HIV-specific cytotoxic cell frequencies measured directly ex vivo by the Lysispot assay can be higher or lower than the frequencies of IFN-gamma-secreting cells: anti-HIV cytotoxicity is not generally impaired relative to other chronic virus responses

CD8(+) T cells in HIV-infected patients are believed to contribute to the containment of the virus and the delay of disease progression. However, the frequencies of HIV-specific CD8(+) T cells, as measured by IFN-gamma secretion and tetramer binding, often do not correlate with a delay in disease progression during chronic infection. Using the Lysispot and ELISPOT assays, we measured the frequencies of cytotoxic and IFN-gamma-secreting T cells responding to overlapping peptides from Gag, Nef, Env, and Pol consensus HIV-1 clade B sequences. PBMC from the majority of HIV-infected subjects have significant frequencies of HIV-specific cells that killed targets within 5 h directly ex vivo. The relative frequencies of IFN-gamma-secreting and cytotoxic cells varied markedly between different HIV peptide pools within the same patient, and some T cells lysed targets without secreting IFN-gamma. These results indicate that measurement of IFN-gamma production alone may be insufficient to evaluate the breadth of the HIV-specific T cell response. Also, neither the CTL to IFN-gamma ratios nor the ex vivo CTL frequencies specific for different HIV proteins were consistently lower than responses specific for two other chronic viral infections, human CMV and EBV, within the same subjects. Thus ex vivo cytotoxic T cell frequencies do not provide evidence for a model of "preterminal differentiation" of HIV-specific CD8(+) T cells during chronic HIV infection. Analysis of the frequency of directly cytotoxic HIV-specific T cells may be of considerable value in the assessment of disease progression and the potential efficacy of HIV vaccines.     

Differential Gag-specific polyfunctional T cell maturation patterns in HIV-1 elite controllers

A small fraction of HIV-infected individuals (<1%), referred to as elite controllers (EC), are able to maintain undetectable viral loads indefinitely without treatment. The role of the maturational phenotype of T cells in the control of HIV infection in these individuals is not well described. We compared the maturational and functional phenotypes of Gag-specific CD4 and CD8 T cells from EC, who maintain undetectable viral loads without treatment; relative controllers (RC), who maintain viral loads of <1,000 copies/ml without treatment; and noncontrollers (NC), who fail to control viral replication. EC maintained higher frequencies of HIV-specific CD4 T cells, less mature polyfunctional Gag-specific CD4 T cells (CD27(+) CD57(-) CD45RO(+)), and Gag-specific polyfunctional CD4 T cells than those observed in NC. In EC, the frequency of polyfunctional Gag-specific CD8 T cells was higher than that observed in RC and NC. RC had a similar functional phenotype to that observed in NC, despite consistently lower viral loads. Finally, we found a direct correlation between the frequency of Gag-specific CD27(+) CD57(-) CD45RO(+) CD4(+) T cells and the frequency of mature HIV-specific CD8 T cells. Altogether, our data suggest that immature Gag-specific interleukin-2 (IL-2)-producing CD4(+) T cells may play an important role in spontaneous control of HIV viremia by effectively supporting HIV-specific CD8 T lymphocytes. This difference appears to differentiate EC from RC.     

Frequencies of ex vivo-activated human immunodeficiency virus type 1-specific gamma-interferon-producing CD8+ T cells in infected children correlate positively with plasma viral load

