Inappropriate Expression of PD-1 and CTLA-4 Checkpoints in Myeloma Patients Is More Pronounced at Diagnosis: Implications for Time to Progression and Response to Therapeutic Checkpoint Inhibitors.

Multiple myeloma (MM) is a hematologic malignancy characterized by severely profound immune dysfunction. Therefore, the efficacy of drugs targeting the immune environments, such as immune checkpoint inhibitors (ICIs), is of high clinical importance. However, several clinical trials evaluating ICIs in MM in different therapeutic combinations revealed underwhelming results showing a lack of clinical efficacy and excessive side effects. The underlying mechanisms of resistance to ICIs observed in the majority of MM patients are still under investigation. Recently, we demonstrated that inappropriate expression of PD-1 and CTLA-4 on CD4 T cells in active MM is associated with adverse clinical outcomes and treatment status. The aim of the current study was to determine the usefulness of immune checkpoint expression assessment as a predictive biomarker of the response to therapeutic inhibitors. For this purpose, along with checkpoint expression estimated by flow cytometry, we evaluated the time to progression (TTP) of MM patients at different clinical stages (disease diagnosis and relapse) depending on the checkpoint expression level; the cut-off point (dividing patients into low and high expressors) was selected based on the median value. Herein, we confirmed the defective levels of regulatory PD-1, CTLA-4 receptors, and the CD69 marker activation in newly diagnosed (ND) patients, whereas relapsed/refractory patients (RR) exhibited their recovered values and reactivity. Additionally, substantially higher populations of senescent CD4+CD28- T cells were found in MM, primarily in NDMM subjects. These observations suggest the existence of two dysfunctional states in MM CD4 T cells with the predominance of immunosenescence at disease diagnosis and exhaustion at relapse, thus implying different responsiveness to the external receptor blockade depending on the disease stage. Furthermore, we found that lower CTLA-4 levels in NDMM patients or higher PD-1 expression in RRMM patients may predict early relapse. In conclusion, our study clearly showed that the checkpoint level in CD4 T cells may significantly affect the time to MM progression concerning the treatment status. Therefore, when considering novel therapies and potent combinations, it should be taken into account that blocking PD-1 rather than CTLA-4 might be a beneficial form of immunotherapy for only a proportion of RRMM patients.

BTLA Expression in CLL: Epigenetic Regulation and Impact on CLL B Cell Proliferation and Ability to IL-4 Production.

In our previous study, while chronic lymphocytic leukemia (CLL) cases showed higher levels of B and T lymphocyte attenuator (BTLA) mRNA compared to controls, lower BTLA protein expression was observed in cases compared to controls. Hence we hypothesize that micro RNA (miR) 155-5p regulates BTLA expression in CLL. In line with earlier data, expression of BTLA mRNA and miR-155-5p is elevated in CLL (p = 0.034 and p = 0.0006, respectively) as well as in MEC-1 cell line (p = 0.009 and 0.016, respectively). Inhibition of miR-155-5p partially restored BTLA protein expression in CLL patients (p = 0.01) and in MEC-1 cell lines (p = 0.058). Additionally, we aimed to evaluate the significance of BTLA deficiency in CLL cells on proliferation and IL-4 production of B cells. We found that secretion of IL-4 is not dependent on BTLA expression, since fractions of BTLA positive and BTLA negative B cells expressing intracellular IL-4 were similar in CLL patients and controls. We demonstrated that in controls the fraction of proliferating cells is lower in BTLA positive than in BTLA negative B cells (p = 0.059), which was not observed in CLL. However, the frequency of BTLA positive Ki67+ B cells in CLL was higher compared to corresponding cells from controls (p = 0.055) while there were no differences between the examined groups regarding frequency of BTLA negative Ki67+ B cells. Our studies suggest that miR-155-5p is involved in BTLA deficiency, affecting proliferation of CLL B cells, which may be one of the mechanisms responsible for CLL pathogenesis.

Deregulated Expression of Immune Checkpoints on Circulating CD4 T Cells May Complicate Clinical Outcome and Response to Treatment with Checkpoint Inhibitors in Multiple Myeloma Patients.

