T-Cell receptor Vbeta repertoire CDR3 length diversity differs within CD45RA and CD45RO T-cell subsets in healthy and human immunodeficiency virus-infected children

The T-cell receptor (TCR) CDR3 length heterogeneity is formed during recombination of individual Vbeta gene families. We hypothesized that CDR3 length diversity could be used to assess the fundamental differences within the TCR repertoire of CD45RA and CD45RO T-cell subpopulations. By using PCR-based spectratyping, nested primers for all 24 human Vbeta families were developed to amplify CDR3 lengths in immunomagnetically selected CD45RA and CD45RO subsets within both CD4(+) and CD8(+) T-cell populations. Umbilical cord blood mononuclear cells or peripheral blood mononuclear cells obtained from healthy newborns, infants, and children, as well as human immunodeficiency virus (HIV)-infected children, were analyzed. All T-cell subsets from newborn and healthy children demonstrated a Gaussian distribution of CDR3 lengths in separated T-cell subsets. In contrast, HIV-infected children had a high proportion of predominant CDR3 lengths within both CD45RA and CD45RO T-cell subpopulations, most commonly in CD8(+) CD45RO T cells. Sharp differences in clonal dominance and size distributions were observed when cells were separated into CD45RA or CD45RO subpopulations. These differences were not apparent in unfractionated CD4(+) or CD8(+) T cells from HIV-infected subjects. Sequence analysis of predominant CDR3 lengths revealed oligoclonal expansion within individual Vbeta families. Analysis of the CDR3 length diversity within CD45RA and CD45RO T cells provides a more accurate measure of disturbances in the TCR repertoire than analysis of unfractionated CD4 and CD8 T cells.     

CD25 expression distinguishes functionally distinct alloreactive CD4 CD134 (OX40) T-cell subsets in acute graft-versus-host disease.

CD134 (OX40) is expressed on activated CD4(+) donor T cells in allogeneic stem cell transplant recipients with acute graft-versus-host disease. The data presented here reveal that differential expression of CD25 by CD4(+) CD134(+) T cells allows separation of these activated cells into 2 phenotypically and functionally distinct alloreactive T-cell subsets. These subsets exhibit distinct tissue associations, with CD4(+) CD134(+) CD25(-) T cells preferentially found in lymphoid tissues and CD4(+) CD134(+) CD25(+) T cells located in lymphoid tissues and inflamed extralymphoid tissues. The CD25(-) T-cell subset exhibited potent proliferative responses to both concanavalin A and allogeneic host leukocytes. By contrast, the CD25(+) T-cell subset proliferated minimally in response to either treatment and inhibited alloantigen-induced proliferation of the CD25(-) subset. Proliferative unresponsiveness associated with the CD25(+) T-cell subset did not extend to cytokine secretion. When stimulated with alloantigen, both CD4(+) CD134(+) T-cell subsets responded by secreting interferon-gamma and interleukin (IL)-10, and neither T-cell subset produced detectable levels of IL-2 or IL-4. Three-day treatment of the CD25(+) T-cell subset with IL-2 restored the proliferative responsiveness of these cells to host alloantigens, suggesting that the proliferative unresponsiveness associated with this T-cell subset reflected a requirement for IL-2. The preferential tissue associations and distinct functional properties associated with these separable alloreactive CD4(+) CD134(+) T-cell subsets suggest that they participate differentially in clinical graft-versus-host disease.     

Role of new population of peripheral CD11c(+)CD8(+) T cells and CD4(+)CD25(+) regulatory T cells during acute and remission stages in rheumatoid arthritis patients.