HIV-specific CD8+ T cells are critical in controlling human immunodeficiency virus (HIV) replication. We present the evaluation of a gamma-interferon (IFN-gamma)-based enzyme linked immunospot (ELISPOT) assay for the quantification of HIV-specific CD8+ T cells from HIV-infected children. We studied 20 HLA-A*0201-positive HIV-infected children. The IFN-gamma production in response to stimulation with two HLA-A*0201-restricted immunodominant CD8 epitopes (SLYNTVATL [SL9] in Gag and ILKEPVHGV [IV9] in Pol) was tested using the ELISPOT assay. The results were compared to labeling with the corresponding tetramers. Among the 20 children, 18 had detectable responses against the SL9 and/or the IV9 epitope using the ELISPOT assay (medians, 351 and 134 spot-forming cells/10(6) peripheral blood mononuclear cells, respectively). Comparison of results from the tetramer and ELISPOT assays suggests that only a fraction of HIV-specific CD8+ T cells were able to produce IFN-gamma. Most importantly, we found that the frequencies of IFN-gamma-producing CD8+ T cells were positively correlated with the viral load whereas the frequencies of tetramer-binding CD8+ T cells were not. The high sensitivity of the ELISPOT assay and the fact that this functional assay provided information different from that of tetramer labeling support its use for measurement of HIV-specific CD8+ T cells. In conclusion, our results show that the ex vivo-activated IFN-gamma-producing HIV-specific CD8+ T-cell subset is dependent upon continuous antigenic stimulation.     

Evidence for in vivo primed and expanded autoreactive T cells as a specific feature of patients with type 1 diabetes

Identifying beta cell autoantigen-reactive T cells that are involved in the pathogenesis of type 1 diabetes has been troublesome for many laboratories. Disease-relevant autoreactive T cells should be in vivo Ag experienced. The aim of this study was to test this hypothesis and then use this principle as a strategy for identifying diabetes-relevant autoreactive T cells. In this study, a CSFE dilution assay was used to detect glutamic acid decarboxylase 65 (GAD65)- and insulin-responsive T cells and HLA-0201*-GAD65(114-122) pentamers were used to detect CD8(+) GAD-responsive T cells in memory CD45RO(+) and naive CD45RO(-) cell populations from patients with type 1 diabetes and healthy control subjects. T cell proliferative history was evaluated by flow cytometry telomere length measurement. CD4(+) and CD8(+) T cells specific for GAD65 and insulin were present in patients with type 1 diabetes and control subjects. Within the naive CD45RO(-) cells, CD4(+) and CD8(+) T cell responses were similar between patients and controls. Within the memory CD45RO(+) cells, CD4(+) T cell responses against whole GAD65 and insulin and HLA-0201*-GAD65(114-122) pentamer-positive CD8(+) T cells were found in patients with type 1 diabetes, but not in control subjects (p < 0.05 for all). Responding cells from the CD45RO(+) T cell population had substantially shorter telomere lengths than responding cells from the CD45RO(-) cell population. Diabetes-specific autoreactive T cells in the circulation have uniquely undergone sustained in vivo proliferation and differentiation into memory T cells. Prior selection of these cells is possible and is a way to identify diabetes-relevant target Ags and epitopes.     

Memory T cells constitute a subset of the human CD8+CD45RA+ pool with distinct phenotypic and migratory characteristics.

Using HLA class I-viral epitope tetramers to monitor herpes virus-specific CD8(+) T cell responses in humans, we have shown that a significant fraction of responding cells revert from a CD45RO(+) to a CD45RA(+) state after priming. All tetramer-binding CD45RA(+) cells, regardless of epitope specificity, expressed a phenotype LFA-1(high)CCR7(low) that was stable for at least 10 years in infectious mononucleosis patients and indefinitely in asymptomatic carriers. CD8(+)CD45RA(+)LFA-1(high) cells were not present in cord blood but in adults account for up to 50% of CD8(+)CD45RA(+) cells. These CD45RA(+)LFA-1(high) cells have significantly shorter telomeres than CD45RA(+)LFA-1(low) cells, suggesting that the latter represent a naive population, while the former are memory cells. CD45RA(+) memory cells are a stable population of noncycling cells, but on stimulation they are potent producers of IFN-gamma, while naive CD8(+) cells produce only IL-2. The chemokine receptor profile and migratory potential of CD45RA(+) memory cells is very similar to CD45RO(+) cells but different to naive CD8 cells. In accord with this, CD45RA(+) memory cells were significantly underrepresented in lymph nodes, but account for virtually all CD8(+)CD45RA(+) T cells in peripheral tissues of the same individuals.     