Unlike solid-tumor patients, a disappointingly small subset of multiple myeloma (MM) patients treated with checkpoint inhibitors derive clinical benefits, suggesting differential participation of inhibitory receptors involved in the development of T-cell-mediated immunosuppression. In fact, T cells in MM patients have recently been shown to display features of immunosenescence and exhaustion involved in immune response inhibition. Therefore, we aimed to identify the dominant inhibitory pathway in MM patients to achieve its effective control by therapeutic interventions. By flow cytometry, we examined peripheral blood (PB) CD4 T cell characteristics assigned to senescence or exhaustion, considering PD-1, CTLA-4, and BTLA checkpoint expression, as well as secretory effector function, i.e., capacity for IFN-γ and IL-17 secretion. Analyses were performed in a total of 40 active myeloma patients (newly diagnosed and treated) and 20 healthy controls. At the single-cell level, we found a loss of studied checkpoints' expression on MM CD4 T cells (both effector (Teff) and regulatory (Treg) cells) primarily at diagnosis; the checkpoint deficit in MM relapse was not significant. Nonetheless, PD-1 was the only checkpoint distributed on an increased proportion of T cells in all MM patients irrespective of disease phase, and its expression on CD4 Teff cells correlated with adverse clinical courses. Among patients, the relative defect in secretory effector function of CD4 T cells was more pronounced at myeloma relapse (as seen in declined Th1/Treg and Th17/Treg cell rates). Although the contribution of PD-1 to MM clinical outcomes is suggestive, our study clearly indicated that the inappropriate expression of immune checkpoints (associated with dysfunctionality of CD4 T cells and disease clinical phase) might be responsible for the sub-optimal clinical response to therapeutic checkpoint inhibitors in MM.

Imbalance in PB IL-17-Secreting and Regulatory Cells in Pars Planitis Is Associated with Dysregulation of IFN-γ-Secreting Cells, Especially in Patients with Clinical Complications.

RESULTS: In patients, an increase in the population of Th17-secreting cells negatively correlated with the abundance of both IFN-γ-producing and T regulatory as well as suppressor cells, regarding all the phenotypes studied. Although a strong dependence of the PB Th1 cell compartment on the duration of the disease was observed, it was limited to the subgroup of patients with macular edema only. The frequency of B regulatory cells was unchanged compared to controls. CONCLUSIONS: In pars planitis, the alterations in lymphocyte cell distribution affect primarily the T cell repertoire. The imbalance in PB Th1/Th17/Treg cells creates proinflammatory conditions, strengthening the suggestion that the immune background may play a role in pars planitis pathogenesis. Also, circulating Th1 level may be of potential clinical relevance in terms of prediction of a more severe course of the disease.

Dysregulation of the immune system as a driver of the critical course of novel coronavirus disease 2019.

Novel coronavirus disease 2019 (COVID­19) is a highly contagious, respiratory disease caused by the newly emerged severe acute respiratory syndrome coronavirus 2 (SARS­CoV­2). Given that inflammatory immune cells may induce severe lung injury, the involvement of immunological factors in the pathogenesis of the disease cannot be overestimated. It has been demonstrated that coronaviruses have developed mechanisms of immune evasion, making themselves invisible to the immune system at an early stage of infection. The mechanism relies on inhibiting the antiviral response of type I interferons, which enhances uncontrolled viral replication in epithelial cells. There has been a growing body of evidence showing that fatal hyperinflammation (cytokine storm) responsible for the severe course of COVID­19 is a consequence of massive SARS­CoV­2 replication rather than inappropriate hyperresponsiveness of the immune system. Therefore, the suppressed antiviral innate immune response seems to be the primary cause of the delayed critical cascade of uncontrolled immune events leading to fulminant systemic inflammation. The occurrence of virus transmission even in asymptomatic individuals infected with SARS­CoV­2 clearly strengthens the evidence for the key role played by the sufficient immune control of viral replication in a subset of cases (eg, in children, a population with a highly effective innate immune response). Although administration of immunomodulatory drugs is recommended under certain conditions by the guidelines for COVID­19 management, controversies regarding treatment protocols in immunocompromised patients infected with SARS­CoV­2 still exist. Extending clinicians' knowledge on the dysregulated immune response, which is a driver of the COVID­19 outcome, may improve both therapeutic strategies and the prognosis of patients infected with SARS­CoV­2.

CD4+CD28null T cells are expanded in moderately active systemic lupus erythematosus and secrete pro-inflammatory interferon gamma, depending on the Disease Activity Index.

