Analysis of bulk and virus-specific CD8+ T cells reveals advanced differentiation of CD8+ T cells in patients with common variable immunodeficiency

Common variable immunodeficiency (CVID) is a heterogeneous antibody deficiency syndrome with alterations in T cell regulation and function in a subgroup of patients. We assessed phenotype and function of bulk and virus-specific CD8+ T cells of a cohort of 34 HLA-A2+ CVID patients by pentamer technology and flow cytometry in relationship to their immunological and clinical phenotypes. Bulk CD8+ T cells displayed a shift toward a more antigen experienced and activated differentiation state. The advanced differentiation pattern was mainly found in a subgroup of CVID patients with lymphadenopathy and granulomatous disease. This effect existed independently of the patients' CMV status even so CMV-associated immunosenescence was more evident in CVID patients than in CMV-positive immunocompetent controls. As the phenotype and function of virus-specific CD8+ T cells were normal in CVID the induction of antiviral immunity by prophylactic immunization appears to be a logical and desirable aim for this group of patients.     

CD4+ T cell help improves CD8+ T cell memory by retained CD27 expression

CD4+ T cell help during the priming of CD8+ T lymphocytes imprints the capacity for optimal secondary expansion upon re-encounter with antigen. Helped memory CD8+ T cells rapidly expand in response to a secondary antigen exposure, even in the absence of T cell help and, are most efficient in protection against a re-infection. In contrast, helpless memory CTL can mediate effector function, but secondary expansion is reduced. How CD4+ T cells instruct CD8+ memory T cells during priming to undergo efficient secondary expansion has not been resolved in detail. Here, we show that memory CTL after infection with lymphocytic choriomeningitis virus are CD27(high) whereas memory CTL primed in the absence of CD4+ T cell have a reduced expression of CD27. Helpless memory CTL produced low amounts of IL-2 and did not efficiently expand after restimulation with peptide in vitro. Blocking experiments with monoclonal antibodies and the use of CD27(-/-) memory CTL revealed that CD27 ligation during restimulation increased autocrine IL-2 production and secondary expansion. Therefore, regulating CD27 expression on memory CTL is a novel mechanism how CD4+ T cells control CTL memory.     

Functional and Vbeta repertoire characterization of human CD8+ T-cell subsets with natural killer cell markers,CD56+ CD57- T cells,CD56+ CD57+ T cells and CD56- CD57+ T cells

We investigated the individual CD8+ populations with natural killer (NK) cell markers (NK-type T cell); CD56 single positive (CD56)-T cells, CD56/CD57 double positive (DP)-T cells and CD57 single positive (CD57)-T cells in the peripheral blood. All NK-type T-cell populations expressed CD122 and intermediate levels of T-cell receptor (TCR; regular CD8+ T cells are CD122- and express high levels of TCR). The number of both DP-T cells and CD57-T cells, but not CD56-T cells, gradually increased with age. All NK-type T-cell populations produced larger amounts of interferon-gamma than did regular CD8+ T cells after stimulation with interleukin (IL)-2, IL-12 and IL-15. However, CD56-T cells and CD57-T cells but not DP-T cells showed a potent antitumour cytotoxity to NK-sensitive K562 cells, whereas only CD56-T cells showed a potent cytotoxity to NK-resistant Raji cells. Furthermore, although NK-type T cells produced large amounts of soluble Fas-ligands, their cytotoxic activities appeared to be mediated by the perforin/granzyme pathway. The oligoclonal or pauciclonal expansions of certain VbetaT cells were found in each NK-type T-cell population. The non-variant CDR3 region(s) for the TCRbeta chain(s) showed CD57-T cells and CD56-T cells to be derived from distinct origins, while the DP-T cell population consisted of a mixture of the clones seen in both CD56-T cells and CD57-T cells. Our results suggest that CD57-T cells and CD56-T cells are functionally and ontogenically different populations while DP-T cells appear to originate from both CD56-T cells and CD57-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.     

