Dissociated production of perforin, granzyme B, and IFN-gamma by HIV-specific CD8(+) cells in HIV infection

CD8(+) T cells play a crucial role in the control of viral infections such as HIV. The functional characterization of HIV-specific CD8(+) T cells has so far been largely restricted to studies of IFN-gamma. The TCR-triggered release of the effector molecules perforin (PFN) and granzyme B (GzB), however, is thought to be a central pathway for the destruction of virus-infected target cells by CD8(+) effector T cells. Here we would like to address two major findings. On the one hand we propose that ex vivo measurements of PFN and GzB secretion via ELISPOT may permit the distinction between in vivo resting versus activated CD8(+) memory T cells in healthy and HIV-infected individuals. Therefore, extending the present standard of IFN-gamma measurements to the analysis of PFN and GzB release in functional T cell assays will provide new insights into CD8(+) effector T cell functions. It should enable the evaluation of therapeutic vaccination efficacy by its ability to reactivate and convert IFN-gamma-positive, but GzB- and PFN-negative memory CD8(+) T cells into PFN/GzB-secreting effector cells. On the other hand, we report on a frequent ex vivo dissociation of the HIV peptide-induced secretion of PFN and GzB in chronic HIV infection underlining CD8(+) effector T cell diversity in this disease--an aspect that also has to be accounted for in immune monitoring approaches.     

Single-cell perforin and granzyme expression reveals the anatomical localization of effector CD8+ T cells in influenza virus-infected mice

Influenza virus infection activates cytolytic T lymphocytes (CTL) that contribute to viral clearance by releasing perforin and granzymes from cytoplasmic granules. Virus-specific, perforin-dependent CD8(+) CTL were detected in freshly isolated cells from the mouse lung parenchyma but not from the mediastinal lymph nodes (MLN), where they are primed, or from the spleen during primary influenza virus infection. To determine whether this difference was due to the low frequency or incomplete maturation of effector CTL in MLN, we measured expression of perforin, granzymes A, B, and C, and IFN-gamma mRNAs in CD8(+) populations and single cells immediately after isolation from virus-infected mice. Quantitative PCR revealed significant expression of perforin, granzyme A, granzyme B, and IFN-gamma in activated CD8(+) cells from MLN, spleen, and lung parenchyma. Granzyme C expression was not detected. Individual activated or nucleoprotein peptide/class I tetramer-binding CD8(+) cells from the three tissues expressed diverse combinations of perforin, granzyme, and IFN-gamma mRNAs. Although cells from lung expressed granzymes A and B at higher frequency, each of the tissues contained cells that coexpressed perforin with granzymes A and/or B. The main difference between MLN and lung was the elevated frequency of activated CD8(+) T cells in the lung, rather than their perforin/granzyme expression profile. The data suggest that some CTL mature into perforin/granzyme-expressing effector cells in MLN but reach detectable frequencies only when they accumulate in the infected lung.     

肝癌患者CD8^＋T淋巴细胞穿孔素表达和脱颗粒特点

目的观察肝癌患者外周血CD8^＋T淋巴细胞穿孔紊表达和脱颗粒特点及与临床分期的相关性。方法采集52例肝癌患者、20例健康人的外周血，用流式细胞仪分析CD8^＋T淋巴细胞穿孔素表达百分比；以抗CD3单抗刺激后CD107a表达量代表CD8^＋T淋巴细胞脱颗粒的数量。结果肝癌患者外周血CD8^＋T淋巴细胞穿孔素表达量（32．3％±17．4％）与健康对照（31．8％±14．1％）差异无统计学意义（P〉0．05）。但是抗CD3单抗刺激5h后CD8^＋T淋巴细胞CD107a表达量（6．8％±4．2％）低于健康对照（15．0％±4．3％），差异有统计学意义（P〈0．01）。结论肝癌患者CD8^＋T淋巴细胞脱颗粒能力明显下降。     

Increase in activated CD8+ T lymphocytes expressing perforin and granzyme B correlates with disease activity in patients with systemic lupus erythematosus

