Decrease in CD4+CD25+ and CD8+CD28+ T cells in interstitial pneumonitis associated with rheumatic disease

Abstract

The expression of CD25 or CD28 on T cells was examined in patients with rheumatic diseases associated with interstitial pneumonitis (IP), in order to investigate the conditions of CD4⁺CD25⁺ regulatory T cells and CD8⁺CD28^? suppressor T cells. Fifty-five patients with various rheumatic diseases and 23 normal controls were enrolled. CD4⁺CD25⁺ T cells of patients with IP were significantly decreased in comparison with non-IP patients, and the ratio of CD8⁺CD28^? T cells in patients with IP was significantly higher than that in non-IP patients or normal controls. These results for CD8⁺CD28^? T cells were in accord with the decrease in CD8⁺CD28⁺ T cells, and may be related to activation-induced CD8⁺CD28⁺ T-cell death. Thus, the abnormality of CD4⁺CD25⁺ regulatory T cells may be related to the pathogenesis of IP, and the survival and activation of CD8⁺ T cells.     

Decrease in CD4+CD25+ and CD8+CD28+ T cells in interstitial pneumonitis associated with rheumatic disease

The expression of CD25 or CD28 on T cells was examined in patients with rheumatic diseases associated with interstitial pneumonitis (IP), in order to investigate the conditions of CD4⁺CD25⁺ regulatory T cells and CD8⁺CD28⁻ suppressor T cells. Fifty-five patients with various rheumatic diseases and 23 normal controls were enrolled. CD4⁺CD25⁺ T cells of patients with IP were significantly decreased in comparison with non-IP patients, and the ratio of CD8⁺CD28⁻ T cells in patients with IP was significantly higher than that in non-IP patients or normal controls. These results for CD8⁺CD28⁻ T cells were in accord with the decrease in CD8⁺CD28⁺ T cells, and may be related to activation-induced CD8⁺CD28⁺ T-cell death. Thus, the abnormality of CD4⁺CD25⁺ regulatory T cells may be related to the pathogenesis of IP, and the survival and activation of CD8⁺ T cells.     

Idiotype-specific T lymphocytes in monoclonal gammopathies: evidence for the presence of CD4+ and CD8+ subsets

Tumour-specific CD4⁺ T helper (Th) and CD8⁺ T cytotoxic (Tc) cells may participate in the control and eradication of tumour cells. In the present study, idiotype-specific stimulation of CD4⁺ and CD8⁺ blood T cells from patients with monoclonal gammopathy of undetermined significance and patients with untreated multiple myeloma stage I was examined. Activation was measured in the CD4⁺ and CD8⁺ subsets enriched by magnetic microbeads as the incorporation of ³H-thymidine and the secretion of interferon (IFN)-γ, interleukin (IL)-2 and IL-4 by single cells using the enzyme-linked immunospot assay. Idiotype-specific T cells were found in four of seven patients. Stimulation was mainly confined to the CD4⁺ subset in three of the four responding patients. This type of response was major histocompatibility complex (MHC) class II restricted as it could be inhibited by monoclonal antibodies against MHC class II (HLA-DR), but not against class I (HLA-ABC) molecules. Idiotype-specific CD8⁺ T cells were also demonstrated in these patients although at a lower frequency. One patient showed a strong and dominating activation of CD8⁺ T cells which could be blocked by antibodies against HLA-ABC but not against HLA-DR. Idiotype-specific CD4⁺ or CD8⁺ T cells were mainly of the type-1 subsets as judged by their secretion of IFN-γ and IL-2. Thus, this study provides evidence for the presence of idiotype-specific and MHC-restricted CD4⁺ and CD8⁺ T cells of the type-1 subsets in patients with monoclonal gammopathies. Such T cells with the potential to control the growth of tumour B cells may be a suitable target for immunotherapeutic interventions in patients.     