BACKGROUND AND PURPOSE: Rheumatoid arthritis (RA) is a CD4(+)-dependent chronic systemic inflammatory disease with autoimmune features. Autoreactive CD4(+) T-cell activation can result in autoimmune diseases. One of the key regulators is the CD4(+)CD25(high) regulatory T (Treg) cell. In an animal arthritis model, CD11c(+)CD8(+) T cells were found to be elevated, and could suppress pathogenic CD4(+) T cells after cross-linking with CD137. The purpose of this study was to compare the expression of CD137, CD4(+)CD25(high) Treg cells, and CD11c(+)CD8(+) in the peripheral blood T lymphocytes of RA patients during active and remissive states, and evaluate the correlation with disease activity. METHODS: Thirty nine RA patients treated at the rheumatology outpatient clinic at the Changhua Christian Hospital were assessed clinically for disease activity and classified as either highly active or remissive by the Disease Activity Score 28. Peripheral blood mononuclear cells were isolated from these patients and compared against normal controls. RESULTS: The presence of CD11c(+)CD8(+) T cells or the expression of CD137 molecules in peripheral blood cells was not related to disease activity. In contrast, CD4(+)CD25(high) Treg cell levels were increased significantly in patients with active RA compared with patients with remissive RA or controls (p<0.05). These lymphocytes were intact, without evidence of apoptosis. CONCLUSIONS: Our results indicate that CD4(+)CD25(high) Treg cells play an important role in modulating RA disease activity and can serve as a parameter of disease activity.     

Circulating lymphocyte subsets in different clinical situations after renal transplantation

Phenotypic characterization of T and B lymphocytes allows the discrimination of functionally different subsets. Here, we questioned whether changes in peripheral lymphocyte subset distribution reflect specific clinical and histopathological entities after renal transplantation. Sixty-five renal transplant recipients with either histologically proven (sub)clinical acute rejection or chronic allograft dysfunction, or without abnormalities were studied for their peripheral lymphocyte subset composition and compared with 15 healthy control individuals. Naive, memory and effector CD8(+) T-cell counts were measured by staining for CD27, CD28 and CD45RO/RA. In addition, we studied the CD25(+) CD4(+) T-cell population for its composition regarding regulatory Foxp3(+) CD45RO(+) CD127(-) cells and activated CD45RO(+) CD127(+) cells. Naive, non-switched and switched memory B cells were defined by staining for IgD and CD27. We found a severe decrease in circulating effector-type CD8(+) T cells in recipients with chronic allograft dysfunction at 5 years after transplantation. Percentages of circulating CD25(+) CD127(low) CD4(+) regulatory T cells after transplantation were reduced, but we could not detect any change in the percentage of CD127(+) CD45RO(+) CD4(+) activated T cells in patients at any time or condition after renal transplantation. Regardless of clinical events, all renal transplant recipients showed decreased total B-cell counts and a more differentiated circulating B-cell pool than healthy individuals. The changes in lymphocyte subset distribution probably reflect the chronic antigenic stimulation that occurs in these transplant recipients. To determine the usefulness of lymphocyte subset-typing in clinical practice, large cohort studies are necessary.     

Programmed cell death in rheumatoid arthritis peripheral blood T-cell subpopulations determined by laser scanning cytometry