OBJECTIVE: Cytotoxic T lymphocyte-mediated killing using granzyme B has recently been proposed to be a preferential and selective source of autoantigens in systemic autoimmune diseases, including systemic lupus erythematosus (SLE), while other reports have indicated that cytolytic activity in SLE patients was decreased. The aim of this study was to examine the phenotypic and functional status of the CD8+ T cells in SLE patients. METHODS: Phenotype analysis of CD8+ T cells was carried out using flow cytometry. The cytotoxic potential of CD8+ T cells and its consequences were examined in redirected-killing experiments. SLE patients with quiescent disease (n = 41) were compared with SLE patients with active disease (n = 20), normal individuals (n = 36), and control patients with vasculitis (n = 14). Cytotoxic CD8+ T cell differentiation was examined by coculture with differentiated dendritic cells (DCs) in the presence of SLE patient sera. RESULTS: Patients with disease flares were characterized by higher proportions of perforin- and/or granzyme B-positive lymphocytes with a differentiated effector phenotype (CCR7- and CD45RA+). The frequency of these cells in peripheral blood correlated with clinical disease activity as assessed by the SLE Disease Activity Index. These cells generated high amounts of soluble nucleosomes as well as granzyme B-dependent unique autoantigen fragments. Finally, the activation of DCs with serum from a patient with active lupus induced granzyme B expression in CD8+ T lymphocytes. CONCLUSION: DCs generated in the presence of sera from SLE patients with active disease could promote the differentiation of CD8+ effector T lymphocytes that are fully functional and able to generate SLE autoantigens. Our data disclose a new and pivotal role of activated CD8+ T lymphocytes in SLE pathogenesis.     

HIV nonprogressors preferentially maintain highly functional HIV-specific CD8+ T cells

Establishing a CD8(+) T cell-mediated immune correlate of protection in HIV disease is crucial to the development of vaccines designed to generate cell-mediated immunity. Historically, neither the quantity nor breadth of the HIV-specific CD8(+) T-cell response has correlated conclusively with protection. Here, we assess the quality of the HIV-specific CD8(+) T-cell response by measuring 5 CD8(+) T-cell functions (degranulation, IFN-gamma, MIP-1beta, TNF-alpha, and IL-2) simultaneously in chronically HIV-infected individuals and elite nonprogressors. We find that the functional profile of HIV-specific CD8(+) T cells in progressors is limited compared to that of nonprogressors, who consistently maintain highly functional CD8(+) T cells. This limited functionality is independent of HLA type and T-cell memory phenotype, is HIV-specific rather than generalized, and is not effectively restored by therapeutic intervention. Whereas the total HIV-specific CD8(+) T-cell frequency did not correlate with viral load, the frequency and proportion of the HIV-specific T-cell response with highest functionality inversely correlated with viral load in the progressors. Thus, rather than quantity or phenotype, the quality of the CD8(+) T-cell functional response serves as an immune correlate of HIV disease progression and a potential qualifying factor for evaluation of HIV vaccine efficacy.     

HIV subtypes induce distinct profiles of HIV-specific CD8(+) T cell responses

CD8(+) T cells play an important role in controlling HIV infection and qualitative differences in HIV-specific CD8(+) responses may determine the degree of immune control. We studied 56 HIV-infected, ARV-naive Ugandans and examined the role of subtypes in modulating their HIV-specific T cell responses. Gag-specific responses were readily detectable in our study population. Interestingly, we found significantly decreased Gag-specific cytolysis (as measured by CD107 expression) in subtype D (n = 21) compared to subtype A (n = 35) HIV infection. Sequence analyses within identified epitopes suggest patterns of conservation that are subtype specific. We conclude that HIV subtypes may promote distinct profiles of T cells responses and immune control.     