Patients with B cell chronic lymphocytic leukaemia have an expanded population of CD4+ perforin expressing T cells enriched for human cytomegalovirus specificity and an effector-memory phenotype

We have previously shown an expansion of cytotoxic antigen‐experienced CD4⁺T cells (CTLs) that express perforin (PF) in the peripheral blood of patients with B cell chronic lymphocytic leukaemia (B‐CLL). Increased frequencies of CD4⁺CTLs have since been attributed to chronic viral infections, particularly, human cytomegalovirus (HCMV). The present study examined the involvement of CD4⁺CTLs in responses to HCMV in B‐CLL, and characterized their differentiation. We studied 36 HCMV seropositive (SP) and seronegative B‐CLL patients and 20 healthy age‐matched individuals. The HCMV reactivity of CD4⁺PF⁺ and CD4⁺PF^? cells was determined by interferon‐gamma expression, and expression of CD45RA and CCR7 was assessed by flow cytometry. Fluorescence in‐situ hybridization was used to measure relative telomere lengths. CD4⁺PF⁺T cell expansion in B‐CLL patients and controls was strongly associated with HCMV seropositivity. CD4⁺PF⁺ compared to CD4⁺PF^? cells from SP B‐CLL patients elicited major histocompatibility complex (MHC) class II‐restricted responses to HCMV. CD4⁺PF⁺T cells from patients and controls were enriched with highly differentiated T‐effector/memory (CCR7^?) and revertant (CCR7^?CD45RA⁺) phenotype. CD4⁺PF⁺T cells from B‐CLL patients had shorter telomeres than CD4⁺PF^?T cells, indicating an extensive replicative history. We conclude that persistent exposure to HCMV antigens in SP B‐CLL patients leads to an expansion of the circulating MHC class II‐restricted CD4⁺PF⁺T cell population with effector/memory phenotype.     

Clonal CD8+ and CD52– T cells are induced in responding B cell lymphoma patients treated with Campath-1H* (anti-CD52)

Abstract: Five patients with non-Hodgkin's lymphoma (NHL) and 4 patients with chronic lymphocytic leukaemia (CLL) were treated with the CDR-grafted (rat × human) monoclonal antibody (mAb) Campath-1H (anti-CD52). Tumour regression was noted preferentially in peripheral blood and in the bone marrow but lymph nodes were less affected. Normal blood B and T cells were profoundly reduced in all patients whereas CD16⁺ NK cells and CD14⁺ monocytes decreased marginally. In all responding CLL patients CD52-negative T but not B cells appeared during treatment and persisted for several months (4–19+) during unmaintained follow-up. Clonal T cells defined as a predominance of a single T cell receptor (TCR) V gene usage, in one case verified by TCR CDR3 fragment analysis and nucleotide sequencing, emerged within the CD52^–/CD8⁺ cell population during Campath-1H therapy in 2 CLL patients, both achieving a long-lasting remission. The increase in CD8⁺ T cell expansions (up to 23-fold) during unmaintained remission and follow-up suggest that the clonal CD8⁺ cells may represent regulatory T cells controlling the growth of the tumour B cell clone. Clonal T cells might thus be a target for an immune therapeutic intervention in B cell tumours.     

Activated CD4+ and CD8+ T-cell subsets in Wegener's granulomatosis

Several lines of evidence argue in favour of an involvement of T cells in the pathogenesis of Wegener's granulomatosis (WG). These include the presence of highly specific IgG autoantibodies to proteinase 3, perivascular T-cell infiltrates and elevated amounts of soluble interleukin-2 (IL-2) receptors in patient's serum. In order to further address this question we evaluated by double immunoflourescence and flow cytometry the expression of several cell surface molecules associated with T-cell activation. As compared to healthy controls (n=15), the CD4⁺ subset was significantly diminished, while the percentage of CD8⁺ T cells was elevated in WG patients (n=24). Within the CD4⁺ T-cell subset we found a highly significant increase in activation/memory markers (CD25, CD29, HLA-DR). Within the CD8⁺ T-cell subset the expression of CD11b, CD29 and CD57 was significantly elevated, while the expression of VD28 was reduced. The use of 10 V-, 1 V-and 1 V-specific monoclonal reagents failed to reveal any significant bias in the peripheral T-cell receptor V-gene repertoire of WG patients. There was also no correlation between T-cell activation markers and laboratory parameters [C-reactive protein (CRP), ESR], disease duration or therapy. A significant correlation was found only for the degree of organ involvement and the increase in CD4⁺ T cells coexpressing HLA-DR, as well as the increase in CD57 expression on CD8⁺ T cells. In conclusion, both CD4⁺ and CD8⁺ T-cell subsets were activated in WG. Cytotoxic CD8⁺ CD57⁺ CD11b⁺ CD28^– T cells may directly contribute to damage of vascular endothelium.     

