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.     

Down-regulation of CXCR4 expression on human CD8+ T cells during peripheral differentiation

Multi-color flow cytometric analysis on human CD8(+) T cell subsets revealed that CXCR4 is predominantly expressed on CD8(+) T cells with the naive CD27(+)CD28(+)CD45RA(+) phenotype, and is down-regulated during differentiation into those with an effector phenotype. The down-regulation of CXCR4 expression during peripheral differentiation was supported by the fact that the expression of CXCR4 on CD8(+) T cells was negatively correlated with that of perforin. The analysis of CCR5, CCR7, and CXCR4 co-expression further showed that CD8(+) T cells expressing a high level of CXCR4 are CCR7(+)CCR5(-) naive or central memory subsets, and those expressing a low level of CXCR4 were included in the CCR7(-)CCR5(+/-) memory/effector and effector subsets. Epstein Barr virus-specific CD8(+) T cells, which mostly express the memory phenotype, expressed CXCR4, while human cytomegalovirus-specific CD8(+) T cells, which mostly express the effector phenotype, partially expressed this receptor, showing that the expression of CXCR4 is also down-regulated during differentiation of viral antigen-specific CD8(+) T cells. The classification of human CD8(+) T cells based on the expression of these chemokine receptors should prove useful for studies that clarify the differentiation of human CD8(+) T cells.     

CD45RA expression on HCMV-specific effector memory CD8+ T cells is associated with the duration and intensity of HCMV replication after transplantation

In this cross-sectional study on 42 solid organ transplant recipients, the association of kinetics of human cytomegalovirus (HCMV) replication and EMRA HCMV-specific CD8+ T cells was investigated. Correlation was observed between the duration of HCMV replication after transplantation and CD45RA+CD27− (r = 0.609; p = 0.004), CD45RA+ CD28− (r = 0.579; p = 0.008) or CD45RA+CCR7− (r = 0.488; p = 0.029) HCMV-specific CD8+ T cells percentages. In the multivariate regression analyses, CD45RA+CD27−, CD45RA+CD28− or CD45RA+CCR7− HCMV-specific CD8+ T cells percentages increased 5.58% (p = 0.001), 5.35% (p = 0.001) or 4.49% (p = 0.012), respectively, with every 10-day increase in the duration of HCMV replication. Moreover, CD45RA+CD27− or CD45RA+CD28− frequencies increased 4.16% (p = 0.024) or 3.58% (p = 0.049), respectively, with every unity increase in log₁₀ genomes/mL. These observations support the major association between the frequency of EMRA HCMV-specific CD8+ T cells and the duration of post-transplant HCMV replication episodes in solid organ transplantation recipients.     

Evidence that human CD8+CD45RA+CD27- cells are induced by antigen and evolve through extensive rounds of division.

We recently showed that circulating human CD8(+) effector cells have a CD45RA+CD27(-) membrane phenotype. In itself this phenotype appeared to pose a paradox: CD45RA, a marker expressed by unprimed cells, combined with absence of CD27, characteristic for chronically stimulated T cells. To investigate whether differentiation towards the CD45RA+CD27(-) phenotype is dependent on antigenic stimulation and involves cellular division, TCR Vbeta usage and telomeric restriction fragment (TRF) length were analyzed within distinct peripheral blood CD8(+) subsets. FACS analysis showed that the TCR Vbeta repertoire of CD8(+)CD45RA+CD27(-) cells differed significantly from that of unprimed CD8(+)CD45RA+CD27(+) cells. Moreover, in two out of six individuals large expansions of particular Vbeta families were observed in the CD8(+)CD45RA+CD27(-) subset. CDR3 spectrotyping and single-strand confirmation analysis revealed that within the CD8(+)CD45RA+CD27(-) population most of the 22 tested Vbeta families were dominated by oligoclonal expansions. The mean TRF length was found to be 2.3+/-1.0 kb shorter in the CD8(+)CD45RA+CD27(-) subset compared with the unprimed CD8(+)CD45RA+CD27(+) population, but did not differ substantially from that of memory type, CD8(+)CD45RA-CD27(+) T cells. These findings indicate that the CD8(+)CD45RA+CD27(-) cytotoxic effector population consists of antigen-induced, clonally expanded cells and confirm that the expression of CD45RA is not a strict marker of antigen non-experienced T cells.     