Because peripheral blood mononuclear cells play an important role in the perpetuation of the autoimmune process in rheumatoid arthritis (RA) and because the maintenance of these cells might be caused by the dysregulation of apoptosis, we investigated the apoptosis susceptibility of peripheral blood mononuclear cells from patients with RA. Freshly separated peripheral blood lymphocytes were stained for apoptosis markers (CD95, Bax, Bcl-2, TNF receptor) and for an activation marker (CD45-RO), and the apoptosis frequency of cells bearing these markers were assessed by the terminal-deoxynucleotidyl transferase-mediated dUTP digoxigenin nick end labeling method and nuclear condensation analysis with laser scanning cytometry. Also, the ability of CD4(+) and CD8(+) T-cell populations to undergo apoptosis was investigated with 24-hour culture in medium alone or with different apoptosis inducers (anti-CD3, anti-CD95, anti-TNF receptor). Laser scanning cytometry analysis was used to enumerate the phenotype and apoptosis ratios of both freshly isolated and cultured lymphocytes. Quantitative ELISA was performed to detect plasma levels of TNF-alpha and soluble Fas ligand. Furthermore, we studied the relationship between marked apoptotic defects in patients with RA and the severity of clinical disease. CD4(+) T-cell counts in patients with RA were elevated compared with controls. A decreased rate of anti-CD95-mediated apoptosis was found within the CD4(+) and CD8(+) lymphocytic subpopulations. In patients with RA, decreased Bax expression and decreased apoptosis rate within the Bax-positive cells were found, whereas Bcl-2 expression was elevated. The CD45-RO expression was higher, whereas the apoptosis within CD45-RO(+) cells were decreased in RA. Evaluation of plasma soluble Fas ligand revealed significantly decreased levels in patients compared with controls. The reduced susceptibility to CD95-mediated apoptosis may contribute to the expansion of an activated CD4(+) lymphocyte subpopulation and thus to the maintenance of peripheral autoreactive T-cell clones in RA. We also revealed a relationship between in vitro demonstrated lymphocyte apoptosis defects and clinical disease activity.     

Accelerated age-dependent transition of human regulatory T cells to effector memory phenotype

We and others recently described a method for isolating viable forkhead boxp3 (FoxP3(+)) T regulatory cells (Tregs) by means of the surface phenotype CD4(+)CD127(lo)CD25(+). In this study, we used the new strategy to measure Treg numbers, phenotype and function at different ages. Mean percentages of CD4(+)CD127(lo)CD25(+) Tregs increased only slightly throughout life, from 6.10% in cord blood to 7.22% in PBMC from adults between 20 and 25 years and 7.50% in PBMC from adults over the age of 60. In all age groups, a higher proportion of Tregs had acquired a CD45RA(-) memory phenotype compared with CD4(+)Foxp3(-) conventional T cells. This increase was entirely attributable to increased Tregs with an effector memory phenotype, whereas central memory phenotype cells were comparably represented within the Treg and conventional CD4(+) T-cell populations. Expression of CD95 also differed between Tregs and conventional CD4(+) T cells at all ages. However there was no difference in the suppressive capacity of the different naive and memory Treg subsets. These results suggest that, compared with their conventional CD4(+) T-cell counterparts, Tregs undergo preferential differentiation from a naive to an effector memory phenotype, driven by their specificity for self- rather than foreign antigen. However, number and function are remarkably stable throughout life.     

Characterization of Effector Memory CD8+ T Cells in the Synovial Fluid of Rheumatoid Arthritis

Little is known about the cellular characteristics of CD8(+) T cells in rheumatoid arthritis (RA). We addressed this by investigating whether the frequency of the CD8(+) T cell subsets and their phenotypic characteristics are altered in the peripheral blood and synovial fluid (SF) from patients with RA. In this study, CD8(+) T cells, mainly CD45RA(-) effector memory (EM) CD8(+) T cells, were increased significantly in the SF, but not in the peripheral blood from RA patients, compared with healthy controls. The synovial EM CD8(+) T cells were activated phenotypes with high levels of CD80, CD86, and PD-1, and had a proliferating signature in vivo upon Ki-67 staining, whereas the Fas-positive cells were prone to apoptosis. In addition, EM CD8(+) T cells in the SF were less cytotoxic, as they expressed less perforin and granzyme B. In particular, the proportions of synovial fluid mononuclear cells that were CCR4(+)CD8(+) T cells and IL-4-producing CD8(+) T cells (i.e., Tc2 cells) were significantly higher than those in peripheral blood mononuclear cells of patients with RA and healthy controls. In addition, the number of IL-10-producing CD8(+) suppressor T (Ts) cells increased significantly in the SF of RA patients. Especially, CD8(+) T cells were inversely correlated with disease activity. These findings strongly suggest that EM CD8(+) T cells in the SF are increased, likely because of inflammation, and they may be involved in modulating inflammation, thereby affecting the development and progression of RA.     

