Role of Interleukin 32 in Human Immunodeficiency Virus Reactivation and Its Link to Human Immunodeficiency Virus-Herpes Simplex Virus Coinfection.

Background: Herpes simplex virus type 2 (HSV-2; herpes) exacerbates human immunodeficiency virus type 1 (HIV) by unclear mechanisms. These studies tested the impact of HSV-2 on systemic T-cells and HIV reservoirs. Methods: Peripheral blood mononuclear cells from HIV-infected women on antiretroviral therapy who were HSV-2 seropositive or seronegative and HIV-uninfected controls were analyzed by flow cytometry. Cell-associated HIV DNA and RNA were quantified in the absence or presence of activating stimuli, recombinant interleukin 32γ (IL-32γ), and a RUNX1 inhibitor. RNA was assessed by nanostring. Results: CD4, but not CD8, T-cell phenotypes differed in HIV+/HSV-2+ versus HIV+/HSV-2- (overall P = .002) with increased frequency of CCR5+, CXCR4+, PD-1+, and CD69+ and decreased frequency of CCR10+ and CCR6+ T-cells. The changes were associated with higher HIV DNA. Paradoxically, IL-32, a proinflammatory cytokine, was lower in subpopulations of CD4+ T-cells in HSV-2+ versus HSV-2- women. Recombinant IL-32γ blocked HIV reactivation in CD4+ T-cells and was associated with an increase in RUNX1 expression; the blockade was overcome by a RUNX1 inhibitor. Conclusions: Herpes is associated with phenotypic changes in CD4+ T-cells, including a decrease in IL-32, which may contribute to increased HIV reservoirs. Blocking IL-32 may facilitate HIV reactivation to improve shock and kill strategies.

Changes in T-cell subsets identify responders to FcR-nonbinding anti-CD3 mAb (teplizumab) in patients with type 1 diabetes.

The mechanisms whereby immune therapies affect progression of type 1 diabetes (T1D) are not well understood. Teplizumab, an FcR nonbinding anti-CD3 mAb, has shown efficacy in multiple randomized clinical trials. We previously reported an increase in the frequency of circulating CD8(+) central memory (CD8CM) T cells in clinical responders, but the generalizability of this finding and the molecular effects of teplizumab on these T cells have not been evaluated. We analyzed data from two randomized clinical studies of teplizumab in patients with new- and recent-onset T1D. At the conclusion of therapy, clinical responders showed a significant reduction in circulating CD4(+) effector memory T cells. Afterward, there was an increase in the frequency and absolute number of CD8CM T cells. In vitro, teplizumab expanded CD8CM T cells by proliferation and conversion of non-CM T cells. Nanostring analysis of gene expression of CD8CM T cells from responders and nonresponders versus placebo-treated control subjects identified decreases in expression of genes associated with immune activation and increases in expression of genes associated with T-cell differentiation and regulation. We conclude that CD8CM T cells with decreased activation and regulatory gene expression are associated with clinical responses to teplizumab in patients with T1D.

Humanized mice as a model for aberrant responses in human T cell immunotherapy.

Immune-deficient mice, reconstituted with human stem cells, have been used to analyze human immune responses in vivo. Although they have been used to study immune responses to xenografts, allografts, and pathogens, there have not been models of autoimmune disease in which the mechanisms of the pathologic process can be analyzed. We have found that reconstituted "humanized" mice treated with anti-CTLA-4 Ab (ipilimumab) develop autoimmune disease characterized by hepatitis, adrenalitis, sialitis, anti-nuclear Abs, and weight loss. Induction of autoimmunity involved activation of T cells and cytokine production, and increased infiltration of APCs. When anti-CTLA-4 mAb-treated mice were cotreated with anti-CD3 mAb (teplizumab), hepatitis and anti-nuclear Abs were no longer seen and weight loss did not occur. The anti-CD3 blocked proliferation and activation of T cells, release of IFN-γ and TNF, macrophage infiltration, and release of IP-10 that was induced with anti-CTLA-4 mAb. We also found increased levels of T regulatory cells (CD25(+)CD127(-)) in the spleen and mesenteric lymph nodes in the mice treated with both Abs and greater constitutive phosphorylation of STAT5 in T regulatory cells in spleen cells compared with mice treated with anti-CTLA-4 mAb alone. We describe a model of human autoimmune disease in vivo. Humanized mice may be useful for understanding the mechanisms of biologics that are used in patients. Hepatitis, lymphadenopathy, and other inflammatory sequelae are adverse effects of ipilimumab treatment in humans, and this study may provide insights into this pathogenesis and the effects of immunologics on autoimmunity.