Functional and phenotypic analysis of human memory CD8+ T cells expressing CXCR3

Several chemokine receptors play an important role in the migration of na?ve, memory, and effector T cells. Flow cytometric analyses showed that human CD8+ T cells with na?ve (CD27+ CD28+ CD45RA+) or memory (CD27+ CD28+/- CD45RA+) phenotypes included a population expressing a high level of CXC chemokine receptor 3 (CXCR3high) and one expressing a low level of it (CXCR3low), but those with the effector phenotype (CD27- CD28- CD45RA+/-) included a population that did not express CXCR3 (CXCR3-) and a CXCR3low population. This relation between the expression level of CXCR3 and memory/effector phenotypes also applied to Epstein-Barr virus- or human cytomegalovirus-specific CD8+ T cells. CXCR3high cells were found predominantly in CC chemokine receptor 7 (CCR7)+ CCR5- and CCR7- CCR5- subsets of CD8+ T cells with the CD27+ CD28+ CD45RA- memory phenotype, suggesting that they are memory cells with intermediate differentiation. Indeed, CXCR3high CD27+ CD28+ CD45RA- CD8+ T cells had the ability to produce interleukin-2 and interferon-gamma. These results together indicate that the expression of CXCR3 is up-regulated on intermediately differentiated memory CD8+ T cells. CXCR3high CD8+ T cells had a greater ability to migrate in response to CXCR3 ligands than CXCR3low ones. As CXCR3high memory CD8+ T cells do not express CCR5, high expression of CXCR3 on these memory CD8+ T cells might play an important role in the migration of these cells to inflammatory sites and in their differentiation.     

Accumulation of memory T cells from childhood to old age: central and effector memory cells in CD4(+) versus effector memory and terminally differentiated memory cells in CD8(+) compartment

Memory T cells can be classified as central memory (T(CM), CD45RA(neg)CCR7(+)), effector memory (T(EM), CD45RA(neg)CCR7(neg)), and terminally differentiated cells (T(TD), CD45RA(+)CCR7(neg)) with different homing and effector capacities. In 101 healthy subjects aged from 5 to 96 years, distinct dynamics were evidenced between circulating CD4(+) and CD8(+) T cell populations. Naive CD4(+) and CD8(+) T cells decreased linearly with age, CD8(+) twice more rapidly. Memory cells outnumbered naive cells on average at 37.4 in the CD4(+) and 29.5 years of age in the CD8(+) pool. CD4(+) T(CM) and T(EM) cells were positively correlated and increased linearly at a similar rate with age, while CD4(+) T(TD) remained rare. CD8(+) T(EM) and T(TD) accumulated linearly with age, while T(CM) increased only slightly, and each memory subset was negatively correlated to the two others. Almost all CD8(+) T(TD) and some CD8(+) T(EM) had lost CD28 expression. Despite different dynamics, each individual CD4(+) naive and memory subset was correlated to the synonymous CD8(+) subset. Half of the subjects aged 65 years or older were characterized by extremely reduced CD8(+) naive and increased CD8(+) T(TD) cell counts, which could indicate an acceleration of the decay of the immune system from this age onward.     

CD8 T cell effector maturation in HIV-1-infected children

HIV-1 infection generates maturational responses in overall CD4 and CD8 T cell populations in adults, with elevated expression of lytic effector molecules perforin and granzyme B, and reduced expression of CCR7 and CD45RA. Here, we have found that these marked effects were significantly less pronounced in children, both in terms of the skewed CCR7/CD45RA expression profile as well as the increased perforin expression. Similar to adults, HIV-specific CD8 cells in children were largely CD27+ CD45RA- and lacked perforin. However, one pediatric subject with late-stage infection displayed robust expansion of Gag 77-85-specific CD8 T cells which were perforin+ and lytic, but lacked expression of CD27 and IFNgamma. Our data indicate that the T cell effector maturation induced by HIV-1 infection is markedly weaker in children as compared to adults. The data also suggest, however, that the perforin-deficient state of HIV-specific CD8 T cells in children may be reversible.     