Associations of circulating CXCR3–PD-1+CD4+T cells with disease activity of systemic lupus erythematosus

Abstract

Objectives: Which helper CD4⁺ T cell subset contributes to autoantibodies generation and severity of end-organ involvement in lupus patients remains to be explored. Our research aims to investigate the roles of circulating Tfh (cTfh) cell subsets and corresponding CXCR5^– Th cells in lupus patients and their correlation with SLE disease activity index 2000 (SLEDAI).

Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from blood of systemic lupus erythematosus (SLE) patients as well as healthy donors. The proportion of Th cell subsets classified from cell surface markers (CD45RO, CXCR5, CXCR3, CCR6, PD-1, ICOS, and CCR7) is detected by flow cytometry.

Results: We found no difference in the frequency of CD45RO⁺CXCR5⁺CD4⁺ T cells between SLE patients and health controls. As previously reported, SLE patients showed an increase in the percentage of CXCR5⁺PD-1⁺, CXCR5⁺ICOS⁺PD-1⁺ and CXCR5⁺CCR7^loPD-1^hi cTfh subset, however, none of these populations had correlation with SLEDAI. Therefore, we further investigated the CXCR5^– subsets, and surprisingly we found that the frequency of CXCR3^–PD-1⁺ subset was correlated with SLEDAI, ds-DNA IgG, anti-nucleosome antibody, C3, and C4 independent of CXCR5. Consistently, CXCR3^–PD-1⁺CD45RO⁺CD4⁺T cells expressed factors associated with B-cell-help for the autoantibody production.

Conclusion: CXCR3^–PD-1⁺CD4⁺T cells are a sensitive indicator to assess SLE disease activity and might contribute B cell help and the generation of autoantibodies in patients.     

The role of CD4+ and CD8+ T cells in protective immunity to the murine nematode parasite Trichuris muris

The role ofCD4⁺ and CD8⁺ T cells in protective immunity to Trichuris muris was studied in CD4⁺ or CD8⁺ or both CD4⁺ and CD8⁺ T cell-depleted BALB/c mice. To assess in vivo depletion of T-cell subsets, CD4⁺ and CD8⁺ T cells in the Peyer's patches, the mesenteric lymph nodes (MLN), and the spleens of mice treated with T cell-specific monoclonal antibodies (MoAbs) were analysed by FACS. CD4⁺ T cells were selectively depleted in mice injected with anti-CD4 MoAb i.p. and injection of anti-CD8 MoAb resulted in selective depletion ofCD8⁺ T cells. The expulsion ofl. muris was inhibited in CD4⁺ T cell-depleted mice and numerous worms were detected in the large intestine on days 14 and 21 after infection, although no suppression of protective immunity to T. muris was observed in CD8⁺ T cell-depleted mice. Moreover, there was no difference in suppression of protective immunity to T. muris between CD4⁺ T cell-depleted and both CD4⁺ and CD8⁺ T cell-depleted mice. These results indicate that CD4⁺ T cells play a central role in protective immunity to T. muris infection.     

Age-associated up-regulation of a negative co-stimulatory receptor PD-1 in mouse CD4 T cells

To explore whether any co-stimulatory receptor(s) for TCR signaling is involved in the age-associated decline in T-cell function, we analyzed changes in these receptors in freshly isolated mouse CD4⁺ T cells during aging. Both the mRNA and protein expression levels of CTLA-4 and PD-1, negative co-stimulatory receptors, increase with aging. No such changes are observed for CD28, a positive regulatory receptor. PD-1 is highly expressed on the surface of old, but not young, mouse T cells, while the level of surface-expressed CTLA-4 is very low regardless of age. PD-1 is preferentially expressed on the surface of effector-memory (CD44^hiCD62L^lo) T cells, a subset that increases with aging. CD4⁺PD-1⁺ T cells from old mice exhibit proliferative hyporesponsiveness. These results suggest that the up-regulation of surface-expressed PD-1 may cause the age-dependent functional decline in effector-memory T cells.     