The duration of exposure to HIV modulates the breadth and the magnitude of HIV-specific memory CD4+ T cells

The impact of exposure to Ag on the development and maintenance of human CD4(+) memory T cells in general and HIV infection in particular is partially understood. In this study, we measured HIV-specific CD4(+) T cell proliferative responses against HIV proteins and derived peptides one year after highly active antiretroviral therapy initiation in 39 HIV-infected patients who initiated therapy at different times following infection. We show that a brief exposure to HIV of <1 month does not allow the generation of significant detectable frequencies of HIV-specific CD4(+) memory T cells. Patients having prolonged cumulative exposure to high viral load due to therapy failures also demonstrated limited HIV-specific CD4(+) T cell responses. In contrast, patients exposed to significant levels of virus for periods ranging from 3 to 18 mo showed brisk and broad HIV-specific CD4(+) T cell responses 1 year following the onset of therapy intervention. We also demonstrate that the nadir CD4(+) T cell count before therapy initiation correlated positively with the breadth and magnitude of these responses. Our findings indicate that the loss of proliferative HIV-specific CD4(+) T cell responses is associated with the systemic progression of the disease and that a brief exposure to HIV does not allow the establishment of detectable frequencies of HIV-specific memory CD4(+) T cells.     

Functional heterogeneity of vaccine-induced CD8(+) T cells

The functional status of circulating vaccine-induced, tumor-specific T cells has been questioned to explain their paradoxical inability to inhibit tumor growth. We enumerated with HLA-A*0201/peptide tetramers (tHLA) vaccine-elicited CD8(+) T cell precursor frequency among PBMC in 13 patients with melanoma undergoing vaccination with the HLA-A*0201-associated gp100:209-217(210 M) epitope. T cell precursor frequency increased from undetectable to 12,400 +/- 3,600 x 10(6) CD8(+) T cells after vaccination and appeared heterogeneous according to previously described functional subtypes: CD45RA(+)CD27(+) (14 +/- 2.6% of tHLA-staining T cells), naive; CD45RA(-)CD27(+) (14 +/- 3.2%), memory; CD45RA(+)CD27(-) (43 +/- 6%), effector; and CD45RA(-)CD27(-) (30 +/- 4.1%), memory/effector. The majority of tHLA(+)CD8(+) T cells displayed an effector, CD27(-) phenotype (73%). However, few expressed perforin (17%). Epitope-specific in vitro stimulation (IVS) followed by 10-day expansion in IL-2 reversed this phenotype by increasing the number of perforin(+) (84 +/- 3.6%; by paired t test, p < 0.001) and CD27(+) (from 28 to 67%; by paired t test, p = 0.01) tHLA(+) T cells. This conversion probably represented a change in the functional status of tHLA(+) T cells rather than a preferential expansion of a CD27(+) (naive and/or memory) PBMC, because it was reproduced after IVS of a T cell clone bearing a classic effector phenotype (CD45RA(+)CD27(-)). These findings suggest that circulating vaccine-elicited T cells are not as functionally active as inferred by characterization of IVS-induced CTL. In addition, CD45RA/CD27 expression may be more informative about the status of activation of circulating T cells than their status of differentiation.     