Reduced IL-7 responsiveness defined by signal transducer and activator of transcription 5 phosphorylation in T cells may be a marker for increased risk of developing cytomegalovirus disease in patients after hematopoietic stem cell transplantation.

Cytomegalovirus (CMV) reactivation may lead to CMV disease associated with high morbidity and mortality in patients after hematopoietic stem cell transplantation (HSCT); the identification of clinically relevant markers may aid in the identification of patients at increased risk for developing CMV-associated complications. We evaluated the phosphorylation of signal transducer and activator of transcription 5 (STAT5) in CD4(+) T cells, CD8(+) T cells, and TCRγδ T cells in response to stimulation with IL-7 or IL-2 after HSCT by analyzing blood samples taken monthly 1 to 6 months after HSCT. Patients were monitored weekly with a quantitative PCR from the time of engraftment for CMV viral load in whole blood until at least day 100 after HSCT. We identified a correlation between clinical outcome regarding CMV replication and the ability to respond to IL-7 and IL-2 defined by STAT5 phosphorylation (pSTAT5). Patients with recurrent or prolonged CMV replications had significantly lower pSTAT5 upon stimulation of T cells with either IL-7 or IL-2 at time points 1 through 3 than those without CMV replication (P < .05). This was also found after stimulation of CD8(+) T cells at time point 2 (P < .05). We conclude that reduced responses to IL-7, reflected by pSTAT5, may represent a clinically relevant functional biomarker for individuals at increased risk for CMV reactivation; our data may also aid in designing better strategies to improve anti-CMV immune responses without increasing the risk of developing graft-versus-host disease.

Reduced plasma levels of soluble interleukin-7 receptor during graft-versus-host disease (GVHD) in children and adults.

BACKGROUND: Interleukin 7 (IL-7) signals via the IL-7 receptor (IL-7R) and drives homeostatic T-cell proliferation in patients after allogeneic hematopoietic stem cell transplantation (aHSCT). PURPOSE: We performed a prospective study in adults (n = 33) and children (n = 29) undergoing aHSCT measuring plasma IL-7 and soluble IL-7R (sIL-7R) concentrations between 1 and 12 months after HSCT in order to investigate the link between sIL-7R and clinical events after aHSCT. RESULTS: sIL-7R, but not IL-7, increased with time after HSCT in plasma from all patients enrolled in the study. sIL-7R values were higher at 2, 3, and 6 months (p < 0.01) if the donor was a sibling as compared to an unrelated donor. Increased sIL-7R levels were also identified in plasma from patients who were not treated with anti-thymocyte globulin (ATG). Low sIL-7R was associated with any grade of acute graft-versus-host disease (GVHD) at 2 and 6 months (p = 0.02) and with a positive CMV PCR at 2 months after HSCT (p < 0.05). Patients with cytomegalovirus (CMV) reactivation had increased IL-7 values at 2 and 3 months (p = 0.02) after HSCT. In multivariate analysis, lower sIL-7R levels were associated with acute GVHD (relative hazard (RH): 0.70, p > 0.01) and sibling donors (RH: 2.23, p = 0.004). Recipients of sibling grafts showed high levels of IL-7 (RH: 1.38, p < 0.05) and bone marrow recipients had low IL-7 levels (RH: 0.73, p = 0.04). CONCLUSIONS: Measurement of the sIL-7R/IL-7 axis will help in guided immune monitoring after HSCT and guided interference with sIL-7R may be explored in GVHD management.

Dysregulation of CD4+CD25(high) T cells in the synovial fluid of patients with antibiotic-refractory Lyme arthritis.