BACKGROUND: Pathogenic CD4+CD28null cells are characterized by inflammatory cytokine synthesis and tropism to the inflamed tissues. Recent studies showed the involvement of CD28null T cells in a severe clinical outcome of lupus. However, their role in moderately active disease is still unresolved. METHODS: We examined the levels of circulating CD4+CD28null cells and CD8+CD28null suppressor T cells. We also compared the CD4+CD28null and CD4+CD28+ T-cell functional properties, including the expression of interferon gamma (IFN-γ) and Ki67 among systemic lupus erythematosus (SLE) patients (n = 20) and healthy controls (n = 20). All the patients were under immunosuppressive treatment and exhibited moderate SLE activity (median SLE Disease Activity Index (SLEDAI) = 6). RESULTS: In patients, we found elevated CD4+CD28null and unchanged levels of suppressor CD8+CD28null T cells. There was no difference between patients and controls in IFN-γ and Ki67-expressing CD4+, CD4+CD28+, and CD4+CD28null T cells, except for higher IFN-γ levels in CD4+CD28+ T cells in SLE. In each studied group, we observed a higher preponderance of IFN-γ- and Ki67-expressing cells among CD4+CD28null T cells and lower levels of IFN-γ in CD4+CD28null T cells compared to the CD28+ subset. Similarly, Ki67 intensity was decreased in healthy CD4+CD28null cells, whereas in patients, comparably high expression was observed in both subsets. IFN-γ intensity in CD4+CD28null T cells correlated with SLEDAI. CONCLUSION: SLE with a moderately active clinical course is characterized by peripheral blood expansion of CD4+CD28null T cells and a normal abundance of suppressor CD8+CD28null T cells. The demonstration that these pathogenic CD4+ T cells, despite the lack of CD28, maintain the ability to produce pro-inflammatory IFN-γ positively correlated with disease activity as well as relatively high proliferative capacity may suggest their potentially predictive role in SLE flares.

CTLA-4 affects expression of key cell cycle regulators of G0/G1 phase in neoplastic lymphocytes from patients with chronic lymphocytic leukaemia.

Previously, we showed that cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) is overexpressed in chronic lymphocytic leukaemia (CLL) and its expression is correlated with the expression of the major regulators of G1 phase progression: cyclins D2 and D3, and cyclin-dependent kinase inhibitory protein 1 (p27 (KIP1) ). In the present study, we blocked CTLA-4 on the surface of both CLL cells and normal B lymphocytes to investigate the impact of CTLA-4 on the expression of the mentioned G1 phase regulators. We found that in CLL patients and in healthy individuals, the median proportions of cyclin D2-positive cells as well as cyclin D3(+) cells significantly decreased following CTLA-4 blockade. Moreover, CTLA-4 blockade led to an increase in the median frequencies of p27 (KIP1) -positive cells, although this increase was marked only in CLL patients. Our study showed that CTLA-4 affects the expression of the key regulators of G1 phase progression in CLL cells as well as in normal B lymphocytes and may contribute to a better understanding of the role of CTLA-4 in the regulation of G1 phase progression.

Patients with chronic lymphocytic leukaemia (CLL) differ in the pattern of CTLA-4 expression on CLL cells: the possible implications for immunotherapy with CTLA-4 blocking antibody.

Recently, systemic administration of a human monoclonal antibody directed against cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) expressed on circulating T cells in patients with chronic lymphocytic leukaemia (CLL) has been considered. Also, CLL cells have been shown to express CTLA-4, increased levels of which in the leukaemic compartment are a predictor of good clinical outcome. Since both CLL and Treg microenvironment cells can be targeted by the CTLA-4 blocking antibody in this immunotherapy approach, the investigation of the functional effect of CTLA-4 blockade on CLL cells might be of potential clinical relevance. The main aim of this study was to examine the effect of CTLA-4 blockade on proliferation activity and apoptosis of CLL cells in patients with low and high CTLA-4 expression. We found that in the high CTLA-4-expressing CLL group, CTLA-4 blockade on the CLL cell surface resulted in a significant increase in the median percentages of Ki67(+) cells and a tendency to decrease in the proportion of apoptotic cells. In contrast, in the low CTLA-4 expressors, CTLA-4 blockade did not affect the proliferation activity or the frequency of apoptosis. This study reports for the first time the different effect of CTLA-4 blockade on CLL cells in CLL patients depending on the levels of CTLA-4 expression. CTLA-4 blockade seems to induce pro-survival signals in leukaemic cells from CLL patients exhibiting high CTLA-4 expression, suggesting that an immunotherapy approach based on the systemic use of monoclonal anti-CTLA-4 antibodies could be an unfavourable strategy for some CLL patients.