Expression and function of NKG2D in CD4+ T cells specific for human cytomegalovirus

The human NKG2D killer lectin-like receptor (KLR) is coupled by the DAP10 adapter to phosphoinositide 3-kinase (PI3 K) and specifically interacts with different stress-inducible molecules (i.e. MICA, MICB, ULBP) displayed by some tumour and virus-infected cells. This KLR is commonly expressed by human NK cells as well as TCRgammadelta(+) and TCRalphabeta(+)CD8(+) T lymphocytes, but it has been also detected in CD4(+) T cells from rheumatoid arthritis and cancer patients. In the present study, we analysed NKG2D expression in human cytomegalovirus (HCMV)-specific CD4(+) T lymphocytes. In vitro stimulation of peripheral blood mononuclear cells (PBMC) from healthy seropositive individuals with HCMV promoted variable expansion of CD4(+)NKG2D(+) T lymphocytes that coexpressed perforin. NKG2D was detected in CD28(-) and CD28(dull )subsets and was not systematically associated with the expression of other NK cell receptors (i.e. KIR, CD94/NKG2 and ILT2). Engagement of NKG2D with specific mAb synergized with TCR-dependent activation of CD4(+) T cells, triggering proliferation and cytokine production (i.e. IFN-gamma and TNF-alpha). Altogether, the data support the notion that NKG2D functions as a prototypic costimulatory receptor in a subset of HCMV-specific CD4(+) T lymphocytes and thus may have a role in the response against infected HLA class II(+) cells displaying NKG2D ligands.     

Ex-Vivo Analysis of CD8+ T Cells Infiltrating Colorectal Tumors Identifies a Major Effector-Memory Subset with Low Perforin Content

Previous studies have indicated that the infiltration of CD8+ T cells in colorectal cancer is an independent predictor of increased survival but clinical observations have suggested that the cytotoxic function of CD8+ T cells infiltrating colorectal cancer may often be limited. In this study, we have assessed the phenotype of colorectal cancer CD8+ tumor-infiltrating lymphocytes (TILs) isolated ex vivo from tumor tissue, and assessed the perforin content of TIL with respect to their location using immunohistochemistry. We found that CD8+ T cells TILs isolated from colorectal cancer are mainly composed of antigen-experienced cells of effector memory type (TEM, CD45RA-CCR7−, and CD27+/CD28− or CD27−/CD28−), and contain only minor proportions of terminally differentiated CD8+ T cells (TEMRA, CD45RA+CCR7−). The perforin content of these TILs, however, is significantly lower than that of antigen-experienced T cells in PBMCs due to the much lower levels of perforin found in the CD27-CD28− subset in TILs compared with CD8+ T cells of similar phenotype in PBMCs.     

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.     

Age-Dependent Association between Low Frequency of CD27/CD28 Expression on pp65 CD8+ T Cells and Cytomegalovirus Replication after Transplantation

