Reduced skin homing by functional Treg in vitiligo

In human vitiligo, cutaneous depigmentation involves cytotoxic activity of autoreactive T cells. It was hypothesized that depigmentation can progress in the absence of regulatory T cells (Treg). The percentage of Treg among skin infiltrating T cells was evaluated by immunoenzymatic double staining for CD3 and FoxP3, revealing drastically reduced numbers of Treg in non-lesional, perilesional and lesional vitiligo skin. Assessment of the circulating Treg pool by FACS analysis of CD4, CD25, CD127 and FoxP3 expression, and mixed lymphocyte reactions in presence and absence of sorted Treg revealed no systemic drop in the abundance or activity of Treg in vitiligo patients. Expression of skin homing receptors CCR4, CCR5, CCR8 and CLA was comparable among circulating vitiligo and control Treg. Treg from either source were equally capable of migrating towards CCR4 ligand and skin homing chemokine CCL22, yet significantly reduced expression of CCL22 in vitiligo skin observed by immunohistochemistry may explain failure of circulating, functional Treg to home to the skin in vitiligo. The paucity of Treg in vitiligo skin is likely crucial for perpetual anti-melanocyte reactivity in progressive disease.     

IL‐17A is not a treatment target in progressive vitiligo

Multiple reports confirm elevated circulating IL‐17 levels and increased numbers of Th17 lymphocytes in patients with non‐segmental vitiligo. Additionally, melanocyte damaging characteristics have been ascribed to IL‐17. A single‐arm pilot study using secukinumab in active non‐segmental vitiligo was conducted. The large majority of patients developed additional skin depigmentations limiting further enrollment. Overall, laboratory analysis revealed no change in secreted chemokines or Th subsets. Th17 lymphocytes correlated with Th2, Th9, and Th22 cells while an inverse link with Th1 cells and serum sCD25 levels was observed. In contrast, Th17.1 cells correlated positively with Th1 lymphocytes. Confirmatory results were found in an independent group of patients with vitiligo showing a significant increase in Th17.1 and Th1 lymphocytes in progressive vitiligo patients compared to healthy controls, which was not found for Th17 cells. These results do not support a direct pathogenic role of IL‐17 or Th17 cells in vitiligo. Nonetheless, a delicate Th17/Th17.1/Th1 balance seems evident which changes markedly according to disease activity. This may offer new treatment options by interfering with cytokines that drive differentiation of Th17 cells toward Th1.     

T cell helper defect in patients with chronic lymphocytic leukemia.

Purified peripheral blood T lymphocytes from normal donors were shown to help allogeneic tonsillar B cells to differentiate and secrete specific anti-SRBC antibody in vitro in a plaque-forming assay. Utilizing this system, a comparison was made between the allogeneic helper activity generated by the T cells of normal individuals and patients with various disease states. Allogeneic helper activity was absent when T lymphocytes from patients with CLL were used. Conversely, relatively normal allogeneic helper function was provided by T cells of patients with a variety of other disorders studied. Thus, a functional deficiency was identified in CLL patients in the subpopulation of regulatory T cells responsible for providing helper activity in allogeneic interactions.     

Successful treatment of metastatic melanoma by adoptive transfer of blood-derived polyclonal tumor-specific CD4+ and CD8+ T cells in combination with low-dose interferon-alpha

A phase I/II study was conducted to test the feasibility and safety of the adoptive transfer of tumor-reactive T cells and daily injections of interferon-alpha (IFNα) in metastatic melanoma patients with progressive disease. Autologous melanoma cell lines were established to generate tumor-specific T cells by autologous mixed lymphocyte tumor cell cultures using peripheral blood lymphocytes. Ten patients were treated with on average 259 (range 38–474) million T cells per infusion to a maximum of six infusions, and clinical response was evaluated according to the response evaluation criteria in solid tumors (RECIST). Five patients showed clinical benefit from this treatment, including one complete regression, one partial response, and three patients with stable disease. No treatment-related serious adverse events were observed, except for the appearance of necrotic-like fingertips in one patient. An IFNα-related transient leucopenia was detected in 6 patients, including all responders. One responding patient displayed vitiligo. The infused T-cell batches consisted of tumor-reactive polyclonal CD8+ and/or CD4+ T cells. Clinical reactivity correlated with the functional properties of the infused tumor-specific T cells, including their in vitro expansion rate and the secretion of mainly Th1 cytokines as opposed to Th2 cytokines. Our study shows that relatively low doses of T cells and low-dose IFNα can lead to successful treatment of metastatic melanoma and reveals a number of parameters potentially associated with this success.     