OBJECTIVE: To examine the role of immune dysregulation in antibiotic-refractory Lyme arthritis by comparing the phenotype, frequency, and function of CD4+ Teff cells and Treg cells in patients with antibiotic- responsive arthritis and patients with antibiotic-refractory arthritis. METHODS: Matched peripheral blood and synovial fluid samples from 15 patients with antibiotic-responsive arthritis were compared with those from 16 patients with antibiotic-refractory arthritis, using flow cytometry, suppression assays, and cytokine assays. RESULTS: Critical differences between the 2 patient groups were observed in the synovial fluid CD4+CD25(high) population, a cell subset usually composed of FoxP3-positive Treg cells. In patients with antibiotic-refractory arthritis, this cell population often had fewer FoxP3-positive cells and a greater frequency of FoxP3-negative (Teff) cells compared with patients with antibiotic-responsive arthritis. Moreover, the expression of glucocorticoid-induced tumor necrosis factor receptor and OX40 on CD4+CD25(high) cells was significantly higher in the antibody-refractory group. Suppression assays showed that CD4+CD25(high) cells in patients with antibiotic-refractory arthritis did not effectively suppress proliferation of CD4+CD25- cells or secretion of interferon-γ and tumor necrosis factor α, whereas those cells in patients with antibiotic-responsive arthritis did suppress proliferation of CD4+CD25- cells and secretion of interferon-γ and tumor necrosis factor α. Finally, in the antibiotic-refractory group, higher ratios of CD25(high) FoxP3-negative cells to CD25(high) FoxP3-positive cells correlated directly with a longer duration of arthritis after antibiotic treatment. CONCLUSION: Patients with antibiotic-refractory Lyme arthritis often have lower frequencies of Treg cells, higher expression of activation coreceptors, and less effective inhibition of proinflammatory cytokines. This suggests that immune responses in these patients are excessively amplified, leading to immune dysregulation and antibiotic-refractory arthritis.

Differential cellular recognition pattern to M. tuberculosis targets defined by IFN-γ and IL-17 production in blood from TB + patients from Honduras as compared to health care workers: TB and immune responses in patients from Honduras.

BACKGROUND: A better understanding of the quality of cellular immune responses directed against molecularly defined targets will guide the development of TB diagnostics and identification of molecularly defined, clinically relevant M.tb vaccine candidates. METHODS: Recombinant proteins (n = 8) and peptide pools (n = 14) from M. tuberculosis (M.tb) targets were used to compare cellular immune responses defined by IFN-γ and IL-17 production using a Whole Blood Assay (WBA) in a cohort of 148 individuals, i.e. patients with TB + (n = 38), TB- individuals with other pulmonary diseases (n = 81) and individuals exposed to TB without evidence of clinical TB (health care workers, n = 29). RESULTS: M.tb antigens Rv2958c (glycosyltransferase), Rv2962c (mycolyltransferase), Rv1886c (Ag85B), Rv3804c (Ag85A), and the PPE family member Rv3347c were frequently recognized, defined by IFN-γ production, in blood from healthy individuals exposed to M.tb (health care workers). A different recognition pattern was found for IL-17 production in blood from M.tb exposed individuals responding to TB10.4 (Rv0288), Ag85B (Rv1886c) and the PPE family members Rv0978c and Rv1917c. CONCLUSIONS: The pattern of immune target recognition is different in regard to IFN-γ and IL-17 production to defined molecular M.tb targets in PBMCs from individuals frequently exposed to M.tb. The data represent the first mapping of cellular immune responses against M.tb targets in TB patients from Honduras.

High content cellular immune profiling reveals differences between rhesus monkeys and men.