The role of CD4+CD25+ regulatory T cells in patients with Rheumatoid Arthritis]

CD4(+)CD25(+) regulatory T cells play an important role in preventing autoimmunity. We investigated the presence of CD4(+)CD25(+) regulatory T cells in the peripheral blood of patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and systemic sclerosis (SSc), using flow cytometry. The percentage of CD4(+)CD25(+) regulatory T cells was significantly decreased in RA, especially in patients with high serum levels of either CRP or MMP-3. In SSc and SLE, the percentage of CD4(+)CD25(+) regulatory T cells was higher in patients than in controls, but not significant. We also investigated the serum levels of IL-10, which influences the function of CD4(+)CD25(+) regulatory T cells and other regulatory T cells. In RA, on contrast to CD4(+)CD25(+) regulatory T cells, the serum levels of IL-10 increased in patients with higher serum levels of CRP, or MMP-3. In SLE and SSc, the serum level of IL-10 increased significantly in patients than in controls. These data thus indicated that CD4(+)CD25(+) regulatory T cells contributes to occurrence and progression of RA, and other regulatory T cells or cytokines contribute to occurrence and progression of SSc and SLE.     

Distribution of lymphocyte subsets in healthy human immunodeficiency virus-negative adult Ethiopians from two geographic locales.

Immunological values for 562 factory workers from Wonji, Ethiopia, a sugar estate 114 km southeast of the capital city, Addis Ababa, Ethiopia, were compared to values for 218 subjects from Akaki, Ethiopia, a suburb of Addis Ababa, for whom partial data were previously published. The following markers were measured: lymphocytes, T cells, B cells, NK cells, CD4(+) T cells, and CD8(+) T cells. A more in depth comparison was also made between Akaki and Wonji subjects. For this purpose, various differentiation and activation marker (CD45RA, CD27, HLA-DR, and CD38) expressions on CD4(+) and CD8(+) T cells were studied in 60 male, human immunodeficiency virus-negative subjects (30 from each site). Data were also compared with Dutch blood donor control values. The results confirmed that Ethiopians have significantly decreased CD4(+) T-cell counts and highly activated immune status, independent of the geographic locale studied. They also showed that male subjects from Akaki have significantly higher CD8(+) T-cell counts, resulting in a proportional increase in each of the CD8(+) T-cell compartments studied: na?ve (CD45RA(+)CD27(+)), memory (CD45RA(-)CD27(+)), cytotoxic effector (CD45RA(+)CD27(-)), memory/effector (CD45RA(-)CD27(-)), activated (HLA-DR(+)CD38(+)), and resting (HLA-DR(-)CD38(-)). No expansion of a specific functional subset was observed. Endemic infection or higher immune activation is thus not a likely cause of the higher CD8 counts in the Akaki subjects. The data confirm and extend earlier observations and suggest that, although most lymphocyte subsets are comparable between the two geographical locales, there are also differences. Thus, care should be taken in extrapolating immunological reference values from one population group to another.     

Cell surface CD28 levels define four CD4+ T cell subsets: abnormal expression in rheumatoid arthritis