Phenotypic, functional, and kinetic parameters associated with apparent T-cell control of human immunodeficiency virus replication in individuals with and without antiretroviral treatment

The human immunodeficiency virus (HIV)-mediated immune response may be beneficial or harmful, depending on the balance between expansion of HIV-specific T cells and the level of generalized immune activation. The current study utilizes multicolor cytokine flow cytometry to study HIV-specific T cells and T-cell activation in 179 chronically infected individuals at various stages of HIV disease, including those with low-level viremia in the absence of therapy ("controllers"), low-level drug-resistant viremia in the presence of therapy (partial controllers on antiretroviral therapy [PCAT]), and high-level viremia ("noncontrollers"). Compared to noncontrollers, controllers exhibited higher frequencies of HIV-specific interleukin-2-positive gamma interferon-positive (IL-2(+) IFN-gamma(+)) CD4(+) T cells. The presence of HIV-specific CD4(+) IL-2(+) T cells was associated with low levels of proliferating T cells within the less-differentiated T-cell subpopulations (defined by CD45RA, CCR7, CD27, and CD28). Despite prior history of progressive disease, PCAT patients exhibited many immunologic characteristics seen in controllers, including high frequencies of IL-2(+) IFN-gamma(+) CD4(+) T cells. Measures of immune activation were lower in all CD8(+) T-cell subsets in controllers and PCAT compared to noncontrollers. Thus, control of HIV replication is associated with high levels of HIV-specific IL-2(+) and IFN-gamma(+) CD4(+) T cells and low levels of T-cell activation. This immunologic state is one where the host responds to HIV by expanding but not exhausting HIV-specific T cells while maintaining a relatively quiescent immune system. Despite a history of advanced HIV disease, a subset of individuals with multidrug-resistant HIV exhibit an immunologic profile comparable to that of controllers, suggesting that functional immunity can be reconstituted with partially suppressive highly active antiretroviral therapy.     

Human immunodeficiency virus type 1 envelope epitope-specific CD4(+) T lymphocytes in simian/human immunodeficiency virus-infected and vaccinated rhesus monkeys detected using a peptide-major histocompatibility complex class II tetramer

A tetrameric recombinant major histocompatibility complex (MHC) class II-peptide complex was used to quantitate human immunodeficiency virus type 1 (HIV-1) envelope (Env)-specific CD4(+) T cells in vaccinated and in simian/human immunodeficiency virus (SHIV)-infected rhesus monkeys. A rhesus monkey MHC class II DR molecule, Mamu-DR*W201, and an HIV-1 Env peptide (p46) were employed to construct this tetrameric complex. A p46-specific proliferative response was seen in sorted, tetramer-binding, but not nonbinding, CD4(+) T cells, directly demonstrating that this response was mediated by the epitope-specific lymphocytes. Although staining of whole blood from 10 SHIV-infected Mamu-DR*W201(+) rhesus monkeys failed to demonstrate tetramer-binding CD4(+) T cells (<0.02%), p46-stimulated peripheral blood mononuclear cells (PBMCs) from 9 of these 10 monkeys had detectable p46 tetramer-binding cells, comprising 0.5 to 15.2% of the CD4(+) T cells. p46-stimulated PBMCs from 7 of 10 Mamu-DR*W201(+) monkeys vaccinated with a recombinant canarypox virus-HIV-1 env construct also demonstrated p46 tetramer-binding cells, comprising 0.9 to 7.2% of the CD4(+) T cells. Thus, Env p46-specific CD4(+) T cells can be detected by tetrameric Mamu-DR*W201-p46 complex staining of PBMCs in both SHIV-infected and vaccinated rhesus monkeys. These epitope-specific cell populations appear to be present in peripheral blood at a very low frequency.     

A significant number of human immunodeficiency virus epitope-specific cytotoxic T lymphocytes detected by tetramer binding do not produce gamma interferon

Despite the seemingly important role of cytotoxic T-lymphocyte (CTL) responses in human immunodeficiency virus (HIV) disease pathogenesis, their measurement has relied on a variety of different techniques. We utilized three separate methodologies for the detection of CTLs in a cohort of HIV-infected individuals who were also human leukocyte antigen A2 (HLA-A2) positive. Among the different CTL assays, a correlation was seen only when the Gag epitope-specific HLA A*0201-restricted tetramer assay was compared with the ELISPOT assay performed after stimulation with the Gag epitope; however, this correlation was of borderline statistical significance. On average, the tetramer reagent detected a 10-fold-higher number of cells than were seen to produce gamma interferon by the ELISPOT assay. The implications of this CTL assay comparison and the possibility of phenotypic differences in HIV-specific CD8(+) T lymphocytes are discussed.     