肝细胞癌患者 Treg 细胞对 CD8＋T 淋巴细胞穿孔素表达的影响

目的：研究肝细胞癌患者免疫抑制性 Treg 细胞对 CD8＋ T 淋巴细胞穿孔素表达的影响。方法采集20例肝细胞癌患者和20名健康人的外周血,用流式分析法检测抗-CD3／CD28刺激48 h 后 CD8＋ T 淋巴细胞的穿孔素表达情况。免疫磁珠分离法分离健康人 CD8＋ T 淋巴细胞和肝细胞癌患者 Treg 细胞,一组 CD8＋ T 淋巴细胞单独培养,另一组CD8＋T 淋巴细胞与 Treg 细胞共同培养,用流式细胞法检测抗-CD3／CD28刺激48 h 后两组 CD8＋ T 淋巴细胞穿孔素表达情况。结果肝细胞癌患者外周血中 CD8＋ T 淋巴细胞穿孔素表达量与健康人相近,分别为（10．74±3．96）％和（12．6±2．48）％,差异无统计学意义（P ＞0．05）。CD8＋ T 淋巴细胞单独培养和与肝癌患者 Treg 细胞共培养后,健康人 CD8＋ T 淋巴细胞穿孔素表达量分别为（34．2±3．65）％和（20．43±4．52）％,差异有统计学意义（t＝11．42,P ＜0．01）。结论肝癌患者免疫抑制性 Treg 细胞可使 CD8＋ T 淋巴细胞穿孔素表达量降低。     

Distribution of functional HIV-specific CD8 T lymphocytes between blood and secondary lymphoid organs after 8-18 months of antiretroviral therapy in acutely infected patients

OBJECTIVES: To assess whether drug-induced suppression of the plasma viral load is associated with selective differential distribution of virus-specific CD8 T cells between the blood and secondary lymphoid organs. METHODS: HIV-specific CD8 T lymphocyte responses were quantified in matched peripheral blood and lymph node samples from seven patients starting treatment shortly after infection, who received antiretroviral therapy (ART) for a median of 14 months. Cells recovered from samples were subjected to IFN-gamma ELISPOT analysis. A series of synthetic peptides corresponding to previously characterized cytotoxic T lymphocyte epitopes restricted by HLA I molecules present in each patient were used as antigens, together with appropriate positive and negative controls. RESULTS: HIV-specific CD8 T lymphocyte responses were found in six of the seven patients. The observed frequencies of HIV-specific CD8 T lymphocytes and the pattern of epitope recognition was identical within the two compartments. These results also confirm the observation that functional HIV-specific CD8 T cells are preserved on ART in most patients initiating treatment at the time of primary HIV-1 infection. CONCLUSION: This investigation demonstrated that patterns of antigenic immunodominance as well as frequencies of HIV-specific CD8 T lymphocytes are similar in blood and lymphoid tissue compartments in HIV-infected individuals. These findings support current approaches to the identification of HIV-specific CD8 T lymphocyte reactivity based on leukocytes isolated from blood even in patients with ART-induced suppression of viral load.     

Perforin is not co-expressed with granzyme A within cytotoxic granules in CD8 T lymphocytes present in lymphoid tissue during chronic HIV infection.

BACKGROUND: Residual HIV-1-infected cells are poorly eliminated from lymphoid tissue (LT) reservoirs by effector cytotoxic T lymphocytes (eCTL) despite antiretroviral therapy. Perforin and granzyme A (grA) constitute major effector molecules within eCTL granules that induce apoptosis and lysis of virally infected cells. OBJECTIVE: Expression of perforin and grA was studied at the single cell level in LT and blood from 16 patients infected with HIV-1 (stage A1-C) who were not taking antiretroviral therapy. METHOD: Immunohistochemical analysis by in situ imaging of cells from blood and LT. RESULTS: Quantitative in situ imaging showed that perforin-expressing CD8 T cells comprised 0.3-1.5% of total cells within the LT from recent HIV-1 seroconverters, while grA was found in 2.1-7.2% of total cells. However, despite high-level grA upregulation (1.5-4.5% of total cells) compared with that in non-infected individuals (0.4-0.9%), perforin expression remained low (< 0.1% of total cells) (P < 0.02) in LT from patients with chronic HIV-1 infection (stage A2-C). This contrasted with findings in peripheral blood mononuclear cells (PBMC) from the same HIV-1 infected cohort where perforin was detected in 13-31% of all PBMC, which was 10- to 100-fold higher than in lymphoid tissue (P < 0.001); grA was found in 14-32% of total PBMC. Two-colour staining showed that granular expression of perforin and grA was restricted to CD8 T cells in over 90% of total cells in both LT and blood. CONCLUSIONS: These findings indicate that cytotoxic perforin expression is impaired at local sites of HIV replication within lymphoid tissue. Since perforin is required together with grA for granule-mediated cytolysis, the low perforin expression in the LT may limit the ability of eCTL to eliminate HIV-1 infected cells in lymphoid tissue.     