[The role of the bone marrow microenvironment in the pathogenesis of multiple myeloma]. / Rola mikrosrodowiska szpiku kostnego w patogenezie szpiczaka plazmocytowego.

Multiple myeloma (MM) is one of the most common hematologic malignancies. It remains an incurable disease, so far. The mainstay of treatment for decades was pointless therapy with cytostatic agents and immunosuppressant's. Because myeloma is most common in the elderly population, vulnerable to aggressive therapy, non-specific treatment approaches led to poor patient survival. Intensive study of MM, allowed identification of the molecular interactions between myeloma cells and bone marrow tumour microenvironment, responsible for the development of the disease and associated complications, such as osteolytic bone lesions. Understanding the molecular mechanisms of action of adhesion molecules, cytokines and signalling pathways involved in the development of myeloma, has led to develop of novel, targeted therapies to improve the quality of patients life and significantly prolong the median survival time. This paper discusses the current state of knowledge of signalling pathways involved in the progression of cancer and the destruction of bone tissue, with particular emphasis on interactions with the bone marrow microenvironment of the tumour.

Pretransplant donor and recipient CTLA-4 mRNA and protein levels as a prognostic marker for aGvHD in allogeneic hematopoietic stem cell transplantation.

BACKGROUND: Cytotoxic T lymphocyte antigen-4 (CTLA-4) is an inhibitory T cells' receptor essential for maintaining T cell homeostasis and immunotolerance. The role of the co-stimulatory pathway in development of aGvHD has been studied mostly in animal models. To the best of our knowledge, there are no published data on the role of CTLA-4 in pathogenesis of aGvHD after hematopoietic stem cell transplantation (HSCT) in humans. Therefore the aim of our study was to determine the association of CTLA-4 mRNA and proteins level in HSCT donor-recipient pairs, prior to and after HSCT, with aGvHD risk. METHODS: Total CTLA-4 mRNA level in 51 donor-recipient pairs prior to and 56 days after HSCT was determined using real time PCR techniques, while membrane (m) and cytoplasmic (c) CTLA-4 expression in CD3+ cells were measured by flow cytometry in 40 donor-recipient pairs at the same time points. RESULTS: We found an association between the risk of aGvHD and high pre-transplant CTLA-4 mRNA expression level both in recipients and in donors, stronger in recipients (OR=2.02, CI95% 1.39-3.01), and less pronounced in donors (OR=1.57, CI95% 1.18-2.0). Moreover, we showed that proportion of CD3+ cells positive for mCTLA-4 in recipients prior to HSCT positively correlated with increased risk of aGvHD (OR=1.175, CI95% 1.024-1.311, p=0.018). CONCLUSION: Our results indicate that both donor and recipient CTLA-4 mRNA as well as recipient membrane protein expression levels measured before transplantation may be considered as prognostic factors for aGvHD development.

Exogenous IL-2 controls the balance in Th1, Th17, and Treg cell distribution in patients with progressive rheumatoid arthritis treated with TNF-alpha inhibitors.

Interleukin-2 (IL-2) has been suggested to control Treg/Th17 balance. Recently, we reported a relationship of rheumatoid arthritis (RA) activity/progression with irreversible systemic Treg and Th1 defects including serum IL-2 shortage. Herein, we explore the role of in vitro stimulation with rIL-2 in the observed immune alterations reversal. Patients with stable or progressive RA were assigned to methotrexate (MTX) group or to TNF-alpha inhibitors (iTNF) group, respectively. Flow cytometric analyses were performed before and after 6 months of treatment. Circulating Th1, Th17, and Treg cells were determined before and after 72-h culture with anti-CD3 + rIL-2. Before therapy, 72-h stimulation restored recently observed phenotypic Th cell alterations, except for the enriched Th17 subset normalized as late as after therapy in all patients. Under 6-month therapy, anti-CD3 stimulation changed the Th cell distribution only in progressive RA; despite Th1 enrichment, it revealed Treg population defects, which were completely reversed by exogenous IL-2 added to the stimulating culture. Our paper shows that in aggressive RA patients exhibiting serum IL-2 shortage despite iTNF therapy, exogenous rIL-2 is capable of promoting Treg differentiation affected by chronic activation, thus supporting its use in the combined strategy of biologic treatment of the progressive form of RA.