CD28 is a costimulatory receptor expressed in most CD4(+) T cells. Despite the long-standing evidence for up- and downregulation of surface CD28 expression in vitro, and the key regulatory role assigned to the upregulation of CD28 counterreceptor [the CD152 (CTLA-4) molecule], in vivo CD28 induction has attracted little attention. We studied CD28 and CD152 expression and function in 33 rheumatoid arthritis (RA) patients, 20 clinically active and 13 inactive, and in 24 healthy donors. Four subsets of CD28(-), CD28(low), CD28(int), and CD28(high) peripheral blood human CD4(+) T cells were defined using three-color flow cytometry. The three CD28(+) subsets displayed a one-, two-, or threefold quantitative difference in their relative number of CD28 antibody binding sites, respectively (P < 0.01). RA patients, whether active or inactive, showed a distinct phenotype when compared to healthy donors: (i) the percentage of CD4(+)CD28(high) cells was increased twofold and the CD4(+)CD28(low) subset was reduced twofold (P < 0.01) and (ii) the CD4(+)CD28(high) cells from RA patients showed an in vivo activated phenotype, CD45RO(+)CD5(high)IL-2Ralpha(+) (P < 0.01). Active RA patients were different from inactive patients. They showed a twofold increase in mean CD28 expression (P < 0.05), whereas each of the CD28(+) subsets in the inactive RA patients showed reduced expression when compared to healthy donors. Notably, both active and inactive RA patients showed abnormal CD28 upregulation when T cells were activated in vitro with CD3 antibodies, but only inactive RA patients showed a hypoproliferative response to TCR/CD3 triggering when compared to healthy donors (P < 0.01). This defective proliferation was normalized by concurrent crosslinking with CD28 antibody. No differences were noted in the expression of CD152 or CD80, a CD28 and CD152 shared ligand. The disregulated in vivo expression of CD28 was related to the RA patients' disease activity and suggests that modulation of CD28 surface levels may be an additional mechanism to finely tune the delicate responsiveness/tolerance balance.     

Alteration in CD29(high) CD4(+) lymphocyte subset is a common feature of early HIV disease and of active tuberculosis

Peripheral CD4 T-cell depletion has been observed in human immunodeficiency virus (HIV)-negative patients with pulmonary tuberculosis (TB). To investigate more accurately this alteration, we studied peripheral blood CD45RA(+) and CD29(high) CD4 subsets in 79 TB patients with (HIV(+)TB(+)) or without (HIV(-)TB(+)) HIV infection, 85 HIV-infected patients without TB (HIV(+)TB(-)), and 43 healthy controls, all living in West Africa. The high proportion of CD4(+)CD29(high) T cells observed in controls was dramatically decreased in CDC-A stage HIV(+)TB(-) patients. CD45RA(+) CD4(+) T cells were depleted during the CDC-B stage. Both the percentage and the absolute count of CD29(high)CD4(+) T cells were decreased in HIV(-)TB(+) and HIV(+)TB(+) patients versus controls, but CD45RA(+)CD4(+) T cells were not decreased in TB patients without HIV-infection. Although distinct alterations in the CD4(+) T-cell homeostasis are involved in TB(-) versus HIV-infected subjects, our data suggest that the CD29(+)CD4(+) T-cell depletion observed during the early HIV disease contributes to the risk of active TB, by reducing the pool of T cells able to relocalize to the sites of the M. tuberculosis multiplication.     

Evidence for extrathymic T cell maturation after thymectomy in infancy

Our previous study showed that children who had been partially or completely thymectomized during heart surgery as infants had lower proportions and numbers of total lymphocytes and reduced proportions of T cells (CD3(+)), helper T cells (CD4(+)) and naive T cells (CD3(+) CD4(+) CD45RA(+)), but normal proportion of cytotoxic T cells (CD8(+)). In this study T lymphocytes from a selected group of eight of these children and age- and gender-matched controls were characterized further using flow cytometry to determine phenotypes of T cells and T cell subsets related to T cell regulation and phenotypes suggestive of extrathymic maturation. Immune function was assessed by measuring autoantibodies and antibodies against vaccines. The study group had significantly lower numbers of all the main subsets of T lymphocytes and the composition was different. Thus, the proportions of lymphocytes with the following phenotypes: CD3(+), CD2(+), CD7(+), CD4(+), CD62L(+), CD4(+) CD62L(+) and CD4(+) CD69(-) were significantly reduced in the study group compared with the control group, but significantly higher proportions were seen of lymphocytes expressing CD8alpha(+) CD8beta(-) and TCRgammadelta(+) CD8alpha(+) CD8beta(-). The absolute number and proportion of CD4(+) CD25(+) cells were reduced but the proportions of the subgroup of naive regulatory T cells (CD4(+) CD25(+) CD62L(+)) and non-activated regulatory T cells (CD4(+) CD25(+) CD69(-)) were not reduced in the thymectomized children. We conclude that the phenotypic characteristics of T lymphocytes of children who have lost their thymus in infancy are indicative of extrathymic maturation. T regulatory cells appear to be less affected than other subsets by the general reduction in T cell numbers.     