A better understanding of similarities and differences in the composition of the cellular immune system in non-human primates (NHPs) compared with human subjects will improve the interpretation of preclinical studies. It will also aid in addressing the usefulness of NHPs as subjects for studying chronic diseases, vaccine development and immune reconstitution. We employed high content colour flow cytometry and analysed simultaneously the expression of CD3, CD4, CD8alpha, CD8beta, CD16/CD56, CD45RA, CCR7, CD27, CD28, CD107a and the interleukin-7 receptor alpha-chain (IL-7Ralpha) in peripheral blood mononuclear cells (PBMCs) of 27 rhesus macaques and 16 healthy human subjects. Regulatory T cells (Tregs) were identified using anti-CD3, -CD4, -CD25, -FoxP3, and -IL-7Ralpha monoclonal antibodies. Responsiveness to IL-7 was gauged in a signal transducer and activation of transcription 5 (STAT-5) phosphorylation assay. Human and NHP PBMCs showed a similar T-cell composition pattern with some remarkable differences. Similarities: human and NHP CD4(+) and CD8(+) cells showed a similar STAT-5 phosphorylation pattern in response to IL-7. Multicolour flow cytometric analysis identified a CD4(+) CD8alphaalpha(+) CD8alphabeta(+) T-cell population in NHPs as well as in human subjects that expressed the degranulation marker CD107a and may represent a unique CD4(+) T-cell subset endowed with cytotoxic capacity. Differences: we identified in PBMCs from NHPs a higher proportion (5.16% in CD3(+) T cells) of CD8alphaalpha(+) T cells when compared with human donors (1.22% in CD3(+) T cells). NHP CD8alphaalpha(+) T cells produced tumour necrosis factor-alpha / interferon-gamma (TNF-alpha/IFN-gamma) or TNF-alpha, whereas human CD8alphaalpha(+) T cells produced simultaneously TNF-alpha/IFN-gamma and IL-2. A minor percentage of human CD8(+) T cells expressed CD25(bright) and FoxP3 (0.01%). In contrast, 0.07% of NHP CD8(+) T cells exhibited the CD25(bright) FoxP3(+) phenotype. PBMCs from NHPs showed less IL-7Ralpha-positive events in all T-cell subsets including CD4(+) Tregs (median 5%) as compared with human (median 12%). The data visualize commonalities and differences in immune cell subsets in humans and NHPs, most of them in long-lived memory cells and cells with suppressive functions. This provides a matrix to assess future efforts to study diseases and vaccines in NHPs.

Microbiota control immune regulation in humanized mice.

The microbiome affects development and activity of the immune system, and may modulate immune therapies, but there is little direct information about this control in vivo. We studied how the microbiome affects regulation of human immune cells in humanized mice. When humanized mice were treated with a cocktail of 4 antibiotics, there was an increase in the frequency of effector T cells in the gut wall, circulating levels of IFN-γ, and appearance of anti-nuclear antibodies. Teplizumab, a non-FcR-binding anti-CD3ε antibody, no longer delayed xenograft rejection. An increase in CD8+ central memory cells and IL-10, markers of efficacy of teplizumab, were not induced. IL-10 levels were only decreased when the mice were treated with all 4 but not individual antibiotics. Antibiotic treatment affected CD11b+CD11c+ cells, which produced less IL-10 and IL-27, and showed increased expression of CD86 and activation of T cells when cocultured with T cells and teplizumab. Soluble products in the pellets appeared to be responsible for the reduced IL-27 expression in DCs. Similar changes in IL-10 induction were seen when human peripheral blood mononuclear cells were cultured with human stool samples. We conclude that changes in the microbiome may impact the efficacy of immunosuppressive medications by altering immune regulatory pathways.

Endothelial apoptosis and circulating endothelial cells after bypass grafting with and without cardiopulmonary bypass.

OBJECTIVE: We compared profiles of the numbers of circulating endothelial cells (CEC) and the apoptosis-inducing capacity of serum samples on human endothelial cells (hEC) in on-pump and off-pump coronary artery bypass grafting (CABG) patients. METHODS: Blood samples from 30 patients undergoing CABG (randomly assigned to two groups: 15 patients off-pump and 15 on-pump (cardiopulmonary bypass, CPB)) were collected after induction of anesthesia (preoperatively), at weaning from CPB/end of bypass grafting (0 h), and 1, 6, 12, 24, and 48 h afterwards. CEC were isolated with immunomagnetic anti-CD146-coated Dynabeads, and counted in a Nageotte chamber. The apoptosis-inducing activity of serum samples on hEC was examined by a tissue culture assay system. Apoptotic and normal cells were identified using phase contrast/fluorescence microscopy after DNA dye staining. RESULTS: CEC numbers and proportions of apoptotic hEC were significantly elevated during and after surgery in both groups (p<0.01). Compared with the on-pump group, CEC and proportions of apoptotic hEC were significantly lower (p=0.04 and p=0.03, respectively) in patients having CABG performed off-pump. Starting at comparable baseline levels, the mean CEC-number was highest at 6h postoperatively with 81.9 ml(-1) (range, 44-141) for on-pump patients and 63.3 ml(-1) (range, 48-105) for off-pump patients. hEC apoptosis peaked also at T4: 16.5+/-2.8% versus 11.3+/-2.2%. In both groups, CEC numbers and proportions of endothelial apoptosis were still elevated at 48 h after surgery. CONCLUSION: The number of circulating endothelial cells and apoptotic endothelial cell death are markers of endothelial activation and damage during CABG. This study provides evidence that CABG with the use of CPB in comparison to OPCAB surgery is associated with a significantly more pronounced endothelial response in the immediate postoperative period.