Assessment of peripheral blood and bone marrow T, NK, NKT and dendritic cells in patients with multiple myeloma.

Symptoms of multiple myeloma (MM) include bone destruction with pathological fractures, kidney failure and frequent infections, which are the major causes of patient mortality. In our recent research, we demonstrated that the degree of dendritic cell (DC) subpopulation deficit could be related to MM progression, which in consequence may contribute to the MM-related impairment of the immune responses. In the present study, we determined by flow cytometry the frequencies of CD4+ and CD8+ T cells, NK, and NKT-like cells as well as their correlation with myeloid and lymphoid populations of DCs in patients with MM. The study involved 50 patients diagnosed with MM at the Department of Hematology in the Holycross Cancer Center in Kielce. The research samples were collected after the MM diagnosis and before the initiation of anticancer therapy. The obtained results revealed the relations between the percentages of DC subpopulations and lymphocyte subsets, especially the activated ones, in the peripheral blood (PB) and bone marrow (BM). The described role of DCs in the process of the immunological response, either adaptive or innate, leads us to conclude that the decrease of the number or percentage of these cells may have a negative impact on the process of activation of effector cells and, consequently, on the effectiveness of a response to foreign as well as neoplastic antigens in patients with MM.

[Daratumumab--breakthrough drug in multiple myeloma therapy]. / Daratumumab--przelomowy lek w terapii szpiczaka plazmocytowego.

Multiple myeloma (MM) remains incurable despite important recent advances in treatment. Over the last 2 years, an anti-CD38 monoclonal antibody daratumumab (DARA) has emerged as a breakthrough targeted therapy for patients with MM. Early-stage clinical trials have found DARA to be safe and to have encouraging clinical activity as a single agent and in combination with lenalidomide in heavily pretreated, relapsed patients in whom other novel agents (such as bortezomib, thalidomide and lenalidomide) as well as stem cell transplant has already failed. This review discusses the preclinical and clinical development of DARA, its pathophysiological basis, and its prospects for future use in MM.

Low-dose interleukin-2 therapy: a driver of an imbalance between immune tolerance and autoimmunity.

For many years, the role of interleukin-2 (IL-2) in autoimmune responses was established as a cytokine possessing strong pro-inflammatory activity. Studies of the past few years have changed our knowledge on IL-2 in autoimmune chronic inflammation, suggesting its protective role, when administered at low-doses. The disrupted balance between regulatory and effector T cells (Tregs and Teffs, respectively) is a characteristic of autoimmune diseases, and is dependent on homeostatic cytokines, including IL-2. Actually, inherent defects in the IL-2 signaling pathway and/or levels leading to Treg compromised function and numbers as well as Th17 expansion have been attributed to autoimmune disorders. In this review, we discuss the role of IL-2 in the pathogenesis of autoimmune diseases. In particular, we highlight the impact of the dysregulated IL-2 pathway on disruption of the Treg/Th17 balance, reversal of which appears to be a possible mechanism of the low-dose IL-2 treatment. The negative effects of IL-2 on the differentiation of follicular helper T cells (Tfh) and pathogenic Th17 cells, both of which contribute to autoimmunity, is emphasized in the paper as well. We also compare the current IL-2-based therapies of animal and human subjects with immune-mediated diseases aimed at boosting the Treg population, which is the most IL-2-dependent cell subset desirable for sufficient control of autoimmunity. New perspectives of therapeutic approaches focused on selective delivery of IL-2 to inflamed tissues, thus allowing local activity of IL-2 to be combined with its reduced systemic and pleiotropic toxicity, are also proposed in this paper.

Peripheral blood Th17/Treg imbalance in patients with low-active systemic lupus erythematosus.