Characteristics of rat CD4(+)CD25(+) T cells and their ability to prevent not only diabetes but also insulitis in an adoptive transfer model in BB rats

Human and mouse CD4(+)CD25(+) T cells have been intensively studied through the last decade. However, little is known about this subset in other species. This study describes the phenotype of rat CD4(+)CD25(+) Foxp3(+) T cells and the site in which they exert regulation in a transfer-induced autoimmune diabetes model. Several proteins and mRNAs are up-regulated in unstimulated rat CD4(+)CD25(+) T cells compared with CD4(+)CD25(-) T cells, including Foxp3, Lag-3, CD80, interleukin 10 (IL-10) and CTLA-4. To investigate CD4(+)CD25(+) T cells in vivo, we transferred three million diabetogenic T cells either alone or in combination with two million CD4(+)CD25(+) T cells to 30-day-old BB rats. The pancreas and the pancreatic lymph nodes were examined as two potential regulatory sites. Time-course analysis of pancreatic histology following diabetogenic T-cell transfers revealed insulitis from about 14 days after transfer. By contrast, rats receiving both diabetogenic T cells and CD4(+)CD25(+) T cells had no insulitis at any time. Moreover, the frequency of diabetogenic T cells in the pancreatic lymph nodes 2 days after transfer was significantly reduced in rats receiving both subsets. These data indicate that the primary site of T-cell regulation is in the draining lymph nodes and not the pancreas in our model.     

Evaluation of a simplified dual-platform flow cytometric method for measurement of lymphocyte subsets and T-cell maturation phenotypes in the population of Nouna, Burkina Faso

In the context of a larger clinical study in Nouna, Burkina Faso, we evaluated a simplified dual-platform (DP) flow cytometric (FCM) method that allows the determination of major lymphocyte subsets in a single test tube. We compared the phenotyping of lymphocytes with DP FCM and simultaneous measurements with standard single-platform (SP) FCM for samples from 177 individuals. Analysis of the comparative measurements revealed that DP FCM systematically underestimates the proportion of NK cells, overestimates the percentage of CD3(+) CD8(+) lymphocytes, and yields proportions of B cells and CD4(+) T cells comparable with the results from SP FCM. Bland-Altman analysis showed a low bias between both methods and an acceptable precision for percent values of CD4(+) T cells (bias +/- precision, -1% +/- 6%) and CD8(+) T cells (-3% +/- 6%). The absolute cell numbers of all lymphocyte subpopulations, however, were systematically biased towards lower values being obtained by DP FCM. Reference values for the distribution of T-cell maturation phenotypes in 177 healthy adults were calculated using DP FCM. The mean +/- standard deviation (SD) CD4(+)-to-CD8(+) T-cell ratio was 1.61 +/- 0.61, the mean percentage +/- SD of CD4(+) T cells was 42% +/- 7%, and that of CD8(+) T cells 29% +/- 7%. Among CD4(+) lymphocytes, 28% +/- 7% were classified as central memory (CD45RA(low) CCR7(+)), 22% +/- 10% as na?ve (CD45RA(high) CCR7(+)), 45% +/- 12% as effector memory (CD45RA(low) CCR7(-)); and 5% +/- 3% as terminally differentiated effector memory expressing CD45RA (CD45RA(high) CCR7(-)). Among CD8(bright) lymphocytes, 3% +/- 2% had a central memory phenotype, 27% +/- 13% were na?ve, 37% +/- 13% had an effector memory phenotype, and 34% +/- 12% were terminally differentiated effector memory cells expressing CD45RA.     