T cell receptor V beta repertoire of the antigen specific CD8 T lymphocyte subset of HIV infected children

OBJECTIVE: Analysis of the T cell receptor V beta repertoire during HIV infection reveals expansions in multiple V beta families of CD8 T cells, but their antigenic specificity is ill-defined. We sought to determine the TCR V beta repertoire of HIV specific CD8 T lymphocytes in infected children. DESIGN/METHODS: We performed flow cytometry to examine TCR V beta families as identified by specific monoclonal antibodies and binding of HIV peptide loaded tetrameric MHC complexes in peripheral blood samples from a group of HIV infected children. RESULTS: Simultaneous assessment of 12 selected expanded V beta families amongst nine HIV infected patients for tetramer binding revealed only one child in whom the expanded V beta population bound HIV Gag or Pol tetramers. In four HIV infected children, percentage tetramer binding cells was determined in 21 TCR V beta families. The tetramer binding cells of three children exhibited a widely distributed TCR V beta repertoire while in the fourth patient they were preferentially localized within two TCR V beta families. Repeat analysis revealed that the TCR V beta repertoire of tetramer binding cells was stable. CONCLUSIONS: These data provide evidence that the HIV-specific CD8 T cell response in children is usually distributed widely among many different TCR V beta families. The heterogeneity of the TCR V beta repertoire usage by the antigen specific CD8 T cells may reflect the dynamic interaction between host and pathogen during the course of HIV infection and may be influenced by the rate of viral mutation, CD4 T cell helper activity, or other factors.     

In patients on prolonged HAART,a significant pool of HIV infected CD4 T cells are HIV-specific

OBJECTIVES: To examine the antigen specificities of HIV reservoir CD4 T cells in patients on prolonged and effective highly active antiretroviral therapy (HAART). DESIGN: Five HIV-infected patients, who were highly adherent to antiretroviral treatment, were selected on the basis of long-term undetectable plasma viral RNA on unmodified HAART. To investigate the antigen specificities of infected memory CD4 T cells, we examined the capacity of recall antigens, including HIV antigens, to induce virus production by peripheral blood mononuclear cells (PBMC). METHODS: To quantify CD4 T cells infected by replication-competent virus, and to determine their antigen specificities, we used a limiting dilution-based culture assay. CD8 T cell-depleted PBMC at several cell densities were activated by using Tuberculin purified protein derivative, cytomegalovirus, or HIV-1 p24 with and without HIV-1 Nef. RESULTS: We found that the pool of infected CD4 T cells includes HIV-specific cells with apparent frequencies between 5- and 100-fold higher than those of the common specificities for cytomegalovirus or Tuberculin. CONCLUSION: Our findings suggest that a significant proportion of replication-competent HIV-infected CD4 T cells in these patients are memory cells directed against HIV determinants. This may provide a rationale for the therapeutic use of recombinant HIV antigens to reduce the pool of HIV-reservoir cells.     