INTRODUCTION: The balance between proinflammatory Th17 cells and regulatory T cells plays an important role in the pathogenesis of autoimmune diseases, including systemic lupus erythematosus (SLE). In particular, an increased ratio of Th17/Treg cells has been shown to correlate with active SLE and specific organ involvement. The aim of our study was to assess Th17 and Treg cell populations in peripheral blood (PB) of patients with clinically quiescent SLE, and to evaluate their correlation with organ involvement. MATERIAL/METHODS: We performed flow cytometric analysis of studied T CD4+ cell subpopulations in PB from 21 patients with SLE and 13 healthy controls. Disease activity was measured with the SELENA-SLEDAI index; organ involvement was divided into renal, neurological and hematological. RESULTS: A statistically significant difference (p<0.01) between the mean percentages of CD4+CD25highFoxP3+ Treg cells in SLE patients (18.57%) and healthy controls (32.08%) was observed. Similarly, proportions of functional CTLA-4+ Treg cells were markedly lower in SLE patients than in healthy controls--19.3% vs. 23.82% (p=0.03). In contrast, SLE patients exhibited a significantly increased frequency of circulating Th17 cells with the phenotype CD4+IL-17+ compared to controls--1.36 % vs 0.19% (p<0.01). Also the ratio of Th17 cells to Th1 cells was markedly higher in SLE patients than in the control group (p<0.01). We did not find any correlation of PB Th cell distribution with organ involvement in SLE patients examined. CONCLUSIONS: Our report showed for the first time that systemic Th17/Treg imbalance occurred also in patients with low disease activity and in remission. We suggest that immunological alterations may precede clinical and laboratory symptoms of the disease activity.

Patients with the most advanced rheumatoid arthritis remain with Th1 systemic defects after TNF inhibitors treatment despite clinical improvement.

Systemic immune defects might reflect severely dysregulated control of chronic inflammation related to disease progression. Th17/Treg cell imbalance has been demonstrated to be involved in rheumatoid arthritis (RA) pathogenesis. Despite controversial results, a growing anti-inflammatory role in this process has been recently attributed to Th1 responses. The aim of the study was to estimate the extent of Th1/Th17/Treg imbalance in peripheral blood (PB) of patients with short- and long-term RA in relation to cytokine milieu and its reversal after therapy with methotrexate and/or TNF inhibitors, respectively. Patients with different duration of RA (median 6 vs. 120 months) in the active phase of RA were enrolled in this study. We performed flow cytometric analysis of PB Th1, Th17, and Treg populations together with estimation of serum cytokine concentrations using cytometric bead array. Disease activity was calculated on the basis of clinical and biochemical indices of inflammation (DAS28, ESR, CRP). All parameters were measured and correlated with each other before and after 6 months therapy. Elevated levels of circulating Th17 cells and IL-6 were found in all active patients, of which Th17 cells were down-regulated by the treatment. Significantly reduced Th1 and functional CTLA-4+ Treg cell frequencies as well as Th1 cytokines observed only in progressive RA seemed to be irreversible. Although therapy induced clinical improvement in almost all patients, those with advanced RA remained with signs of inflammation. Our report demonstrates that both the extent of systemic immune abnormalities and their restoration are dependent on duration of the active RA.

Alterations in both the activatory and inhibitory potential of peripheral blood CD4+ T cells in rheumatoid arthritis patients correlate with disease progression.

The chronic nature of rheumatoid arthritis (RA) suggests immune dysfunction, including persistent systemic activation. Therefore, we evaluated the activatory and inhibitory potential as well as proliferative activity of peripheral blood (PB) CD4+ T cells from RA patients in different stages of the disease and after different therapeutic interventions. We found that CD4+ T cells from RA patients were activated in vivo concerning decreased CD28 expression and increase of CD40L, CD69, and CTLA-4 expression; however, the extent of stimulation was suboptimal when compared to healthy controls. Consequently, impaired proliferative activities of these cells were found in all patients irrespective of the active disease duration. Treatment with methotrexate (MTX) and/or inhibitors of TNF-alpha (iTNF) did not significantly influence systemic activation in RA patients, which corresponded with the maintenance of inflammation markers; however, partial restoration of CD28 and CTLA-4 expression as well as clinical improvement were observed. In patients with early disease (the MTX group), we noted higher capacity of CD4+ T cells for restoration of T cell function, whereas cells from the iTNF group with progressive disease remained with a proliferative defect after the treatment. In conclusion, our study demonstrates that the dysregulated expression of molecules interfering with CD4+ T cell signaling may result in functional impairment of the effector T cells and correlates with disease progression.