Phenotypic and functional characteristics of CD4+ CD39+ FOXP3+ and CD4+ CD39+ FOXP3neg T-cell subsets in cancer patients

Human CD4(+) CD39(+) regulatory T (Treg) cells hydrolyze exogenous adenosine triphosphate (ATP) and participate in immunosuppressive adenosine production. They contain two T-cell subsets whose role in mediating suppression is not understood. Frequencies of both CD4(+) CD39(+) subsets were evaluated in peripheral blood lymphocytes of 57 cancer patients and in tumor infiltrating lymphocytes (TILs) of 6 patients. CD4(+) CD39(+) and CD4(+) CD39(neg) T cells isolated using immunobeads and cell sorting were cultured under various conditions. Their conversion into CD39(+) FOXP3(+) CD25(+) or CD39(+) FOX(neg) CD25(neg) cells was monitored by multiparameter flow cytometry. Hydrolysis of exogenous ATP was measured in luminescence assays. Two CD4(+) CD39(+) cell subsets differing in expression of CD25, FOXP3, CTLA-4, CD121a, PD-1, latency associated peptide (LAP), glycoprotein A repetitions predominant (GARP), and the cytokine profile accumulated with equal frequencies in the blood and tumor tissues of cancer patients. The frequency of both subsets was significantly increased in cancer. CD39 expression levels correlated with the subsets' ability to hydrolyze ATP. Conventional CD4(+) CD39(neg) T cells incubated with IL-2 + TGF-β expanded to generate CD4(+) CD39(+) FOXP3(+) Treg cells, while CD4(+) CD39(+) FOXP3(neg) CD25(neg) subset cells stimulated via the TCR and IL-2 converted to FOXP3(+) CTLA4(+) CD25(+) TGF-β-expressing Treg cells. Among CD4(+) CD39(+) Treg cells, the CD4(+) CD39(+) FOXP3(neg) CD25(neg) subset serves as a reservoir of cells able to convert to Treg cells upon activation by environmental signals.     

Aberrant T-cell antigen receptor-mediated responses in autoimmune lymphoproliferative syndrome

Autoimmune Lymphoproliferative Syndrome (ALPS) is a disorder of defective lymphocyte apoptosis due to mutations of the Fas receptor and other molecules in the Fas signaling pathway. In addition to accumulation of CD4(-) CD8(-) double-negative (DN) T cells, many patients display a dysregulated cytokine pattern with dysfunctional T cells, suggesting Fas defects may impact pathways of T-cell activation/differentiation. Here, we report two novel mutations in the Fas receptor resulting in an ALPS phenotype. Utilizing flow cytometry, we found anti-CD3 activated CD4(+) T cells from these patients were incapable of fully upregulating activation markers (CD25, CD69, and CD40L) or producing interferon-gamma and IL-2. Additionally, DN T cells were unable to transduce proximal T-cell antigen receptor signals or produce cytokines. Furthermore, DN T cells overexpressed CD57 and phenotypically resembled end-stage effector cells. As DN T cells were essentially anergic, the clinical manifestations of autoimmunity are more likely to be a consequence of aberrant cytokine secretion within the CD4(+) T-cell subpopulation.     

Increase in Gamma Interferon-Secreting CD8+, as Well as CD4+, T Cells in Lungs following Aerosol Infection with Mycobacterium tuberculosis

Although it is well established that CD4(+) T cells are required for the protective immune response against tuberculosis (TB), there is some evidence that CD8(+) T cells are also involved in the host response to Mycobacterium tuberculosis. There is, however, a paucity of information on the pulmonary CD8(+) T-cell response during infection. We therefore have compared the changes in both CD8(+) and CD4(+) T cells following aerosol infection with M. tuberculosis. There was an observed delay between the peak of infection and the activated T-cell response in the lung. The kinetics of CD8(+) and CD4(+) T-cell responses in the lung were identical, both peaking at week 8, 4 weeks later than the peak of cellular response in draining lymph nodes. Similar changes in activation/memory phenotypes occurred on the pulmonary CD8(+) and CD4(+) T cells. Following in vitro restimulation, both subsets synthesized gamma interferon, a cytokine essential for controlling M. tuberculosis infection. Since lung CD8(+) T cells are actively expanded during aerosol M. tuberculosis infection, it is important that both CD8(+) and CD4(+) T cells be targeted in the design of future TB vaccines.     