The fraction of perforin-expressing HIV-specific CD8 T cells is a marker for disease progression in HIV infection

OBJECTIVE: Perforin is an important component of the death machinery of cytotoxic T cells (CTL). To evaluate functional differences between HIV- and cytomegalovirus (CMV)-specific CTL of coinfected patients, the frequencies of the respective perforin-expressing T cells were analysed in a rapid whole blood assay. METHODS: Whole blood of HIV- and CMV-infected individuals was specifically stimulated by HIV-1 Pr55(gag) or complete CMV antigen, and activation-induced intracellular cytokine and perforin expression in CD8 T cells was analysed by flow cytometry. RESULTS: Perforin-expressing HIV-1- and CMV-specific CD8 T cells can be quantified simultaneously. Within a patient, the frequency of such HIV-specific CD8 T cells in peripheral blood was lower than the frequency of the respective CMV-specific cells. The number of the perforin-expressing HIV-specific CD8 T cells inversely correlated with the peripheral blood CD4 T cell count. CONCLUSIONS: The differential fractions of perforin-expressing virus-specific CD8 T cells in HIV and CMV double infection might be caused by differences in priming and trafficking to or from replication sites. However, without knowing the underlying mechanism, the fraction of perforin-expressing HIV-specific CD8 T cells provides another surrogate marker for disease progression.     

Effect of long-term infection with nef-defective attenuated HIV type 1 on CD4+ and CD8+ T lymphocytes: increased CD45RO+CD4+ T lymphocytes and limited activation of CD8+ T lymphocytes

Members of the Sydney Blood Bank Cohort (SBBC) have been infected with an attenuated strain of HIV-1 with a natural nef/LTR mutation and have maintained relatively stable CD4+ T lymphocyte counts for 14-18 years. Flow cytometric analysis was used to examine the phenotype of CD4+ and CD8+ T lymphocytes in these subjects, including the immunologically important naive (CD45RA+CD62L+), primed (CD45RO+), and activated (CD38+HLA-DR+ and CD28-) subsets. The median values were compared between the SBBC and control groups, comprising age-, sex-, and transfusion-matched HIV-1-uninfected subjects; transfusion-acquired HIV-1-positive LTNPs; and sexually acquired HIV-1-positive LTNPs. Members of the SBBC not only had normal levels of naive CD4+ and CD8+ T lymphocytes, but had primed CD45RO+ CD4+ T lymphocytes at or above normal levels. Furthermore, these primed cells expressed markers suggesting recent exposure to specific antigen. SBBC members exhibited variable activation of CD8+ T lymphocytes. In particular, SBBC members with undetectable plasma HIV-1 RNA had normal levels of activated CD8+ T lymphocytes. Therefore, the result of long-term infection with natural nef/LTR mutant HIV-1 in these subjects suggests a decreased cytopathic effect of attenuated HIV-1 on susceptible activated CD4+ T lymphocyte subsets in vivo, and minimal activation of CD8+ T lymphocytes.     

Cord blood T lymphocytes lack constitutive perforin expression in contrast to adult peripheral blood T lymphocytes

Perforin is the cytolytic pore-forming protein, which alone can be responsible for the lethal hit in one of the killing mechanisms used by natural killer (NK) cells or cytotoxic T lymphocytes. In this study, perforin expression was investigated in cord blood (CB) lymphocytes to determine their killing potential in vivo. The majority of CB CD3- NK cells had the protein. Compared with adult perforin-positive NK cells, a significantly lower percentage of cells expressing CD56 and CD57, the related neural cell adhesion molecules, was found (P = .0001). Perforin was also present in a unique immature CB NK-cell subset, characterized by cytoplasmic CD3 antigen (Ag) expression. In CB, very few CD8 perforin-positive T lymphocytes could be detected, but they were in significant numbers in adult peripheral blood (P = .02). A substantial proportion of these cells (70% +/- 23%) lacked the CD28 T-cell coactivation Ag, and they were able to exert NK-like, major histocompatibility complex nonrestricted cytolytic activity. CD4+ and gamma delta-T cells expressing perforin were absent from CB, but low numbers of such cells were detected in adult peripheral blood (P = .0001). Therefore, the spontaneous cytolytic activity of CB lymphocytes appeared to be dependent on well-represented perforin-positive NK cells, which were shown to efficiently lyse NK-sensitive target cells.