Both high and low levels of cellular Epstein-Barr virus DNA in blood identify failure after hematologic stem cell transplantation in conjunction with acute GVHD and type of conditioning.

The level of Epstein-Barr virus DNA in blood has proven to be a biomarker with some predictive value in allogeneic hematopoietic stem cell transplantation patients (HSCT). We evaluated the impact of EBV load on survival of 51 patients (32M/19F, median age: 32 years, from < 1 to 68 years old), who had received HSCT for different types of malignancies (49 cases) or non-malignancies (2 cases). The overall survival [1]was compared between patients with extreme and moderate cell bound EBV DNA levels. Different sources of stem-cells (peripheral blood stem, n = 39; bone marrow, n = 9; or umbilical cord blood, n = 3) were used. Twenty patients received reduced-intensity conditioning regimen while the other 31 received myeloablative conditioning. Patients with high or very low level of cell bound EBV-DNA levels had a shorter OS than those with moderate EBV load: OS at 5 years was 67% vs 90% (p < 0.03). There was a conspicuous relationship between EBV load and the reconstitution dynamics of total and EBV-specific T cells, CD4+ and CD4- CD8- (double negative) T cells in the few patients where it was analyzed. This was not statistically significant. Two other factors were associated to early mortality in addition to high or low EBV load: acute GVHD II-IV (p < 0.02) and pre-transplant conditioning with total body irradiation (TBI) ≥6 Gy, (p < 0.03). All the patients meeting all three criteria died within two years after transplantation. This points to a subgroup of HSCT patients which deserve special attention with improvement of future, personalized treatment.

Sodium chloride inhibits the suppressive function of FOXP3+ regulatory T cells.

FOXP3+ Tregs are central for the maintenance of self-tolerance and can be defective in autoimmunity. In multiple sclerosis and type-1 diabetes, dysfunctional self-tolerance is partially mediated by a population of IFNγ-secreting Tregs. It was previously reported that increased NaCl concentrations promote the induction of proinflammatory Th17 cells and that high-salt diets exacerbate experimental models of autoimmunity. Here, we have shown that increasing NaCl, either in vitro or in murine models via diet, markedly impairs Treg function. NaCl increased IFNγ secretion in Tregs, and reducing IFNγ - either by neutralization with anti-IFNγ antibodies or shRNA-mediated knockdown - restored suppressive activity in Tregs. The heightened IFNγ secretion and loss of Treg function were mediated by the serum/glucocorticoid-regulated kinase (SGK1). A high-salt diet also impaired human Treg function and was associated with the induction of IFNγ-secreting Tregs in a xenogeneic graft-versus-host disease model and in adoptive transfer models of experimental colitis. Our results demonstrate a putative role for an environmental factor that promotes autoimmunity by inducing proinflammatory responses in CD4 effector cells and Treg pathways.

Treatment of new onset type 1 diabetes with teplizumab: successes and pitfalls in development.

INTRODUCTION: Type 1 diabetes is an organ-specific autoimmune disease, characterized by selective destruction of insulin-producing pancreatic ß-cells by T-cell-mediated inflammation. Beginning with studies of cyclosporin A in the 1980s, but with more activity in the past decade, there have been a number of clinical trials to test whether immunotherapies can arrest the decline in C-peptide, which is associated with progression of type 1 diabetes leading to the metabolic instability that characterizes the disease. One of the most promising agents, teplizumab , is an FcR-nonbinding anti-CD3 monoclonal antibody that has been tested in Phase II - III clinical trials and was shown to preserve the C-peptide levels and reduce the need for exogenous insulin. AREAS COVERED: In this review, we discuss the recent update on clinical data obtained from trials of teplizumab in type 1 diabetes, the drug's postulated mechanism of action and the identification of responders to therapy. We highlight the results of recent trials as well as the lessons that have been learned from the clinical trials involving selection of end points and the inclusion of diverse study populations. EXPERT OPINION: Teplizumab has been shown to preserve ß cell function in patients; however, it does not represent a 'cure' for patients, and its efficacy does entail a significant advance in arresting the progression of the disease toward complete insulin deficiency and reliance on exogenous insulin.