Increases in soluble CD8 antigen in plasma, and CD8+ and CD8+CD38+ cells in human immunodeficiency virus type-1 infection.

Increases in plasma levels of soluble CD8 (SCD8) antigen and expansion of the CD8+ CD38+ lymphocyte compartment were early immunologic alterations frequently observed prior to detection of antibodies against human immunodeficiency virus type 1 (HIV-1) and diminution of CD4+ cells in subjects at risk to develop AIDS. These increases identified in the 49 seronegative homosexual men were manifest in all 164 homosexual subjects and 45 intravenous drug users (IVDU) positive for HIV-1 antibodies (HIV-1+), 19 patients with ARC, and 29 AIDS patients. Augmentation of plasma sCD8 antigen correlated with increases in both CD8+ and CD8+ CD38+ cells in HIV-1(-) homosexual men (r = 0.35, P less than 0.013; r = 0.48, P less than 0.0005; respectively) and the 258 HIV-1+ subjects (r = 0.25, P less than 0.0003; r = 0.33, P less than 0.0001, respectively). In vitro examination of unstimulated peripheral blood lymphocytes from HIV-1+ homosexuals and IVDU confirmed the fivefold higher constitutive levels of cellular release of sCD8 antigen in these subjects compared to heterosexual controls. Inclusion of radiolabeled amino acids during the 3-day culture period in the presence or absence of phytohemagglutinin resulted in negligible levels of radioactivity associated with the sCD8 antigen indicative of a lack of de novo synthesis. Throughout clinical progression to AIDS, sCD8 antigen levels continued to escalate relative to the numbers of CD8+ cells bearing CD38+ antigen. The data confirm the interrelationship between sCD8+ antigen and CD8+ and CD8+ CD38+ cells.     

Persistence of herpes simplex virus type 2 VP16-specific CD4+ T cells

Patients with genital herpes have frequent viral reactivations. The repeated antigenic rechallenges can modulate the CD4+ memory T-cell repertoires during the course of infection. In this study, the CD4+ T-cell responses against the herpes simplex virus type 2 (HSV-2) tegument protein VP16 were studied in two HSV-2-infected subjects at two different time points that spanned a 5-year period. Although the VP16-specific T cells did exhibit variation of T-cell receptor Vbeta usages at the two time points, T cells that used identical Vbeta and CDR3 junction sequences were also observed at the two time points. These experiments demonstrate that the CD4+ T cells that are directed against HSV-2 VP16 protein in chronically infected individuals are oligoclonal and that T cells of specific clonotypes can be maintained throughout the course of the disease.     

Functional expression of chemokine receptor CCR6 on human effector memory CD8+ T cells

Since CCR6 is a receptor for the chemokine CCL20, which is produced in tissues such as intestine and colon, it is thought that T cells expressing CCR6 are involved in mucosal immunity. The expression and function of CCR6 on human CD8+ T cells have not well been analyzed, although it is known that this receptor is expressed on a subset of human CD8+ T cells. We here characterize human CCR6+ CD8+ T cells. Multi-color flow cytometric analysis demonstrated that CCR6+ cells are predominantly found among CD8+ T cells having the memory phenotype. The expression of CCR6 is positively and negatively correlated with that of CCR5 and CCR7, respectively. CCR6+ CD8+ T cells express granzyme A and a low level of perforin but not granzyme B. In addition, a major population among these cells has the ability to produce IFN-gamma and TNF-alpha but not IL-2. These results indicate that CCR6+ CD8+ T cells have characteristics of early effector memory cells rather than effector or central memory cells. A chemotaxis assay revealed that CCR6+ CD8+ T cells have the ability to migrate in response to CCL20, suggesting that these T cells migrate to tissues such as colon and are involved in mucosal immunity.     

Inverse relationship of CD8+CD11+ suppressor T cells with human immunodeficiency virus (HIV)-specific cellular cytotoxicity and natural killer cell activity in HIV infection.