In this cross-sectional study of 42 solid organ transplant recipients, the association of human cytomegalovirus (HCMV) replication and age with the phenotype of the HCMV-specific CD8⁺ T cells was analyzed by using the CMV pp65 HLA-A*0201 pentamer. A correlation between the proportion of CD28⁻ HCMV-specific CD8⁺ T cells and age was observed in patients without HCMV replication (r = 0.50; P = 0.02) but not in patients with HCMV replication (r = −0.05; P = 0.83), a finding which differs from that observed for total CD8⁺ T cells. Within the group of patients younger than 50 years of age, patients with HCVM replication after transplantation had higher percentages of CD28⁻ HCMV-specific CD8⁺ T cells (85.6 compared with 58.7% for patients without HCMV replication; P = 0.004) and CD27⁻ HCMV-specific CD8⁺ T cells (90.7 compared with 68.8% for patients without HCMV replication; P = 0.03). However, in patients older than age 50 years, a high frequency of these two subpopulations was observed in patients both with and without previous HCMV replication (for CD28⁻ HCMV-specific CD8⁺ T cells, 84.4 and 80.9%, respectively [P = 0.39]; for CD27⁻ HCMV-specific CD8⁺ T cells 86.6 and 81.5%, respectively [P = 0.16]). In conclusion, the present study shows that in the group of recipients younger than age 50 years, HCMV replication after transplantation is associated with a high percentage of CD27⁻ and CD28⁻ HCMV-specific CD8⁺ T cells. These results suggest that the increased percentage of CD27⁻ or CD28⁻ HCMV-specific subsets can be considered a biomarker of HCMV replication in solid organ transplant recipients younger than age 50 years but not in older patients. Further studies are necessary to define the significance of these changes in HCMV-associated clinical complications posttransplantation.Although a robust innate immune response is quickly induced just after the entry of human cytomegalovirus (HCMV) (21), the adaptive response of CD8⁺ T cells plays a major role in the control of HCMV infection (9, 19, 36). The development of HLA multimer technology and the analysis of surface markers (CD45RA, CCR7, CD27, CD28) have allowed the study of the CMV-specific CD8⁺ T-cell immune response and its phenotypic characteristics. Naïve cells (CD45RA⁺ CCR7⁺), which lose the ability to express CD45RA after interaction with the antigen, become memory cells and comprise two subpopulations: central memory cells (CD45RA⁻ CCR7⁺) and effector memory cells (CD45RA⁻ CCR7⁻). Subsequently, a fraction of the HCMV-specific effector memory cells may revert to RA expression (CD45RA⁺ CCR7⁻) after the acute phase of infection (32, 34). CD28 and CD27 costimulatory molecules, however, are also useful markers in determining the phenotype of CD8⁺ T cells and allow their classification into early memory cells (CD27⁺ CD28⁺), intermediate memory cells (CD27⁻ CD28⁺), and late memory cells (CD27⁻ CD28⁻), which progressively increase their levels of perforin production (3, 32).Latent HCMV infection is associated with the massive clonal expansion of virus-specific memory CD8⁺ T cells (47), which is especially pronounced in HCMV-seropositive elderly patients. In these patients, the population of HCMV-specific CD8⁺ T cells shows a highly differentiated phenotype with a high percentage of CD28⁻ lymphocytes and a series of functional changes associated with a process of immune senescence. It has been suggested that HCMV may contribute significantly to this senescence process (29, 33, 45, 50). In fact, the combination of HCMV seropositivity with a high percentage of CD28⁻ cells and inversion of the CD4⁺/CD8⁺ ratio define the so-called immune risk phenotype, which some longitudinal studies have associated with decreased survival in individuals older than 80 years of age (1, 23, 24, 29, 53).Because of immunosuppression, there is a higher frequency of CMV reactivation in CMV-seropositive transplant recipients and of primary infection in CMV-seronegative recipients receiving a CMV-seropositive organ (donor positive and recipient negative [D⁺/R⁻] patients) (13-15). HCMV replication may induce the accumulation of HCMV-specific CD8⁺ T cells observed in some studies, which may account for as much as 10 to 50% of the total CD8⁺ T-cell pool (26, 27). Furthermore, other studies show that these cell clones may have a highly differentiated phenotype (5, 6, 18) and may show functional changes similar to those observed in HCMV-seropositive elderly individuals (10, 17, 26). These observations led us to propose the hypothesis that transplant patients with HCMV replication after transplantation may have a population of HCMV-specific CD8⁺ T cells with a more differentiated phenotype and a higher percentage of CD28⁻ and/or CD27⁻ cells than transplant patients without replication. In order to test this hypothesis, a cross-sectional study was carried out with patients who had received a solid organ transplant at least 1 year earlier to determine the frequency and phenotype of the HCVM-specific CD8⁺ T-cell population and its relationship to age and HCMV replication posttransplantation.     

Distribution of human CMV-specific memory T cells among the CD8pos. subsets defined by CD57, CD27, and CD45 isoforms.