TCR+CD4-CD8- T cells in antigen-specific MHC class I-restricted T-cell responses after allogeneic hematopoietic stem cell transplantation.

Human TCRαß(+) CD4(-)CD8(-) double-negative (DN) T cells represent a minor subset in peripheral blood, yet are important in infectious diseases and autoimmune responses. We examined the frequency of DN T cells in 17 patients after allogeneic hematopoietic stem cell transplantation (aHSCT) at 1, 2, 3, 6, and 12 months post-aHSCT and show that these cells increase early after aHSCT and decrease with time after aHSCT. DN T cells reside in the terminally differentiated effector (CD45RA(+)CCR7(-)) T-cell population and are polyclonal, determined by T-cell receptor Vß CDR3 analysis. Gene expression analysis of ex vivo sorted DN T cells showed a distinct set of gene expression, including interleukin-8, as compared with CD4(+) or CD8(+) T cells. DN T cells contributed to MHC class I-restricted EBV-directed immune responses, defined by antigen-specific cytokine production and by detection of HLA-A*02:01-restricted EBV BMLF-1 (GLCTLVAML), LMP-2A (CLGGLLTMV), and HLA-A*24:02-restricted EBV BRLF-1 (DYCNVLNKEF) and EBNA3 (RYSIFFDY)-specific T cells. We created retroviral-transfected Jurkat cell lines with a Melan-A/MART-1-specific TCR(+) and the CD8α chain to study TCR(+) DN T cells in response to their nominal MHC class I/peptide ligand. We show that DN T cells exhibit increased TCRζ chain phosphorylation as compared with the TCR(+)CD8(+) transgenic T-cell line. DN T cells contribute to antigen-specific T-cell responses and represent an effector T-cell population that may be explored in immunotherapeutic approaches against viral infections or transformed cells.

A carbon nanotube-polymer composite for T-cell therapy.

Clinical translation of cell therapies requires strategies that can manufacture cells efficiently and economically. One promising way to reproducibly expand T cells for cancer therapy is by attaching the stimuli for T cells onto artificial substrates with high surface area. Here, we show that a carbon nanotube-polymer composite can act as an artificial antigen-presenting cell to efficiently expand the number of T cells isolated from mice. We attach antigens onto bundled carbon nanotubes and combined this complex with polymer nanoparticles containing magnetite and the T-cell growth factor interleukin-2 (IL-2). The number of T cells obtained was comparable to clinical standards using a thousand-fold less soluble IL-2. T cells obtained from this expansion were able to delay tumour growth in a murine model for melanoma. Our results show that this composite is a useful platform for generating large numbers of cytotoxic T cells for cancer immunotherapy.

Delayed anti-CD3 therapy in a mouse heart transplant model induced tolerance and long-term survival of allograft: achieving tolerance.

Goto R, You S, Zaitsu M, Chatenoud L, Wood KJ. Delayed anti-CD3 therapy results in depletion of alloreactive T cells and the dominance of Foxp3(+) CD4(+) graft infiltrating cells. Am. J. Transplant. 13(7), 1655-1664 (2013). Humanized Fc receptor nonbinding anti-CD3 monoclonal antibodies have been tested in patients with autoimmune diseases with the goal of inducing immune tolerance. However, the timing of drug administration may be an important determinant of the biologic effects, since not all T cells are equally affected, and there may be different subsets of cells involved during the evolution of immune responses. The study by Goto et al. showed that delayed administration of anti-CD3 therapy was more effective in depleting alloreactive T cells than administration at the time of transplant, and resulted in long-term survival of the graft by promoting infiltration of CD4 Tregs into the graft.

Alternative splicing of interleukin-7 (IL-7) and interleukin-7 receptor alpha (IL-7Ralpha) in peripheral blood from patients with multiple sclerosis (MS).

IL-7 and IL-7Ralpha (IL-7R) form a non-redundant ligand receptor system which plays a crucial role in human T cell immunity. Both IL-7 and IL-7R are multi-exonal genes and exhibit alternative splicing. We measured the relative distribution of IL-7 and IL-7R spliced mRNA from patients with MS and healthy individuals and observed extensive alternative splicing of both genes with marked differences in proportional transcript expression levels. We report here for the first time that the IL-7 transcript, lacking exon 4, and not the full length IL-7 represents the dominant IL-7 RNA transcript in human PBMCs and a novel IL-7R splice variant lacking exons 5, 6 and 7.