Profiles of CD8+CD11+ T suppressor cells, human immunodeficiency virus (HIV)-env-specific cytotoxic T-lymphocyte (CTL) activities, and natural killer (NK) cell activity were studied in 12 asymptomatic untreated HIV-infected patients. These patients were followed for 4-7 months. NK activity, HIV-env-specific CTL activities mediated by CD4+, CD8+ T cells and CD8+CD11+ T-suppressor cell number remained stable in seven patients during the study period. Alternatively, NK and HIV-specific CTL activities decreased and CD8+CD11+ cell number increased in five patients whose CD4+ T-cell number fell, and in four of these five patients serum p24 antigen level increased, and they developed minor clinical signs of disease progression during the study period. CD8+CD11+ cells are present in higher percentage (10-45% of peripheral blood mononuclear cells) in these HIV-infected patients as compared to those in normal individuals (3-5%). Our results suggest that CD8+CD11+ cells, NK, and HIV-specific cytotoxic activities may be helpful in monitoring prognosis of HIV infection. These observations also suggest that CD8+CD11+ cells may play an important role in the failure of host immune defences against HIV.     

Persistent human immunodeficiency virus-1 antigenaemia affects the expression of interleukin-7Ralpha on central and effector memory CD4+ and CD8+ T cell subsets

Interleukin (IL)-7 and its receptor (IL-7Ralpha) play important roles in regulating lymphopoiesis. Previous studies have reported that human immunodeficiency virus-1 (HIV-1) viraemia affects the expression of IL-7Ralpha, but its effects on CD4+ and CD8+ T cell memory subsets have not been studied. Using eight-colour flow cytometry, we compared the immunophenotypic patterns of CD4+ and CD8+ T cell subsets expressing IL-7Ralpha and activation markers, as well as circulating IL-7 levels, in three well-defined groups of HIV-1-infected subjects: successfully treated, viraemic and long-term non-progressor (LTNP). Compared with successfully treated and LTNP subjects, viraemic patients had reduced expression of IL-7Ralpha on both CD4+ and CD8+ T cells, particularly on central and effector memory T cell compartments, and substantially elevated expression of activation markers on CD8+ T cell subsets. Circulating IL-7 levels were correlated negatively with the number of CD4+ and CD8+ T cell subsets expressing IL-7Ralpha; these associations were stronger with CD4+ T cell subsets and mainly with central and effector memory cells. The expression of activation markers on CD4+ and CD8+ cell T subsets was not related to circulating IL-7 levels. A strong negative correlation was observed between central memory CD4+ or CD8+ T cells expressing IL-7Ralpha and those expressing activation markers, independently of IL-7 levels. Collectively, these results provide further insight on the role of unsuppressed viral load in disrupting the IL-7/IL-7Ralpha system and contributing to HIV-1 disease progression.     

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.     

CD8+ CD28- and CD8+ CD57+ T cells and their role in health and disease

Chronic antigenic stimulation leads to gradual accumulation of late-differentiated, antigen-specific, oligoclonal T cells, particularly within the CD8(+) T-cell compartment. They are characterized by critically shortened telomeres, loss of CD28 and/or gain of CD57 expression and are defined as either CD8(+) CD28(-) or CD8(+) CD57(+) T lymphocytes. There is growing evidence that the CD8(+) CD28(-) (CD8(+) CD57(+)) T-cell population plays a significant role in various diseases or conditions, associated with chronic immune activation such as cancer, chronic intracellular infections, chronic alcoholism, some chronic pulmonary diseases, autoimmune diseases, allogeneic transplantation, as well as has a great influence on age-related changes in the immune system status. CD8(+) CD28(-) (CD8(+) CD57(+)) T-cell population is heterogeneous and composed of various functionally competing (cytotoxic and immunosuppressive) subsets thus the overall effect of CD8(+) CD28(-) (CD8(+) CD57(+)) T-cell-mediated immunity depends on the predominance of a particular subset. Many articles claim that CD8(+) CD28(-) (CD8(+) CD57(+)) T cells have lost their proliferative capacity during process of replicative senescence triggered by repeated antigenic stimulation. However recent data indicate that CD8(+) CD28(-) (CD8(+) CD57(+)) T cells can transiently up-regulate telomerase activity and proliferate under certain stimulation conditions. Similarly, conflicting data is provided regarding CD8(+) CD28(-) (CD8(+) CD57(+)) T-cell sensitivity to apoptosis, finally leading to the conclusion that this T-cell population is also heterogeneous in terms of its apoptotic potential. This review provides a comprehensive approach to the CD8(+) CD28(-) (CD8(+) CD57(+)) T-cell population: we describe in detail its origins, molecular and functional characteristics, subsets, role in various diseases or conditions, associated with persistent antigenic stimulation.     