Modulation of chronic lymphocytic leukemia (CLL) lymphocyte phenotypes by in vitro incubation with alpha 1 thymosin

In this study an attempt was made to elucidate (1) the level(s) of differentiation arrest of B cells, and (2) whether T-cell functional defects in CLL patients are related to their defective maturation. In addition, an attempt was also made to induce and/or correct maturation of T cells in CLL patients by in vitro incubation with alpha 1 thymosin. In CLL patients and controls, we determined the percentage of T and B cells with T11, T8, T4, C3, and mouse erythrocyte (ME) receptors, along with T-cell functional reactivity (measured by local xenogeneic graft vs host reaction), before and after incubation with alpha 1 thymosin. In about 60% of stable CLL patients, and in 80% of those in the progressive phase of disease, T cells possess receptors for ME and/or C3. After incubation with alpha 1 thymosin, separate analysis of surface markers on T and B cells revealed (along with the induction of T11 receptors on T-cell surface) induction of ME receptors on T and B cells in stable phase and selective loss of ME receptors on B cells in the progressive phase of CLL. After incubation of normal lymphocytes with alpha 1 thymosin, we observed an increase of T8 receptors, no change in expression of T11, and a decrease of T4 receptors along with the increase of the intensity of T-cell functional reactivity. In contrast, in CLL patients following incubation with alpha 1 thymosin, the induction of T8 receptors was less prominent in the progressive than in the stable phase of disease. Furthermore, induction of T8 receptors in CLL patients in the stable phase was accompanied by recovery of impaired or increase of preserved functional T-cell reactivity. In the progressive phase, however, T-cell functional areactivity remained unchanged. The findings suggest that different levels of B-cell-differentiation arrest along with defective maturation of T cells might be responsible for the spectrum of disease evolution in CLL.     

Loss of immunological tolerance in Gimap5-deficient mice is associated with loss of Foxo in CD4+ T cells

Previously, we reported the abrogation of quiescence and reduced survival in lymphocytes from Gimap5(sph/sph) mice, an ENU germline mutant with a missense mutation in the GTPase of immunity-associated protein 5 (Gimap5). These mice showed a progressive loss of peripheral lymphocyte populations and developed spontaneous colitis, resulting in early mortality. In this study, we identify the molecular pathways that contribute to the onset of colitis in Gimap5(sph/sph) mice. We show that CD4(+) T cells become Th1/Th17 polarized and are critically important for the development of colitis. Concomitantly, regulatory T cells become reduced in frequency in the peripheral tissues, and their immunosuppressive capacity becomes impaired. Most importantly, these progressive changes in CD4(+) T cells are associated with the loss of Forkheadbox group O (Foxo)1, Foxo3, and Foxo4 expression. Our data establish a novel link between Gimap5 and Foxo expression and provide evidence for a regulatory mechanism that controls Foxo protein expression and may help to maintain immunological tolerance.     

Global activation of CD8+ cytotoxic T lymphocytes correlates with an impairment in regulatory T cells in patients with generalized vitiligo

Melanocyte-specific CD8(+) cytotoxic T lymphocytes (CTLs) play a pivotal role in vitiligo-induced depigmentation. Yet, the mechanisms underlying the high frequency of generalized autoimmune disorders associated with generalized vitiligo (GV) are unknown. We hypothesized that an imbalance between activated CD8(+) CTLs and regulatory T cells (Tregs) exists in patients with GV . Assessment of the circulating CD8(+) CTLs and Tregs by flow cytometric analysis revealed an obvious expansion of CD8(+) CTLs and a concomitant decrease in Treg cells in GV patients. The percentages of skin infiltrating CD8(+) CTLs and Tregs were evaluated by immunohistochemistry and revealed dramatically increased numbers of both CD8(+) CTLs and Tregs in the perilesional skin of GV patients. However, peripheral Tregs were impaired in their ability to suppress the proliferation and cytolytic capacity of autologous CD8(+) T cells, suggesting that a functional failure of Tregs and the hyper-activation of CD8(+) CTLs may contribute to progressive GV. Our data indicate that reduced numbers and impaired function of natural Tregs fail to control the widespread activation of CD8(+) CTLs, which leads to the destruction of melanocytes and contributes to the elevated frequency of various associated autoimmune diseases. This knowledge furthers our understanding of the mechanisms of immune tolerance that are impaired in GV patients and may aid in the future development of effective immunotherapy for GV patients.     

Melanocyte-specific, cytotoxic T cell responses in vitiligo: the effective variant of melanoma immunity?