Identification of major epitopes of Mycobacterium tuberculosis AG85B that are recognized by HLA-A*0201-restricted CD8+ T cells in HLA-transgenic mice and humans

CD8(+) T cells are thought to play an important role in protective immunity to tuberculosis. Although several nonprotein ligands have been identified for CD1-restricted CD8(+) CTLs, epitopes for classical MHC class I-restricted CD8(+) T cells, which most likely represent a majority among CD8(+) T cells, have remained ill defined. HLA-A*0201 is one of the most prevalent class I alleles, with a frequency of over 30% in most populations. HLA-A2/K(b) transgenic mice were shown to provide a powerful model for studying induction of HLA-A*0201-restricted immune responses in vivo. The Ag85 complex, a major component of secreted Mycobacterium tuberculosis proteins, induces strong CD4(+) T cell responses in M. tuberculosis-infected individuals, and protection against tuberculosis in Ag85-DNA-immunized animals. In this study, we demonstrate the presence of HLA class I-restricted, CD8(+) T cells against Ag85B of M. tuberculosis in HLA-A2/K(b) transgenic mice and HLA-A*0201(+) humans. Moreover, two immunodominant Ag85 peptide epitopes for HLA-A*0201-restricted, M. tuberculosis-reactive CD8(+) CTLs were identified. These CD8(+) T cells produced IFN-gamma and TNF-alpha and recognized Ag-pulsed or bacillus Calmette-Guérin-infected, HLA-A*0201-positive, but not HLA-A*0201-negative or uninfected human macrophages. This CTL-mediated killing was blocked by anti-CD8 or anti-HLA class I mAb. Using fluorescent peptide/HLA-A*0201 tetramers, Ag85-specific CD8(+) T cells could be visualized in bacillus Calmette-Guérin-responsive, HLA-A*0201(+) individuals. Collectively, our results demonstrate the presence of HLA class I-restricted CD8(+) CTL against a major Ag of M. tuberculosis and identify Ag85B epitopes that are strongly recognized by HLA-A*0201-restricted CD8(+) T cells in humans and mice. These epitopes thus represent potential subunit components for the design of vaccines against tuberculosis.     

Direct detection and magnetic isolation of Chlamydia trachomatis major outer membrane protein-specific CD8+ CTLs with HLA class I tetramers

We recently identified HLA class I-presented epitopes in the major outer membrane protein (MOMP) of Chlamydia trachomatis that elicit CTL responses in human genital tract infections. T cells possessing cytolytic activities specific for these epitopes could be detected following in vitro stimulation of peripheral blood CD8(+) T cells with peptides. In the present study we used HLA-A2 tetramers for detailed characterization of MOMP-specific CTL responses. Ex vivo tetramer analysis detected MOMP-specific T cells in the peripheral blood of infected individuals at significant frequencies (0.01-0.20% of CD8(+) T cells). After in vitro stimulation with peptides, the frequencies of MOMP peptide-specific T cells increased up to 2.34% of CD8(+) T cells in bulk cultures. In contrast, HLA-A2/MOMP tetramer-binding T cells were virtually undetectable in the peripheral blood from uninfected individuals, either ex vivo or after 3 wk of in vitro peptide stimulation of their T cells. Magnetically sorted, tetramer-bound T cells specifically lysed peptide-pulsed targets as well as C. trachomatis-infected epithelial cells with nearly 50-fold greater per cell efficiency than that of unsorted populations. This study provides conclusive evidence of in vivo induction of HLA class I-restricted CD8(+) CTL responses to C. trachomatis MOMP. Direct detection of these cells with tetramers will allow their further characterization without prior manipulation and facilitate monitoring of CTL responses during infections and in immunization trials with MOMP-based vaccines.     