Tolerogenic dendritic cells impede priming of naïve CD8+ T cells and deplete memory CD8+ T cells

Type 1 diabetes is a T‐cell‐mediated autoimmune disease in which autoreactive CD8⁺ T cells destroy the insulin‐producing pancreatic beta cells. Vitamin D3 and dexamethasone‐modulated dendritic cells (Combi‐DCs) loaded with islet antigens inducing islet‐specific regulatory CD4⁺ T cells may offer a tissue‐specific intervention therapy. The effect of Combi‐DCs on CD8⁺ T cells, however, remains unknown. To investigate the interaction of CD8⁺ T cells with Combi‐DCs presenting epitopes on HLA class I, naive, and memory CD8⁺ T cells were co‐cultured with DCs and proliferation and function of peptide‐specific T cells were analyzed. Antigen‐loaded Combi‐DCs were unable to prime naïve CD8⁺ T cells to proliferate, although a proportion of T cells converted to a memory phenotype. Moreover, expansion of CD8⁺ T cells that had been primed by mature monocyte‐derived DCs (moDCs) was curtailed by Combi‐DCs in co‐cultures. Combi‐DCs expanded memory T cells once, but CD8⁺ T‐cell numbers collapsed by subsequent re‐stimulation with Combi‐DCs. Our data point that (re)activation of CD8⁺ T cells by antigen‐pulsed Combi‐DCs does not promote, but rather deteriorates, CD8⁺ T‐cell immunity. Yet, Combi‐DCs pulsed with CD8⁺ T‐cell epitopes also act as targets of cytotoxicity, which is undesirable for survival of Combi‐DCs infused into patients in therapeutic immune intervention strategies.     

Anti-human immunodeficiency virus-gag CD8+ memory T cells generated in vitro from Listeria-immunized mice

The goal of vaccination is the generation of immune memory, an immune state that permits rapid and intense recall responses to a pathogen. Considerable effort is being made to understand the nature of memory T cells. We report here that by extending the length of in vitro culture following a single restimulation with specific peptide, preparations of highly enriched, highly active antigen-specific CD8+ memory T cells could be obtained. These cultures were begun with splenocytes from mice primed by infection either with an attenuated strain of Listeria monocytogenes or vaccinia virus, both expressing the human immunodeficiency virus-1-gag gene. In the cultures, antigen-specific cytotoxic T lymphocyte (CTL) activity reached a maximum at about 9 days and thereafter fell to negligible values. Concomitant with the fall of CTL activity, however, we observed enrichment for a subset of CD11ahigh antigen-specific gag-tetramerpos CD8+ T cells. The cells showed little or no 4-hr CTL activity, but had high delayed (18-hr) CTL activity, and very high cytolytic activity after restimulation. They rapidly expressed interferon-gamma production. Their growth and survival after sorting was completely dependent on interleukin-2 or -15. As few as 5000 of the fluorescence-activated cell sorting-purified cells protected recipients against challenge 3 months after transfer. In response to the challenge, the cells repopulated lymphoid and non-lymphoid organs and showed a sizeable increase in number. The cells therefore demonstrate high protective activity for long periods of time. These cultured cells are thus a potential source of enriched natural memory T cells for reperfusion studies and in which the mechanisms that underlie the generation, differentiation and persistence of memory can be examined.     