Increased numbers of IL-7 receptor molecules on CD4+CD25-CD107a+ T-cells in patients with autoimmune diseases affecting the central nervous system.

BACKGROUND: High content immune profiling in peripheral blood may reflect immune aberrations associated with inflammation in multiple sclerosis (MS) and other autoimmune diseases affecting the central nervous system. METHODS AND FINDINGS: Peripheral blood mononuclear cells from 46 patients with multiple sclerosis (MS), 9 patients diagnosed with relapsing remitting MS (RRMS), 13 with secondary progressive multiple sclerosis (SPMS), 9 with other neurological diseases (OND) and well as 15 healthy donors (HD) were analyzed by 12 color flow cytometry (TCRalphabeta, TCRgammadelta, CD4, CD8alpha, CD8beta, CD45RA, CCR7, CD27, CD28, CD107a, CD127, CD14) in a cross-sectional study to identify variables significantly different between controls (HD) and patients (OND, RRMS, SPMS). We analyzed 187 individual immune cell subsets (percentages) and the density of the IL-7 receptor alpha chain (CD127) on 59 individual immune phenotypes using a monoclonal anti-IL-7R antibody (clone R34.34) coupled to a single APC molecule in combination with an APC-bead array. A non-parametric analysis of variance (Kruskal-Wallis test) was conducted in order to test for differences among the groups in each of the variables. To correct for the multiplicity problem, the FDR correction was applied on the p-values. We identified 19 variables for immune cell subsets (percentages) which allowed to segregate healthy individuals and individuals with CNS disorders. We did not observe differences in the relative percentage of IL-7R-positive immune cells in PBMCs. In contrast, we identified significant differences in IL-7 density, measured on a single cell level, in 2/59 variables: increased numbers of CD127 molecules on TCRalphabeta+CD4+CD25 (intermed) T-cells and on TCRalphabeta+CD4+CD25-CD107a+ T-cells (mean: 28376 Il-7R binding sites on cells from HD, 48515 in patients with RRMS, 38195 in patients with SPMS and 33692 IL-7 receptor binding sites on cells from patients with OND). CONCLUSION: These data show that immunophenotyping represents a powerful tool to differentiate healthy individuals from individuals suffering from neurological diseases and that the number of IL-7 receptor molecules on differentiated TCRalphabeta+CD4+CD25-CD107a+ T-cells, but not the percentage of IL-7R-positive cells, segregates healthy individuals from patients with neurological disorders.

Tumor antigen-specific T-cells are Present in the CD8alphaalpha+ T-cell effector-memory pool.

CD8+ T-cell memory formation has recently been demonstrated to be associated with CD8alphaalpha homodimer expression by T-cells in mice. Up to now, the knowledge about the clinical significance of CD8alphaalpha+ T-cells in humans is limited. We assessed in longitudinally collected blood samples from patients with melanoma, who underwent a peptide-based vaccination, the role of CD8alphaalpha+ T-cells in tumor-specific cellular immune responses. Phenotypic analysis showed that the expression of CD8alphaalpha+ by T-cells was stable over time and associated with a CD45RA+/-CCR7- effector-memory profile. Melan-A/MART-1-specific T-cells were identified in the CD8alphaalpha+ T-cell compartment by tetramer technology. Detection of intracellular cytokine production (interleukin-2, interferon-gamma, and tumor necrosis factor-alpha) upon phorbol 12-myristate 13-acetate-ionomycin stimulation in CD8alphaalpha+ and CD8alphabeta+ T-cells revealed that CD8alphaalpha+ T-cells show a unique cytokine production pattern (tumor necrosis factor-alpha and interferon-gamma production) as compared with CD8alphabeta+ T-cells. T-cell receptor-CDR3 length analysis revealed that Melan-A/MART-1-specific CD8alphaalpha+ T-cells showed a similar T-cell receptor-repertoire as compared with Melan-A/MART-1-specific CD8alphabeta+ T-cells. Our results show that CD8alphaalpha+ T-cells represent a compartment of CD45RA+/- effector-memory cells in the peripheral circulation of patients with melanoma and suggest that CD8alphaalpha T-cells may originate from CD8+ T-cells that have down-regulated the expression of the CD8beta chain. CD8alphaalpha+ and tetramer-specific T-cells may represent a valuable marker to gauge long-term antigen-specific T-cell memory.