Quantitative alterations of CD8+ T cells in juvenile idiopathic arthritis patients in remission

The study was aimed to investigate whether quantities of CD8⁺ T cell subsets are normal in juvenile idiopathic arthritis (JIA) patients with disease remission compared to age-matched healthy donors (HD) and whether chronological age may have an impact on proportions of naive CD8⁺ T cells. CD8⁺ T cell subsets were analyzed in 17 JIA patients and 32 age-matched HD by flow cytometry. JIA patients showed lower CD3⁺CD8⁺ T cells compared to HD. Total counts of CD8⁺CD28⁺ and CD8⁺CD28⁺CD45RA⁺ T cells were inversely correlated to chronological age in JIA patients and HD. In JIA patients, percentages of CD8⁺CD28⁺CD45RA⁺ T cells and of CD62L-expressing CD8⁺CD28⁺CD45RA⁺ T cells showed a negative correlation with age. The trend to lower CD28⁺CD45RA⁺ T cell proportions in aged JIA patients in remission may reflect a disturbed T cell homeostasis independently of disease activity and may be due to an intrinsic effect in reconstitution of the peripheral T cells.     

Increased surface receptor Fas (CD95) levels on CD4+ lymphocytes in patients with primary intestinal lymphangiectasia

Objective. Exudative enteropathy secondary to primary intestinal lymphangiectasia (PIL) is characterized by lymphopenia, hypogammaglobulinemia and hypoalbuminemia resulting from leakage of lymph fluid into the intestinal tract. The objective of this study was to better characterize the lymphopenia of PIL-confirmed patients. Material and methods. T-cell markers and T-cell proliferation/capacities (differentiation, activation and death) were analyzed for phenotype in 9 patients (6 F, 3 M, aged from 18 to 72 years). Results. Mean counts of CD4 and CD8 subsets were significantly decreased, 174±123/µl and 134±77/µl compared with controls, 858±260/µl and 482±164/µl, respectively (p<0.0001). Significant depletion of naïve (CD45RA⁺ CD62L⁺) CD4⁺ T cells was noted, with a mean expression of 7±4% compared with controls, 45±1% (p<0.0001). Both CD4⁺ and CD8⁺ T-cell mean subsets were activated as assessed by their proportion expressing the late activation markers HLA-DR, 18±7% and 19±9% compared with controls, 6±3% and 10±6%, respectively (p<0.0001 and p<0.01). The mean expression of CD95/Fas on CD4⁺ T cells was significantly higher in patients than in controls, 83±16% versus 45±13% (p<0.0001). No major abnormality of T-cell proliferation/capacities was observed. Conclusions. Our results suggest that the T-cell loss in PIL patients is probably due to various mechanisms including enteric lymphocytes loss and activation of residual T cells, leading to death. Moreover, this loss is not compensated by a sufficient increase in T-cell thymic production.     

CD8+/CD57+ cells and apoptosis suppress T-cell functions in multiple myeloma

The aim of this study was to evaluate the role of CD8⁺/CD57⁺ lymphocytes in the immune dysregulation of multiple myeloma (MM). Cytofluorimetry of peripheral blood lymphocytes (PBL) purified from 39 MM patients showed an inverse relationship between the percentage of CD8⁺/CD57⁺ cells and CD4/CD8 ratio. Analysis of their activation antigens revealed that they were prevalently HLA-DR⁺ and Fas⁺. Removal of CD8⁺/CD57⁺ cells from MM PBL significantly improved cell proliferation and pokeweed mitogen (PWM)-induced polyclonal Ig production in vitro, whereas the addition of supernatants from patients' CD8⁺/CD57⁺ cell cultures to normal PBL suppressed both the PWM-driven Ig synthesis and the proliferative rate of stimulated PBL, supporting the contention that CD8⁺/CD57⁺ cells release in vitro an inhibitory factor that is directly involved in T-cell regulatory function. However, since the proliferative recovery of PWM- and phytohaemagglutinin (PHA)-stimulated MM PBL in the absence of CD8⁺/CD57⁺ lymphocytes was only partial, a dysregulated activation-induced apoptosis was anticipated. In fact, patients' PBL displayed an increased susceptibility to apoptosis and this was significantly enhanced after PWM and, even more, after PHA stimulation. Analysis of CD57 antigen expression on apoptotic or viable cells demonstrated a substantial defect of apoptosis in the CD8⁺/CD57⁺ population. Our results indicate that both the immunosuppressive effect of CD8⁺/CD57⁺ cells and the enhanced susceptibility to apoptosis of PBL could be involved in the pathogenesis of the immunodeficiency observed in this disease.     

