization activity in untreated HIV infection in our cohort. Furthermore, we found that within the peripheral TFH population, the expression level of TFH-associated genes more closely resembles a memory, non-TFH population, as opposed to a TFH population. Overall, our data identify a heterogeneous population of circulating CD4 T cells that provides in vitro help to B cells, and challenges the origin of these cells as memory TFH cells.

CD4 T follicular helper (TFH) cells interact with and stimulate the generation of antigen-specific B cells. TFH cell interaction with B cells correlates with production of SIV-specific immunoglobulins. However, the fate of TFH cells and their participation in SIV-induced antibody production is not well understood. We investigated the phenotype, function, location, and molecular signature of TFH cells in rhesus macaques. Similar to their human counterparts, TFH cells in rhesus macaques represented a heterogeneous population with respect to cytokine function. In a highly differentiated subpopulation of TFH cells, characterized by CD150lo expression, production of Th1 cytokines was compromised while IL-4 production was augmented, and cells exhibited decreased survival, cycling, and trafficking capacity. TFH cells exhibited a distinct gene profile that was markedly altered by SIV infection. TFH cells were infected by SIV; yet, in some animals, these cells actually accumulated during chronic SIV infection. Generalized immune activation and increased IL-6 production helped drive TFH differentiation during SIV infection. Accumulation of TFH cells was associated with increased frequency of activated germinal center B cells and SIV-specific antibodies. Therefore, chronic SIV does not disturb the ability of TFH cells to help B cell maturation and production of SIV-specific immunoglobulins.

Purpose: To extensively characterize the complex network of cytokines present in uveitis aqueous humor (AqH), and the relationships between cytokines and the cellular infiltrate., Methods: AqH from noninflammatory control subjects and patients with idiopathic, Fuchs' heterochromic cyclitis (FHC), and herpes-viral or Behçet's uveitis were analyzed for IL-1beta, -2, -4, -5, -7, -8, -10, -12, -13, -15, TNFalpha, IFNgamma, CCL2 (MCP-1), CCL5 (RANTES), CCL11 (Eotaxin), TGFbeta2, and CXCL12 (SDF-1), using multiplex bead immunoassays. The cellular infiltrate was also determined for each sample., Results: Idiopathic uveitis AqH, compared with noninflammatory controls, was characterized by high levels of IL-6, IL-8, CCL2 and IFNgamma, the levels of which correlated with each other. For IL-6 and IL-8 these levels were proportional to the number of neutrophils present. By contrast, the levels of both TGFbeta2 and CXCL12 decreased in idiopathic uveitis AqH with increasing inflammation. Cluster analysis showed a degree of segregation between noninflammatory and idiopathic uveitis AqH. Further examination using random forest analysis yielded a complete distinction between these two groups. The minimum cytokines required for this classification were IL-6, IL-8, CCL2, IL-13, TNFalpha, and IL-2., Conclusions: Application of multiplex bead immunoassays has allowed us to identify distinct patterns of cytokines that relate to both clinical disease and the cellular infiltrates present. Bioinformatics analysis allowed identification of cytokines that differentiate idiopathic uveitis from noninflammatory control AqH and are likely to be important for the pathogenesis of uveitis.

A fundamental mechanism of immune privilege in the eye is the induction of T lymphocyte apoptosis. Intraocular inflammation in uveitis implies compromise of immune privilege. This study sought to determine whether apoptosis of T cells is actively inhibited in patients with uveitis and by what pathways this may occur. Apoptotic lymphocytes were found to be absent from aqueous humor (AqH) of virtually all patients with recent-onset uveitis. However, T cells removed from the eye were highly susceptible to both spontaneous and Fas ligand-induced apoptosis in vitro. AqH from patients with uveitis had no modulatory effect on Fas ligand-induced apoptosis, but strongly suppressed survival factor deprivation-induced apoptosis. In contrast, noninflammatory AqH from patients undergoing cataract surgery had no modulatory effects on apoptosis at all. These data suggest that triggering of the Fas pathway is diminished in uveitis, and also that homeostatic resolution through survival factor deprivation-induced apoptosis is inhibited by factors present in AqH. The most widely recognized pathways, common gamma-chain cytokines and type I IFNs, did not contribute to AqH-mediated T cell survival. High levels of both IL-6 and soluble IL-6R were found in AqH. IL-6 alone did not induce T cell survival, because IL-6R expression on T cells in AqH was too low to facilitate signaling. However, combinations of IL-6 and soluble IL-6R were highly effective inhibitors of T cell apoptosis, suggesting that the trans-signaling pathway is likely to be a key mediator of T cell apoptosis inhibition mediated by uveitis AqH.

The migration and infiltration of cells into the eye whether blood-borne leucocytes, endothelial or epithelial cells occurs in many ocular diseases. Dysregulation of this process is apparent in chronic inflammation, corneal graft rejection, allergic eye disease and other sight-threatening conditions. Under normal and inflammatory conditions, chemokines and their receptors are important contributors to cell migration. To date, 47 chemokines and 19 chemokine receptors have been identified and characterised. In recent years, investigations into the role of chemokines and their receptors in ocular disease have generated an increasing number of publications. In the eye, the best understood action of these molecules has arisen from the study of their ability to control the infiltration of leucocytes in uveitis. However, the involvement of chemokines in angiogenesis in several ocular conditions and in the survival of corneal transplants demonstrates the multifaceted nature of their effects. Interestingly, the constitutive expression of chemokines and their receptors in ocular tissues suggests that certain chemokines have a homeostatic function. In this review, we discuss the nature and function of chemokines in health and disease, and describe the role of chemokines in the pathogenesis of different ocular conditions.

Overexpression of the constitutive chemokine receptor CXCR4 has been shown to contribute to the accumulation of leukocytes at sites of chronic inflammation. Glucocorticoids are widely used to treat inflammatory disorders such as uveitis to considerable effect, yet paradoxically have been reported to increase CXCR4 expression in vitro. We show here that ocular lymphocytes isolated from patients with uveitis who had been treated with topical glucocorticoids expressed highly elevated levels of CXCR4. The up-regulation of CXCR4 could be reproduced in vitro by culture of CD4(+) T cells with aqueous humor (AqH), indicating a role for the ocular microenvironment rather than preferential recruitment of CXCR4(+) cells. Untreated uveitis and noninflammatory AqH up-regulated CXCR4 to a limited extent; this was dependent on TGF-beta2. However, the highest levels of CXCR4 both in vivo and in vitro were found in the glucocorticoid-treated patients. Glucocorticoids appeared to be directly responsible for the induction of CXCR4 in treated patients, as the glucocorticoid receptor antagonist RU38486 inhibited the in vitro up-regulation by AqH from these patients. Dexamethasone selectively up-regulated CXCR4 in vitro, but not any of a wide range of other chemokine receptors. CXCL12, the ligand for CXCR4, was present in AqH under noninflammatory conditions, but the levels were low in untreated uveitis and undetectable in treated uveitis AqH. The importance of these results for the treatment of HIV patients with glucocorticoids is discussed as well as a role for glucocorticoid-induced CXCR4 up-regulation and CXCL12 down-regulation in controlling the migration of lymphocyte populations, resulting in resolution of inflammation.