Human CD8+ CTL specific for the mycobacterial major secreted antigen 85A

The role of CD8(+) CTL in protection against tuberculosis in human disease is unclear. In this study, we stimulated the peripheral blood mononuclear cells of bacillus Calmette-Guérin (BCG)-vaccinated individuals with live Mycobacterium bovis BCG bacilli to establish short-term cell lines and then purified the CD8(+) T cells. A highly sensitive enzyme-linked immunospot (ELISPOT) assay for single cell IFN-gamma release was used to screen CD8(+) T cells with overlapping peptides spanning the mycobacterial major secreted protein, Ag85A. Three peptides consistently induced a high frequency of IFN-gamma responsive CD8(+) T cells, and two HLA-A*0201 binding motifs, P(48-56) and P(242-250), were revealed within the core sequences. CD8(+) T cells responding to the 9-mer epitopes were visualized within fresh blood by ELISPOT using free peptide or by binding of HLA-A*0201 tetrameric complexes. The class I-restricted CD8(+) T cells were potent CTL effector cells that efficiently lysed an HLA-A2-matched monocyte cell line pulsed with peptide as well as autologous macrophages infected with Mycobacterium tuberculosis or recombinant vaccinia virus expressing the whole Ag85A protein. Tetramer assays revealed a 6-fold higher frequency of peptide-specific T cells than IFN-gamma ELISPOT assays, indicating functional heterogeneity within the CD8(+) T cell population. These results demonstrate a previously unrecognized, MHC class I-restricted, CD8(+) CTL response to a major secreted Ag of mycobacteria and supports the use of Ag85A as a candidate vaccine against tuberculosis.     

Identification of naive or antigen-experienced human CD8(+) T cells by expression of costimulation and chemokine receptors: analysis of the human cytomegalovirus-specific CD8(+) T cell response

Human CMV (HCMV) infection provides an informative model of how long term human CD8(+) T cell memory is maintained in the presence of Ag. To clarify the phenotypic identity of Ag-experienced human CD8(+) T cells in vivo, we determined the expression of costimulation and chemokine receptors on Ag-specific CD8(+) T cells by quantifying individual virus-specific clones in different cell populations using TCR clonotypic probing. In healthy HCMV carriers, expanded CD8(+) clones specific for either HCMV tegument protein pp65 or immediate-early protein IE72 are found in both CD45RO(high) cells and the subpopulation of CD45RA(high) cells that lack the costimulatory molecule CD28. In contrast to previous suggested models of CD8(+) T cell memory, we found that in healthy virus carriers highly purified CD28(-)CD45RA(high)CCR7(-) cells are not terminally differentiated, because following stimulation in vitro with specific HCMV peptide these cells underwent sustained clonal proliferation, up-regulated CD45RO and CCR5, and showed strong peptide-specific cytotoxic activity. In an individual with acute primary HCMV infection, HCMV pp65-specific CD8(+) T cells are predominantly CD28(-)CD45RO(high)CCR7(-). During convalescence, an increasing proportion of pp65-specific CD8(+) T cells were CD28(-)CD45RA(high)CCR7(-). We conclude that naive human CD8(+) T cells are CD28(+)CD45RA(high), express CCR7 but not CCR6, and are predominantly CD27(+) and L-selectin CD62 ligand-positive. The phenotype CD27(+)CD45RA(high) should not be used to identify naive human CD8(+) T cells, because CD27(+)CD45RA(high) cells also contain a significant subpopulation of CD28(-)CD27(+) Ag-experienced expanded clones. Thus CD8(+) T cell memory to HCMV is maintained by cells of expanded HCMV-specific clones that show heterogeneity of activation state and costimulation molecular expression within both CD45RO(high) and CD28(-)CD45RA(high) T cell pools.     