人类CD8+记忆T细胞体外扩增方法的研究

 目的： 研究体外扩增人类CD8+记忆T细胞的新方法,为抗病毒与抗肿瘤的过继性免疫治疗提供新的手段。方法：将anti-CD3抗体、anti-CD28抗体、CD70、白细胞介素（IL）-2、IL-7和IL-15进行排列组合,设计出63种刺激方式,对体外分离得到的正常人外周血CD8+ T细胞进行体外扩增;培养14 d后进行细胞计数,并检测CD8+ T细胞的纯度以及CD8+中枢记忆T细胞（TCM）和CD8+效应记忆T细胞（TEM）所占的比例,进而计算出CD8+ T细胞、CD8+ TCM和CD8+ TEM的体外扩增倍数,从而确定理想的刺激方法。结果：体外扩增CD8+ T细胞、CD8+ TCM和CD8+ TEM的理想刺激方式均为anti-CD3抗体、IL-2和IL-7三者的组合;该刺激方式使3种细胞在培养14 d后分别扩增了13.19、13.28和15.27倍。结论：Anti-CD3抗体、IL-2和IL-7三者的组合,是刺激人类CD8+记忆T细胞体外扩增的相对理想方法。     

Definition of two new epitopes on human immunodeficiency virus type 1 gag protein recognized by human CD8+ cytotoxic T lymphocyte clones.

We have established 3 CD8+ cytotoxic T lymphocyte (CTL) clones from the peripheral blood mononuclear cells of two HIV-1-seropositive asymptomatic donors. The epitopes recognized by these CTL clones were defined using synthetic peptides. The epitopes were located on HIV-1gag protein between amino acid (a.a.) 145 and 155 (QAISPRTLNAW), a.a.193 and 201 (GHQAAMQML), and a.a.260 and 267 (EIYKRWII), and were presented by HLA-A25, HLA-B38 and HLA-B8, respectively. The former 2 epitopes have not been previously defined.The HLA-A25-restricted epitope overlapped with HLA-B57-restricted and HLA-Cw3-restricted epitopes previously reported. In addition, this epitope overlapped with an HLA-DQ-restricted epitope recognized by CD4+ CTL. The HLA-B38-restricted epitope overlapped with HLA-A2-restricted and HLA-Bw52-restricted epitopes that were previously reported. The HLA-B38-restricted epitope between a.a.193 and 201 was highly conserved among HIV-1 strains. The results demonstrate that two new epitopes were defined in a region of gag protein that includes multiple epitopes presented by multiple HLA.     

Large clonal expansions of human virus-specific memory cytotoxic T lymphocytes within the CD57+ CD28- CD8+ T-cell population

The proportion of human peripheral blood CD8+ T cells that are CD57+ CD28- is low at birth but increases with age and in individuals infected with human cytomegalovirus (HCMV) or human immunodeficiency virus (HIV). These CD57+ CD28- CD8+ T cells contain large oligoclonal T-cell expansions whose antigen specificity is unknown. We identified clonal expansions of virus-specific memory cytotoxic T-lymphocyte precursors (CTLp) in both healthy carriers of HCMV and in asymptomatic HIV-infected subjects. In each subject, from the T-cell receptor (TCR) beta-chain hypervariable sequence of each immunodominant CTL clone, we designed complementary oligonucleotide probes to quantify the size and phenotypic segregation of individual virus-specific CTL clones in highly purified populations of peripheral blood CD8+ T cells. We found large clonal expansions of virus-specific CTL clonotypes in CD57+ CD28- CD8+ T cells. Using limiting dilution analysis, we found functional peptide-specific CTLp at high frequency in CD57+ CD28- cells. Thus, memory CTL specific for persistent viruses account for many oligoclonal expansions within CD57+ CD28- CD8+ T cells.