Chronic antigenic stimulation has been associated with peripheral blood expansions of CD8pos. T cells characterized by CD57 expression, loss of CD27 expression, and reversal of the CD45RO(bright) /RA(dim) phenotype usually associated with immunological memory towards a CD45RO(dim) /RA(bright) phenotype. However, the relationship and functional significance of these subset(s) has remained controversial. Here, this issue was addressed using a novel flow cytometric technique that allows simultaneous detection of human cytomegalovirus (HCMV)-specific CD8pos. memory T cells by rapid (< 6 h) HCMV peptide-specific induction of cytokine synthesis, and their phenotypic characterization, including CD57, CD27 and CD45RA/RO. The vast majority of resting CD8(pos.) T cells capable of rapid induction of IFN-gamma and TNF-alpha synthesis in response to HCMV peptides were found in a subset characterized by intermediate to high expression of CD57, down-regulation/loss of CD27, and varying degrees of reversal of the classical "memory" CD45RO(bright) /RA(dim) phenotype. This subpopulation likely includes the fully differentiated memory cells responsible for the long-term immune defense against HCMV reactivation.     

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 heterogeneity of antigen-specific CD4 T cells under different conditions of antigen persistence and antigen load

The factors responsible for the phenotypic heterogeneity of memory CD4 T cells are unclear. In the present study, we have identified a third population of memory CD4 T cells characterized as CD45RA(+)CCR7(-) that, based on its replication history and the homeostatic proliferative capacity, was at an advanced stage of differentiation. Three different phenotypic patterns of memory CD4 T cell responses were delineated under different conditions of antigen (Ag) persistence and load using CD45RA and CCR7 as markers of memory T cells. Mono-phenotypic CD45RA(-)CCR7(+) or CD45RA(-)CCR7(-) CD4 T cell responses were associated with conditions of Ag clearance (tetanus toxoid-specific CD4 T cell response) or Ag persistence and high load (chronic HIV-1 and primary CMV infections), respectively. Multi-phenotypic CD45RA(-)CCR7(+), CD45RA(-)CCR7(-) and CD45RA(+)CCR7(-) CD4 T cell responses were associated with protracted Ag exposure and low load (chronic CMV, EBV and HSV infections and HIV-1 infection in long-term nonprogressors). The mono-phenotypic CD45RA(-)CCR7(+) response was typical of central memory (T(CM)) IL-2-secreting CD4 T cells, the mono-phenotypic CD45RA(-)CCR7(-) response of effector memory (T(EM)) IFN-gamma-secreting CD4 T cells and the multi-phenotypic response of both IL-2- and IFN-gamma-secreting cells. The present results indicate that the heterogeneity of different Ag-specific CD4 T cell responses is regulated by Ag exposure and Ag load.     

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.     

Trypanosoma cruzi modulates the profile of memory CD8+ T cells in chronic Chagas' disease patients

We present a cross-sectional analysis of the maturation and migratory properties of the memory CD8(+) T cell compartment, in relation to the severity of heart disease in individuals with chronic Trypanosoma cruzi infection removed from endemic areas for longer than 20 years. Subjects with none or mild heart involvement were more likely to mount T. cruzi-specific memory IFN-gamma responses than subjects with more advanced cardiac disease, and the T. cruzi-specific CD8(+) T cell population was enriched in early-differentiated (CD27(+)CD28(+)) cells in responding individuals. In contrast, the frequency of CD27(+)CD28(+)CD8(+) T cells in the total memory CD8(+) T cell population decreases, as disease becomes more severe, while the proportion of fully differentiated memory (CD27(-)CD28(-)) CD8(+) T cells increases. The analysis of CCR7 expression revealed a significant increase in total effector/memory CD8(+) T cells (CD45RA(-)CCR7(-)) in subjects with mild heart disease as compared with uninfected controls. Altogether, these results are consistent with the hypothesis of a gradual clonal exhaustion in the CD8(+) T cell population, perhaps as a result of continuous antigenic stimulation by persistent parasites.     

CD4(+)CD8(dim) T lymphocytes exhibit enhanced cytokine expression, proliferation and cytotoxic activity in response to HCMV and HIV-1 antigens