Vitiligo is a relatively common progressive depigmentary condition that is believed to be due to the autoimmune-mediated loss of epidermal melanocytes. An interesting aspect of vitiligo is its relation to melanoma: cytotoxic T lymphocytes directed to self-antigens shared by normal melanocytes and melanoma cells are found in both conditions and might prove important in melanocyte destruction, yet the resulting immune reactions are completely different. From this standpoint, the selective destruction of pigment cells that occurs in cases of vitiligo is the therapeutic goal sought in melanoma research. In the present article, we will address these issues by reviewing current literature on the subject as well as by posing some speculations.     

Melanocyte‐specific,cytotoxic T cell responses in vitiligo: the effective variant of melanoma immunity?

Vitiligo is a relatively common progressive depigmentary condition that is believed to be due to the autoimmune‐mediated loss of epidermal melanocytes. An interesting aspect of vitiligo is its relation to melanoma: cytotoxic T lymphocytes directed to self‐antigens shared by normal melanocytes and melanoma cells are found in both conditions and might prove important in melanocyte destruction, yet the resulting immune reactions are completely different. From this standpoint, the selective destruction of pigment cells that occurs in cases of vitiligo is the therapeutic goal sought in melanoma research. In the present article, we will address these issues by reviewing current literature on the subject as well as by posing some speculations.     

Evidence for a selective migration of fetus-specific CD4+CD25bright regulatory T cells from the peripheral blood to the decidua in human pregnancy

During pregnancy, the maternal immune system has to tolerate the persistence of fetal alloantigens. Many mechanisms contribute to the prevention of a destructive immune response mediated by maternal alloreactive lymphocytes directed against the allogeneic fetus. Murine studies suggest that CD4(+)CD25(+) T cells provide mechanisms of specific immune tolerance to fetal alloantigens during pregnancy. Previous studies by our group demonstrate that a significantly higher percentage of activated T cells and CD4(+)CD25(bright) T cells are present in decidual tissue in comparison with maternal peripheral blood in human pregnancy. In this study, we examined the phenotypic and functional properties of CD4(+)CD25(bright) T cells derived from maternal peripheral blood and decidual tissue. Depletion of CD4(+)CD25(bright) T cells from maternal peripheral blood demonstrates regulation to third party umbilical cord blood cells comparable to nonpregnant controls, whereas the suppressive capacity to umbilical cord blood cells of her own child is absent. Furthermore, maternal peripheral blood shows a reduced percentage of CD4(+)CD25(bright)FOXP3(+) and CD4(+)CD25(bright)HLA-DR(+) cells compared with peripheral blood of nonpregnant controls. In contrast, decidual lymphocyte isolates contain high percentages of CD4(+)CD25(bright) T cells with a regulatory phenotype that is able to down-regulate fetus-specific and fetus-nonspecific immune responses. These data suggest a preferential recruitment of fetus-specific regulatory T cells from maternal peripheral blood to the fetal-maternal interface, where they may contribute to the local regulation of fetus-specific responses.     

Systemic analyses of immunophenotypes of peripheral T cells in non-segmental vitiligo: implication of defective natural killer T cells

Although it is widely believed that non‐segmental vitiligo (NSV) results from the autoimmune destruction of melanocytes, a clear understanding of defects in immune tolerance, which mediate this uncontrolled self‐reactivity, is still lacking. In the present study, we systemically evaluated circulating regulatory T (Treg) cells, including CD4⁺CD25⁺FoxP3⁺ Treg cells and invariant natural killer T (iNKT) cells, as well as naïve and memory CD4⁺ and CD8⁺ T cells and their cytokine production, in a cohort of 43 progressive NSV patients with race‐, gender‐, and age‐matched healthy controls. We found that the general immunophenotypes of CD4⁺ and CD8⁺ T cells and the percentage of CD4⁺CD25⁺FoxP3⁺ Tregs were comparable between NSV and healthy controls. However, percentages of peripheral iNKT cells were significantly decreased in NSV patients compared to that in healthy controls. Our data confirm the previous notion that the percentage of peripheral CD4⁺CD25⁺FoxP3⁺ Tregs remains unaltered in NSV and suggests the involvement of defective iNKT cells in the pathogenesis of NSV.     