Impaired Mitogenic Response of Peripheral Blood T Cells in Ulcerative Colitis Is Not Due to Apoptosis

An abnormal immune response may play apathogenic role in ulcerative colitis (UC). Animalmodels suggest that T-cell regulation may be of centralimportance in the inflammatory process. Our aims werethe characterization of the phenotype andfunctional status of circulating T-cells in ulcerativecolitis patients and to determine if activation-inducedcell death in CD4⁺ and CD8⁺lymphocytes in patients differs from healthy controls.Forty-eight patients (24 women and 24 men) fulfillingthe histopathological, clinical, and immunologicalcriteria for UC were studied. T-cell phenotype andfunction were studied in blood lymphocytes from patientswith ulcerative colitis and healthy donors by flowcytometric analysis, as well as [³H]thymidineincorporation. There were no significant differences in the percentage of T-cell subpopulations(CD3, CD4, CD8) and NK cells in the different groups.The percentage of cells in growth phase S+G₂Mat two and three days of phytohemagglutinin (PHA) stimulation was significantly decreased in UCpatients, but the percentage of CD4⁺ andCD8⁺ cells in UC patients that showedapoptosis was not significantly different than that inthe control group. Proliferative responses to IL-4 also suggested that a reducedresponsiveness to this cytokine may be involved in UC.In conclusion, the impaired proliferative response toPHA of T lymphocytes from UC patients is not associated with an in vitro increase in theapoptotic response in CD4⁺ or CD8⁺cells. A reduced IL-4 response may be involved in thispeculiar mitogenic response. These changes may be pathogenic or a favorable adaptivemechanism.     

Increased CD4+CD16+ and CD4+CD56+ T cell subsets in Behçet's disease

Behçet's disease is a systemic vasculitis of unknown etiology. Various immune abnormalities have previously been shown in Behçet's disease. We investigated T lymphocyte subsets associated with cytotoxic activity and natural killer (NK) cells by flow cytometry in 37 patients with Behçet's disease, 38 healthy controls, and 17 diseased control patients. Compared to the healthy controls, CD4⁺CD16⁺ and CD4⁺CD56⁺ subsets were found to be higher in the Behçet's disease group as well as in the disease control group (CD4⁺CD16⁺: BD=5?±?3, DC=14?±?14, HC= 3?±?2, P=0.001; CD4⁺CD56⁺: BD=11?±?5, DC= 18?±?17, HC=8?±?6, P=0.01). CD8⁺CD16⁺ and CD8⁺CD56⁺ T cell subsets were at normal levels in Behçet's disease but found to be elevated in disease controls. Similarly, NK cells (CD16⁺CD56⁺) were high only in the disease control group. Significant increases in CD4⁺CD16⁺ and CD4⁺CD56⁺ cell subsets in Behçet's patients and disease controls suggest that T cell activation patterns of these subsets in Behçet's disease are similar to those in other inflammatory disorders.     

Enhanced SARS-CoV-2-Specific CD4+ T Cell Activation and Multifunctionality in Late Convalescent COVID-19 Individuals

Background: Examination of CD4⁺ T cell responses during the natural course of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection offers useful information for the improvement of vaccination strategies against this virus and the protective effect of these T cells. Methods: We characterized the SARS-CoV-2-specific CD4⁺ T cell activation marker, multifunctional cytokine and cytotoxic marker expression in recovered coronavirus disease 2019 (COVID-19) individuals. Results: CD4⁺ T-cell responses in late convalescent (>6 months of diagnosis) individuals are characterized by elevated frequencies of activated as well as mono, dual- and multi-functional Th1 and Th17 CD4⁺ T cells in comparison to early convalescent (<1 month of diagnosis) individuals following stimulation with SARS-CoV-2-specific antigens. Similarly, the frequencies of cytotoxic marker expressing CD4⁺ T cells were also enhanced in late convalescent compared to early convalescent individuals. Conclusion: Our findings from a low-to middle-income country suggest protective adaptive immune responses following natural infection of SARS-CoV-2 are elevated even at six months following initial symptoms, indicating the CD4⁺ T cell mediated immune protection lasts for six months or more in natural infection.     