Phenotypic classification of human CD4+ T cell subsets and their differentiation

CD4(+) T cells have T(h) cell function and include two major functional subsets, T(h)1 and T(h)2. However, there are a restricted number of studies concerning phenotypic classification of human CD4(+) T cells. Here by using seven- and eight-color flow cytometric analysis, we investigated the function of the subsets classified by four markers, CD27, CD28, CD45RA and CCR7. Five major subsets were identified by using these markers. These subsets showed different patterns of cytokine production after they were stimulated with phorbol myristate acetate and ionomycin. The analyses of cytokine production suggested that CCR7(+)CD45RA(+)CD27(+)CD28(+), CCR7(+)CD45RA(-)CD27(+)CD28(+) and CCR7(-)CD45RA(-)CD27(+)CD28(+) subsets were naive, central memory and effector memory T cells, respectively, whereas CCR7(-)CD45RA(-)CD27(-)CD28(+) and CCR7(-)CD45RA(-)CD27(-)CD28(-) subsets included T(h)1 and T(h)2 cells. The analysis of cytokine production by these subsets stimulated with anti-CD3 and anti-CD28 mAbs or with human cytomegalovirus antigens showed that IFN-gamma production was significantly higher in the CCR7(-)CD45RA(-)CD27(-)CD28(-) subset than in other subsets and that both CCR7(-)CD45RA(-)CD27(-)CD28(+) and CCR7(-)CD45RA(-)CD27(-)CD28(-) subsets produced a higher level of IL-4 than did other subsets. Our analyses demonstrated that the CCR7(-)CD45RA(-)CD27(-)CD28(-) subset predominantly included T(h)1 effector cells and that CCR7(-)CD45RA(-)CD27(-)CD28(+) subsets included T(h)1 and T(h)2 effector memory/effector cells as well as unclassified cells. The analysis of classification by using these four markers also suggested the differentiation pathway of human CD4(+) T cells.     

Age-related changes in the occurrence and characteristics of thymic CD4(+) CD25(+) T cells in mice

Natural regulatory CD4(+) CD25(+) T cells play an important role in preventing autoimmunity by maintaining self-tolerance. They express CD25 constitutively and are produced in the thymus as a functionally mature T-cell population. Changes in the potential of these cells to regulate the activity of conventional effector lymphocytes may contribute to an increased susceptibility to infection, cancer and age-associated autoimmune diseases. In this study we demonstrated that the thymi of aged mice are populated by a higher percentage of CD4(+) CD25(+) thymocytes than in young animals. The expression of several surface markers (CD69, CD5, CD28, CTLA-4, CD122, FOXP3), usually used to characterize the phenotype of CD4(+) CD25(+) T regulatory cells, was compared between young and aged mice. We also examined the ability of sorted thymus-deriving regulatory T cells of young and aged BALB/c mice to inhibit the proliferation of lymph node lymphocytes activated in vitro. Natural regulatory T cells isolated from the thymi of young mice suppress the proliferation of responder lymph node cells. We demonstrated that thymus-deriving CD4(+) CD25(+) T cells of old mice maintain their potential to suppress the proliferation of activated responder lymphocytes of young mice. However, their potential to inhibit the proliferation of old responder T cells is abrogated. Differences in the occurrence and activity of CD4(+) CD25(+) thymocytes between young and old animals are discussed in relation to the expression of these surface markers.