Augmentation of regulatory T cells (CD4+CD25+Foxp3+) correlates with tumor stage in patients with colorectal cancer

Recent evidence suggests that decline of regulatory T cells (Tregs) play a critical role in the prevalence of autoimmune diseases inhibiting the maintenance of peripheral self tolerance, while its augmentation leads to insufficient antitumor response, accompanied with poor prognosis in various malignancies. Increased number of Tregs (CD4+CD25+FoxP3+) were noticed in peripheral blood mononuclear cells (PBMCs), tumor-infiltrating lymphocytes (TILs) and/or regional lymph nodes lymphocytes (LNLs) of patients with gastrointestinal tumors. The aim of our study was to investigate the correlation between the percentage of Tregs in peripheral blood of patients with colorectal carcinoma, using flow cytometric technique and tumor stages, classified as Dukes' A, B, C or D and by stage of differentiation. Peripheral blood venous samples were obtained from 92 patients with colorectal cancer and from 30 healthy adult volunteers. Statistical analysis: Linear regression equations were generated using a least-squares method and analyzed for differences of covariance. Statistical significance was calculated by Mann Whitney U-test. Our data has shown that 15% patients with colorectal cancer were classified as Dukes' A, 41% were Dukes' B, 35% were Dukes' C and 9% were Dukes' D. 54% patients with CRC were well differentiated, 11% were poorly differentiated, 20 were moderately differentiated, tage, 4% were mucinous carcinoma and rest of 11% were partly good differentiated with mucinous components. The increased percentage of Tregs in colorectal cancer patients correlates with tumor stage. These results indicate a possible involvement of regulatory T cells in disease progression. New strategies using inhibition or depletion of Tregs are necessary to elucidate the complexity of defective tumor immunity.     

JC virus-specific cytotoxic T lymphocytes in individuals with progressive multifocal leukoencephalopathy

Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease of the central nervous system caused by a reactivation of the polyomavirus JC (JCV) within a setting of immunosuppression. The nature of the immune response that contains replication of this virus is unknown. We have explored JCV-specific cellular immune responses in patients with PML and control subjects. JCV antigen-stimulated peripheral blood mononuclear cells (PBMC) of four human immunodeficiency virus (HIV)-infected patients who were survivors of PML and one HIV-uninfected patient recently diagnosed with PML lysed autologous B-lymphoblastoid cell lines expressing either the JCV T regulatory protein or the VP1 major capsid protein. This lysis was mediated by CD8(+) T lymphocytes and was major histocompatibility complex class I restricted. These cells were therefore cytotoxic T lymphocytes (CTL). JCV-specific CTL could not be detected in PBMC of three HIV-infected PML patients who had progressive neurologic disease and an eventual fatal outcome. These data suggest that the JCV-specific cellular immune response may play a crucial role in the containment of PML. This finding may also prove useful as a favorable prognostic marker in the clinical management of these patients.     

Initiation and regulation of CD8+T cells recognizing melanocytic antigens in the epidermis: implications for the pathophysiology of vitiligo

Antigen-specific CD8+T lymphocytes play an important role in defense against cutaneous microbial infection and skin cancer as well as in the pathophysiology of autoimmune skin disease such as lupus erythematodes and vitiligo. We have explored the role of CD8+ cytotoxic T lymphocytes (CTL) in an experimental mouse model of vitiligo, a pigmentation disorder characterized by focal loss of melanocytes in the skin. Using genetic immunization techniques we found that pigment cells in the epidermis can be destroyed by CD8+ T cells specifically recognizing a single H2-Kb-binding peptide derived from the model melanocytic self antigen tyrosinase-related protein 2 (TRP2), a melanosomal enzyme involved in pigment synthesis. Experimental evidence suggests that peripheral tolerance of pigment cell-specific cytotoxic CD8+T cells is regulated in two steps. In the induction phase, stimulation and expansion of these T cells in vivo strictly depends on CD4+ T cell help. In the effector phase, autoimmune destruction of melanocytes in the skin depends on local inflammation facilitating the migration of T cells into the epidermis and supporting effector functions. Our results suggest that accidental stimulation of CD8+ CTL recognizing MHC class I-binding peptides derived from melanocytic proteins in the context of an inflammatory skin disease may play an important role in the pathophysiology of vitiligo. Further investigations will address the role of chemokines, chemokine receptors and adhesion molecules in this experimental system and will reveal the role of keratinocytes and Langerhans cells in regulating cutaneous CD8+ T cell responses.     