IFN-γ and TNF-α secretion by CD4+ and CD8+ TCRαβ+ T-cell clones derived early after allogeneic bone marrow transplantation

Abstract: Secretion of the potentially antileukaemic cytokines IFN-γ and TNF-α was investigated for CD4 ⁺ and CD8 ⁺ TCRαβ⁺ T-cell clones derived from 4 leukaemia patients 3–6 weeks after allogeneic BMT. We investigated cytokine secretion in response to the activation signal accessory cells + phytohaemagglutinin + Interleukin 2. All clones derived after BMT were capable of IFN-γ and TNF-α secretion, and both for CD4⁺ (n = 96) and CD8⁺ (n = 8) T cells quantities of IFN-γ and TNF-α were significantly correlated with one another. When comparing the overall results for posttransplant and normal T-cell clones derived from 2 bone marrow donors (n = 65), both CD4⁺ and CD8⁺ TCRαβ⁺ T-cell clones showed increased IFN-γ production, and CD4⁺ but not CD8⁺ clones showed a decreased TNF-α secretion. The results suggest that noncytotoxic T cells derived after allogeneic BMT can produce IFN-γ and TNF-α and may thus be capable of mediating antileukaemic effects.     

Impact of CD39 expression on CD4+ T lymphocytes and 6q deletion on outcome of patients with chronic lymphocytic leukemia

Objective/Background

Chronic lymphocytic leukemia is one of the commonest leukemias affecting adults. CD39 inhibits T-cell and Natural killer (NK) cell responses by hydrolyzing adenosine triphosphate and adenosine diphosphate, suppressing the immune system. We investigated expression of CD39 on CD4+ T Lymphocytes in chronic lymphocytic leukemia (CLL) patients and its relationship with deletion 6q, its association with disease stage and survival.

Identification of activated T cells and the suppressor/inducer subset in patients suffering from severe aplastic anemia

Summary In patients with severe aplastic anemia (SAA), lymphocyte subpopulations were examined for the presence of HLA-DR and 2H4 (suppressor/inducer subset) antigen-expressing cells by flow cytometric analysis. Investigations were performed on peripheral blood lymphocytes before and after therapy with antithymocyte globulin (ATG) and methylprednisolone (MP), as well as on bonemarrow lymphocytes before therapy. Before treatment, only the absolute numbers of CD4⁺ T cells and the CD4⁺HLA^–DR⁺/CD8⁺HLA-DR⁺ activated T cell ratio were significantly decreased (p<0.01 and p<0.001, respectively). Following successful ATG/MP treatment, a decrease in the CD4⁺/CD8⁺T cell ratio was found. Regarding the suppressor/inducer subset, only absolute numbers of CD4⁺/2H4⁺ cells were somewhat higher in treated patients; the percentages were the same in all groups of patients. Studies performed on bonemarrow lymphocytes showed significantly decreased percentages of CD4⁺ and CD8⁺ T lymphocytes, which also express HLA-DR antigen. No significant changes in the distribution of activated T cells following ATG/MP therapy were found, suggesting that these cells play no major role in the pathogenesis of the disease.     

NKG2D expression in CD4+ T lymphocytes as a marker of senescence in the aged immune system

Human aging is characterized by changes in the immune system which have a profound impact on the T-cell compartment. These changes are more frequently found in CD8+ T cells, and there are not well-defined markers of differentiation in the CD4+ subset. Typical features of cell immunosenescence are characteristics of pathologies in which the aberrant expression of NKG2D in CD4+ T cells has been described. To evaluate a possible age-related expression of NKG2D in CD4+ T cells, we compared their percentage in peripheral blood from 100 elderly and 50 young adults. The median percentage of CD4+ NKG2D+ in elders was 5.3% (interquartile range (IR): 8.74%) versus 1.4% (IR: 1.7%) in young subjects (p < 0.3 × 10⁻¹⁰). CD28 expression distinguished two subsets of CD4+ NKG2D+ cells with distinct functional properties and differentiation status. CD28+ cells showed an immature phenotype associated with high frequencies of CD45RA and CD31. However, most of the NKG2D+ cells belonged to the CD28^null compartment and shared their phenotypical properties. NKG2D+ cells represented a more advanced stage of maturation and exhibited greater response to CMV (5.3 ± 3.1% versus 3.4 ± 2%, p = 0.037), higher production of IFN-γ (40.56 ± 13.7% versus 24 ± 8.8%, p = 0.015), lower activation threshold and reduced TREC content. Moreover, the frequency of the CD4+ NKG2D+ subset was clearly related to the status of the T cells. Higher frequencies of the NKG2D+ subset were accompanied with a gradual decrease of NAIVE and central memory cells, but also with a higher level of more differentiated subsets of CD4+ T cells. In conclusion, CD4+ NKG2D+ represent a subset of highly differentiated T cells which characterizes the senescence of the immune system.