CD4(+)CD8(dim) T cells represent a minor subset of the total CD3(+) T cell population in peripheral blood. Although transient and persistent expansions of these cells have been reported in both healthy and diseased individuals, the functional properties of the CD4(+)CD8(dim) population are largely unknown. In this study, we examined antigen-specific cytokine and proliferative responses of the CD4(+)CD8(dim) subset. In whole blood cultures stimulated with the viral antigens HCMV and HIV-1, a significant fraction of the CD4(+)CD8(dim) subset exhibited cytokine expression and proliferation in response to antigen activation. Typically, the CD4(+)CD8(dim) population contained two- to eightfold higher frequencies of antigen-specific cytokine producing cells than the CD4(+)CD8(-) population. Phenotypic analysis of the cytokine expressing CD4(+)CD8(dim) population indicated that these cells are memory T cells, with a high frequency of this population expressing the cytotoxic markers CD56 and perforin. Furthermore, the CD4(+)CD8(dim) cytokine responses to CMV were shown to be MHC class II dependent. Significantly, purified CD4(+)CD8(dim) T cells were found to possess higher CMV-specific cytotoxic activity than purified CD4(+)CD8(-) T cells in a standard (51)Cr-release CTL assay. Thus, CD4(+)CD8(dim) T cells appear to be MHC class II dependent, are capable of cytolytic effector activity, and are highly enriched within the CD4(+) cell populations specific for HCMV and HIV-1.     

A subset of functional effector-memory CD8+ T lymphocytes in human immunodeficiency virus-infected patients

CD8(+) T cells provide protective immune responses via both cytolytic and non-cytolytic mechanisms in subjects infected with human immunodeficiency virus (HIV). In the present study, we investigated the CD28 expression of CD8(+) T cells present in the peripheral blood lymphocyte subset isolated from chronically HIV-infected subjects. Using flow cytometric analysis, a continuous spectrum of CD28 intensity ranging from negative to high, which could be separated into CD28-negative, intermediate (int) and high, was seen for CD8(+) T cells. Our study focused mostly on the CD28(int) CD8(+) T cells. The CD28(int) CD8(+) T cells are CD57(-) CD27(+) CD45RO(+) CD45RA(-) CCR7(low) CD62L(int). The proliferative capacity of CD28(int) CD8(+) T cells was intermediate between those of CD28(-) CD8(+) T cells and CD28(high) CD8(+) T cells. The CD28(int) CD8(+) T cells are specific for HIV, cytomegalovirus (CMV) and Epstein-Barr virus (EBV) antigens as measured by human leucocyte antigen pentamer binding and produce both intracellular interferon-gamma and tumour necrosis factor-alpha in response to their cognate viral peptides. The CD28(int) CD8(+) T cells have HIV-specific, CMV-specific and EBV-specific cytotoxic activity in response to their cognate viral peptides. These findings indicate that a subset of functional effector-memory CD8(+) T cells specific for HIV, CMV and EBV antigens may contribute to an efficient immune response in HIV-infected subjects.     

Loss of CD28 expression on CD8(+) T cells is induced by IL-2 receptor gamma chain signalling cytokines and type I IFN, and increases susceptibility to activation-induced apoptosis

CD8(+)CD28(-) T cells are selectively expanded during viral infections, indicating their importance in anti-viral immune responses. Since little is known about the differentiation of CD8(+)CD28(-) cells, we investigated the generation, function and survival characteristics of this subset. In healthy individuals CD8(+)CD28(-) T cells contained more elevated levels of perforin and IFN-gamma than the CD8(+)CD28(+) subset, indicating that they can have an effector function. CD8(+)CD28(-) cells were selectively expanded when activated CD8(+)CD28(+) T cells were cultured in IL-2, IL-7 or IL-15. Moreover, the generation of CD8(+)CD28(-) cells was accelerated by type I IFN suggesting that these cytokines which are released during viral infections influence CD8(+) T cell differentiation. We did not observe re-expression of CD28 by CD8(+)CD28(-) T cells in any of the experiments performed. Activated T cells are susceptible to activation-induced cell death (AICD) if re-stimulated in the absence of co-stimuli. AICD was induced in both CD28(+) and CD28(-) subsets of activated T cells when stimulated with anti-CD3 antibody in the absence of co-stimuli but the magnitude of death was greater in the CD28(-) subset. While co-stimulation through LFA-1 (CD11a and CD18) significantly reduced AICD in the CD8(+)CD28(+) subset, death was not prevented in CD8(+)CD28(-) cells. These results suggest that CD8(+)CD28(-) T cells are more functionally differentiated than the CD8(+)CD28(+) subset and indicate they may represent a terminally differentiated effector population which is destined for clearance by apoptosis at the end of the immune response.