Vaccine-specific local T cell reactivity in immunotherapy-associated vitiligo in melanoma patients

The occurrence of vitiligo in patients with melanoma is especially reported for patients undergoing immunotherapy. While vitiligo in these patients is thought to be related to an immune response directed against melanoma cells, solid evidence is lacking. Here we report local cytotoxic T cell reactivity in three melanoma patients who developed vitiligo, after experimental immunotherapy using dendritic cell vaccinations. Tetramer analysis showed that vaccine-induced T cells recognizing gp100 and tyrosinase are present at the vitiligo lesions. These T cells secrete IFN-γ and IL-2 upon peptide specific stimulation as well as upon recognition of the autologous tumor. We show that functional CD8⁺ T cells specific for melanoma differentiation antigens used in a melanoma immunotherapy trial, do not only invade the tumor, but also the vitiligo lesions. This directly links vitiligo to the immuno-therapeutic intervention and supports the hypothesis that vitiligo is a marker of immunity against melanoma cells. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Regulation of the natural killer activity of lymphocytes from healthy donors and patients with chronic lympholeukemia through the interaction of T- and non-T cells

The role of T- and B-cell cooperative interaction in the regulation of natural killer (NK) activity in human peripheral blood lymphocytes was studied. It has been shown that preincubation of normal donor mononuclear cells (MNC) for 48 h is followed by the loss of NK activity, while the incubation of the isolated T- and non-T-cell subsets does not lead to an analogous fall in the killer lymphocyte function. NK activity of MNC and isolated lymphocyte subsets in normal donors is shown to exceed that of CLL patients. The absence of preincubation effect on NK activity level of MNC, T- and non-T-cells in CLL patients has been also found. The findings obtained suggest that as a result of T- and B-cell interaction during preincubation differentiation of young T lymphocytes with NK cellular properties takes place. It is followed by the loss in NK activity. B-cell defect in CLL patients might cause the absence of preincubation effect on NK activity of T cells.     

Dermal Mesenchymal Stem Cells (DMSCs) Inhibit Skin-Homing CD8+ T Cell Activity,a Determining Factor of Vitiligo Patients’ Autologous Melanocytes Transplantation Efficiency

We here investigated the efficiency of autologous melanocyte transplantation of 23 vitiligo patients by focusing on perilesional skin homing CD8+ T lymphocytes, and studied the potential effect of dermal mesenchymal stem cells (DMSCs) on CD8+ T cell activities in vitro. Out of 23 patients with the autologous melanocyte transplantation, 12 patients (52.17%) had an excellent re-pigmentation, 6 patients (26.09%) had a good re-pigmentation, 5 patients (21.74%) had a fair or poor re-pigmentation. CD8+ T cells infiltrating was observed in the perilesional vitiligo area of all patients. Importantly, the efficiency of the transplantation was closely associated with skin-homing CD8+ T cell activities. The patients with high number of perilesional CD8+ T cells or high level of cytokines/chemokines were associated with poor re-pigmentation efficiency. For in-vitro experiments, we successfully isolated and characterized human DMSCs and skin-homing CD8+ T cells. We established DMSCs and CD8+ T cell co-culture system, where DMSCs possessed significant inhibitory effects against skin homing CD8+ T lymphocytes. DMSCs inhibited CD8+ T cells proliferation, induced them apoptosis and regulated their cytokines/chemokines production. Our results suggest that vitiligo patients’ autologous melanocytes transplantation efficiency might be predicted by perilesional skin-homing CD8+ T cell activities, and DMSCs might be used as auxiliary agent to improve transplantation efficacy.     

Th17 cells and activated dendritic cells are increased in vitiligo lesions

Background

Vitiligo is a common skin disorder, characterized by progressive skin de-pigmentation due to the loss of cutaneous melanocytes. The exact cause of melanocyte loss remains unclear, but a large number of observations have pointed to the important role of cellular immunity in vitiligo pathogenesis.

Methodology/Principal Findings

In this study, we characterized T cell and inflammation-related dermal dendritic cell (DC) subsets in pigmented non-lesional, leading edge and depigmented lesional vitiligo skin. By immunohistochemistry staining, we observed enhanced populations of CD11c+ myeloid dermal DCs and CD207+ Langerhans cells in leading edge vitiligo biopsies. DC-LAMP+ and CD1c+ sub-populations of dermal DCs expanded significantly in leading edge and lesional vitiligo skin. We also detected elevated tissue mRNA levels of IL-17A in leading edge skin biopsies of vitiligo patients, as well as IL-17A positive T cells by immunohistochemistry and immunofluorescence. Langerhans cells with activated inflammasomes were also noted in lesional vitiligo skin, along with increased IL-1ß mRNA, which suggest the potential of Langerhans cells to drive Th17 activation in vitiligo.

Conclusions/Significance

These studies provided direct tissue evidence that implicates active Th17 cells in vitiligo skin lesions. We characterized new cellular immune elements, in the active margins of vitiligo lesions (e.g. populations of epidermal and dermal dendritic cells subsets), which could potentially drive the inflammatory responses.