Differential Expression of Activation Markers by Mycobacterium tuberculosis-specific CD4+ T Cell Distinguishes Extrapulmonary From Pulmonary Tuberculosis and Latent Infection.

BACKGROUND: Diagnosis of active tuberculosis (ATB) currently relies on detection of Mycobacterium tuberculosis (Mtb). Identifying patients with extrapulmonary TB (EPTB) remains challenging because microbiological confirmation is often not possible. Highly accurate blood-based tests could improve diagnosis of both EPTB and pulmonary TB (PTB) and timely initiation of anti-TB therapy. METHODS: A case-control study was performed using discriminant analyses to validate an approach using Mtb-specific CD4+T-cell activation markers in blood to discriminate PTB and EPTB from latent TB infection (LTBI) as well as EPTB from PTB in 270 Brazilian individuals. We further tested the effect of human immunodeficiency virus (HIV) coinfection on diagnostic performance. Frequencies of interferon-γ +CD4+T cells expressing CD38, HLADR, and/or Ki67 were assessed by flow cytometry. RESULTS: EPTB and PTB were associated with higher frequencies of CD4+T cells expressing CD38, HLADR, or Ki67 compared with LTBI (all P values < .001). Moreover, frequencies of HLADR+ (P = .03) or Ki67+ (P < .001) cells accurately distinguished EPTB from PTB. HIV infection did not affect the capacity of these markers to distinguish ATB from LTBI or EPTB from PTB. CONCLUSIONS: Cell activation markers in Mtb-specific CD4+T cells distinguished ATB from LTBI and EPTB from PTB, regardless of HIV infection status. These parameters provide an attractive approach for developing blood-based diagnostic tests for both active and latent TB.

High Frequencies of Caspase-3 Expressing Mycobacterium tuberculosis-Specific CD4+ T Cells Are Associated With Active Tuberculosis.

Antigen-specific CD4+ T cell responses to Mycobacterium tuberculosis (Mtb) infection are important for host defense against tuberculosis (TB). However, Mtb-specific IFN-γ-producing T cells do not distinguish active tuberculosis (ATB) patients from individuals with asymptomatic latent Mtb infection (LTBI). We reasoned that the immune phenotype of Mtb-specific IFN-γ+CD4+ T cells could provide an indirect gauge of Mtb antigen load within individuals. We sought to identify immune markers in Mtb-specific IFN-γ+CD4+ T cells and hypothesized that expression of caspase-3 Mtb-specific CD4+ T cells would be associated with ATB. Using polychromatic flow cytometry, we evaluated the expression of caspase-3 in Mtb-specific CD4+ T cells from LTBI and ATB as well as from ATB patients undergoing anti-TB treatment. We found significantly higher frequencies of Mtb-specific caspase-3+IFN-γ+CD4+ T cells in ATB compared to LTBI. Caspase-3+IFN-γ+CD4+ T cells were also more activated compared to their caspase-3-negative counterparts. Furthermore, the frequencies of caspase-3+IFN-γ+CD4+ T cells decreased in response to anti-TB treatment. Our studies suggest that the frequencies of caspase-3-expressing antigen-specific CD4+ T cells may reflect mycobacterial burden in vivo and may be useful for distinguishing Mtb infection status along with other host biomarkers.

CD4+ Foxp3+ T cells promote aberrant immunoglobulin G production and maintain CD8+ T-cell suppression during chronic liver disease.

Persistent hepatotropic viral infections are a common etiologic agent of chronic liver disease. Unresolved infection can be attributed to nonfunctional intrahepatic CD8+ T-cell responses. In light of dampened CD8+ T-cell responses, liver disease often manifests systemically as immunoglobulin (Ig)-related syndromes due to aberrant B-cell functions. These two opposing yet coexisting phenomena implicate the potential of altered CD4+ T-cell help. Elevated CD4+ forkhead box P3-positive (Foxp3+) T cells were evident in both human liver disease and a mouse model of chemically induced liver injury despite marked activation and spontaneous IgG production by intrahepatic B cells. While this population suppressed CD8+ T-cell responses, aberrant B-cell activities were maintained due to expression of CD40 ligand on a subset of CD4+ Foxp3+ T cells. In vivo blockade of CD40 ligand attenuated B-cell abnormalities in a mouse model of liver injury. A phenotypically similar population of CD4+ Foxp3+, CD40 ligand-positive T cells was found in diseased livers explanted from patients with chronic hepatitis C infection. This population was absent in nondiseased liver tissues and peripheral blood. CONCLUSION: Liver disease elicits alterations in the intrahepatic CD4+ T-cell compartment that suppress T-cell immunity while concomitantly promoting aberrant IgG mediated manifestations. (Hepatology 2017;65:661-677).

INTIMATE PARTNER VIOLENCE IS ASSOCIATED WITH INCREASED CD4+ T-CELL ACTIVATION AMONG HIV-NEGATIVE HIGH-RISK WOMEN.