Breast milk-derived antigen-specific CD8+ T cells: an extralymphoid effector memory cell population in humans

Although mouse studies have demonstrated the presence of an effector memory population in nonlymphoid tissues, the phenotype of human CD8(+) T cells present in such compartments has not been characterized. Because of the relatively large number of CD8(+) T cells present in breast milk, we were able to characterize the phenotype of this cell population in HIV-infected and uninfected lactating women. CMV, influenza virus, EBV, and HIV-specific CD8(+) T cells as measured by the IFN-gamma ELISPOT and MHC class I tetramer staining were all present at greater frequencies in breast milk as compared with blood. Furthermore, a greater percentage of the breast milk CD8(+) T cells expressed the intestinal homing receptor, CD103, and the mucosal homing receptor CCR9. Breast milk T cells were predominantly CD45RO(+)HLADR(+) and expressed low levels of CD45RA, CD62L, and CCR7 consistent with an effector memory population. Conversely, T cells derived from blood were mainly characterized as central memory cells (CCR7(+)CD62L(+)). These results demonstrate a population of extralymphoid CD8(+) T cells with an effector memory phenotype in humans, which could contribute to enhanced local virologic control and the relative lack of HIV transmission via this route.     

Regulation of trafficking receptor expression in human forkhead box P3+ regulatory T cells

Forkhead Box P3(+) (FOXP3(+)) T cells are regulatory cells important for maintaining immune tolerance. While chemokine- and other homing-receptors are important for T cell migration, it has been unclear how they are regulated in FOXP3(+) T cells. We thoroughly investigated, ex vivo and in vitro, the regulation of chemokine receptor expression on human FOXP3(+) T cells in neonatal cord blood, adult peripheral blood, and tonsils. We found that human FOXP3(+) T cells undergo changes in trafficking receptors according to their stages of activation and differentiation. FOXP3(+) T cells are divided into CD45RA(+) (naive type) and CD45RO(+) (memory type) FOXP3(+) T cells in neonatal blood, adult blood, and tonsils. CD45RA(+)FOXP3(+) T cells mainly express lymphoid tissue homing receptors (CD62L, CCR7, and CXCR4), while CD45RO(+)FOXP3(+) T cells highly express both Th1 and Th2-associated trafficking receptors along with the lymphoid tissue homing receptors at reduced frequencies. Up-regulation of Th1/Th2-associated trafficking receptors begins with activation of CD45RA(+)FOXP3(+) T cells and is completed after their differentiation to CD45RO(+) cells. Some chemokine receptors such as CXCR5 and CXCR6 are preferentially expressed by many FOXP3(+) cells at a specific stage (CD69(+)CD45RO(+)) in tonsils. Our in vitro differentiation study demonstrated that CD45RA(+)FOXP3(+) T cells indeed undergo chemokine receptor switch from CD45RA(+) (secondary lymphoid tissue homing) to CD45RO(+) type (lymphoid and nonlymphoid tissue homing). The orderly regulation of trafficking receptors in FOXP3(+) T cells according to stages of differentiation and activation is potentially important for their tissue-specific migration and regulation of immune responses in humans.     

Unusually high frequencies of HIV-specific cytotoxic T lymphocytes in humans

CTL specific for the HIV belong to the CD8 subset of T lymphocytes, and their activity is restricted by class I HLA transplantation Ag. In this report, HIV-specific CTL and their precursor cells were quantified by limiting dilution analysis. CTL were recovered from the lungs, lymph nodes, and blood of asymptomatic seropositive carriers and of patients with AIDS. HIV was found to be very immunogenic. High frequencies of both HIV-specific CTL and CTL precursor cells were detected in infected individuals. These CTL killed autologous HIV-infected macrophages and T4 lymphoblasts. They also killed doubly transfected P815-A2-env-LAV mouse tumor cells, which express the human HLA-A2 gene and the HIV-1 env gene. In the longitudinal studies of two HIV-infected patients, CTL and CTL precursor cell frequencies decreased as the clinical and immunologic status of the patients deteriorated. Most surprisingly, PBL from seronegative donors also responded to HIV stimulation in vitro and generated large numbers of HLA-restricted, HIV-specific CTL.     

Decreased CXCR3+ CD8 T cells in advanced human immunodeficiency virus infection suggest that a homing defect contributes to cytotoxic T-lymphocyte dysfunction

To exert their cytotoxic function, cytotoxic T-lymphocytes (CTL) must be recruited into infected lymphoid tissue where the majority of human immunodeficiency virus (HIV) replication occurs. Normally, effector T cells exit lymph nodes (LNs) and home to peripheral sites of infection. How HIV-specific CTL migrate into lymphoid tissue from which they are normally excluded is unknown. We investigated which chemokines and receptors mediate this reverse homing and whether impairment of this homing could contribute to CTL dysfunction as HIV infection progresses. Analysis of CTL chemokine receptor expression in the blood and LNs of untreated HIV-infected individuals with stable, chronic infection or advanced disease demonstrated that LNs were enriched for CXCR3(+) CD8 T cells in all subjects, suggesting a key role for this receptor in CTL homing to infected lymphoid tissue. Compared to subjects with chronic infection, however, subjects with advanced disease had fewer CXCR3(+) CD8 T cells in blood and LNs. CXCR3 expression on bulk and HIV-specific CD8 T cells correlated positively with CD4 count and negatively with viral load. In advanced infection, there was an accumulation of HIV-specific CD8 T cells at the effector memory stage; however, decreased numbers of CXCR3(+) CD8 T cells were seen across all maturation subsets. Plasma CXCL9 and CXCL10 were elevated in both infected groups in comparison to the levels in uninfected controls, whereas lower mRNA levels of CXCR3 ligands and CD8 in LNs were seen in advanced infection. These data suggest that both CXCR3(+) CD8 T cells and LN CXCR3 ligands decrease as HIV infection progresses, resulting in reduced homing of CTL into LNs and contributing to immune dysfunction.     

Trafficking of human immunodeficiency virus type 1-specific CD8+ T cells to gut-associated lymphoid tissue during chronic infection

Gut-associated lymphoid tissue (GALT) is a significant but understudied lymphoid organ, harboring a majority of the body's total lymphocyte population. GALT is also an important portal of entry for human immunodeficiency virus (HIV), a major site of viral replication and CD4(+) T-cell depletion, and a frequent site of AIDS-related opportunistic infections and neoplasms. However, little is known about HIV-specific cell-mediated immune responses in GALT. Using lymphocytes isolated from rectal biopsies, we have determined the frequency and phenotype of HIV-specific CD8(+) T cells in human GALT. GALT CD8(+) T cells were predominantly CD45RO(+) and expressed CXCR4 and CCR5. In 10 clinically stable, chronically infected individuals, the frequency of HIV Gag (SL9)-specific CD8(+) T cells was increased in GALT relative to peripheral blood mononuclear cells by up to 4.6-fold, while that of cytomegalovirus (CMV)-specific CD8(+) T cells was significantly reduced (P = 0.012). Both HIV- and CMV-specific CD8(+) T cells in GALT expressed CCR5, but only HIV-specific CD8(+) T cells expressed alpha E beta 7 integrin, suggesting that mucosal priming may account for their retention in GALT. Chronically infected individuals exhibited striking depletion of GALT CD4(+) T cells expressing CXCR4, CCR5, and alpha E beta 7 integrin, but CD4(+)/CD8(+) T-cell ratios in blood and GALT were similar. The percentage of GALT CD8(+) T cells expressing alpha E beta 7 was significantly decreased in infected individuals, suggesting that HIV infection may perturb lymphocyte retention in GALT. These studies demonstrate the feasibility of using tetramers to assess HIV-specific T cells in GALT and reveal that GALT is the site of an active CD8(+) T-cell response during chronic infection.