BACKGROUND: Biological pathways mediating the link between intimate partner violence (IPV) and increased HIV risk remain unexplored. We hypothesized that IPV-induced stress negatively affects HIV systemic immune defenses and aimed to evaluate whether IPV was associated with immune profiles linked to HIV susceptibility: CD4 activation and diminished regulatory T-cell (Treg) frequency. METHODS: Seventy-five HIV-negative high-risk women were surveyed regarding their IPV experience. They provided blood, urine, and (if present) genital ulcer samples for cortisol, immune assays, and STI testing. Using flow cytometry, we assessed activated CD4+ T-cell (%HLA-DR+/CD38+) and Treg (%CD4+CD25+FoxP3+) frequencies and phenotyping. Nonparametric tests evaluated the association between IPV and immune outcomes. Multivariate regression explored confounding and moderation of the IPV-CD4 activation pathway. RESULTS: Lifetime IPV was associated with increased CD4+ activation (r = 0.331, P = 0.004), a shift in CD4+ phenotype from naïve to effector memory (r = 0.343, P = 0.003), and a decrease in naive (%HLA-DR+/CD45RA-) Treg frequency (r = -0.337, P = 0.003). Experiencing IPV over the past year had similar trends. After controlling for sexual IPV, lifetime physical and psychological abuse remained significantly associated with CD4+ activation (P = 0.004 and P = 0.033, respectively). After controlling for race (the only covariate linked to activation), the lifetime IPV-CD4 activation association remained significant (P = 0.012). Alcohol use and depression were identified as potential pathway moderators. CONCLUSION: Our data is the first to suggest an immune link between IPV and HIV, and may help explain differences at the individual level in HIV susceptibility and response to biological HIV prevention strategies. The association of psychological and physical abuse with CD4 activation independent of sexual abuse further supports the existence of a stress-induced immune pathway.

Induction of Th1-Biased T Follicular Helper (Tfh) Cells in Lymphoid Tissues during Chronic Simian Immunodeficiency Virus Infection Defines Functionally Distinct Germinal Center Tfh Cells.

Chronic HIV infection is associated with accumulation of germinal center (GC) T follicular helper (Tfh) cells in the lymphoid tissue. The GC Tfh cells can be heterogeneous based on the expression of chemokine receptors associated with T helper lineages, such as CXCR3 (Th1), CCR4 (Th2), and CCR6 (Th17). However, the heterogeneous nature of GC Tfh cells in the lymphoid tissue and its association with viral persistence and Ab production during chronic SIV/HIV infection are not known. To address this, we characterized the expression of CXCR3, CCR4, and CCR6 on GC Tfh cells in lymph nodes following SIVmac251 infection in rhesus macaques. In SIV-naive rhesus macaques, only a small fraction of GC Tfh cells expressed CXCR3, CCR4, and CCR6. However, during chronic SIV infection, the majority of GC Tfh cells expressed CXCR3, whereas the proportion of CCR4(+) cells did not change, and CCR6(+) cells decreased. CXCR3(+), but not CXCR3(-), GC Tfh cells produced IFN-γ (Th1 cytokine) and IL-21 (Tfh cytokine), whereas both subsets expressed CD40L following stimulation. Immunohistochemistry analysis demonstrated an accumulation of CD4(+)IFN-γ(+) T cells within the hyperplastic follicles during chronic SIV infection. CXCR3(+) GC Tfh cells also expressed higher levels of ICOS, CCR5, and α4ß7 and contained more copies of SIV DNA compared with CXCR3(-) GC Tfh cells. However, CXCR3(+) and CXCR3(-) GC Tfh cells delivered help to B cells in vitro for production of IgG. These data demonstrate that chronic SIV infection promotes expansion of Th1-biased GC Tfh cells, which are phenotypically and functionally distinct from conventional GC Tfh cells and contribute to hypergammaglobulinemia and viral reservoirs.

Humoral and Cell-Mediated Immune Responses to Alternate Booster Schedules of Anthrax Vaccine Adsorbed in Humans.

Protective antigen (PA)-specific antibody and cell-mediated immune (CMI) responses to annual and alternate booster schedules of anthrax vaccine adsorbed (AVA; BioThrax) were characterized in humans over 43 months. Study participants received 1 of 6 vaccination schedules: a 3-dose intramuscular (IM) priming series (0, 1, and 6 months) with a single booster at 42 months (4-IM); 3-dose IM priming with boosters at 18 and 42 months (5-IM); 3-dose IM priming with boosters at 12, 18, 30, and 42 months (7-IM); the 1970 licensed priming series of 6 doses (0, 0.5, 1, 6, 12, and 18 months) and two annual boosters (30 and 42 months) administered either subcutaneously (SQ) (8-SQ) or IM (8-IM); or saline placebo control at all eight time points. Antibody response profiles included serum anti-PA IgG levels, subclass distributions, avidity, and lethal toxin neutralization activity (TNA). CMI profiles included frequencies of gamma interferon (IFN-γ)- and interleukin 4 (IL-4)-secreting cells and memory B cells (MBCs), lymphocyte stimulation indices (SI), and induction of IFN-γ, IL-2, IL-4, IL-6, IL-1ß, and tumor necrosis factor alpha (TNF-α) mRNA. All active schedules elicited high-avidity PA-specific IgG, TNA, MBCs, and T cell responses with a mixed Th1-Th2 profile and Th2 dominance. Anti-PA IgG and TNA were highly correlated (e.g., month 7,r(2)= 0.86,P< 0.0001, log10 transformed) and declined in the absence of boosters. Boosters administered IM generated the highest antibody responses. Increasing time intervals between boosters generated antibody responses that were faster than and superior to those obtained with the final month 42 vaccination. CMI responses to the 3-dose IM priming remained elevated up to 43 months. (This study has been registered at ClinicalTrials.gov under registration no. NCT00119067.).

Biomarkers on patient T cells diagnose active tuberculosis and monitor treatment response.

BACKGROUND: The identification and treatment of individuals with tuberculosis (TB) is a global public health priority. Accurate diagnosis of pulmonary active TB (ATB) disease remains challenging and relies on extensive medical evaluation and detection of Mycobacterium tuberculosis (Mtb) in the patient's sputum. Further, the response to treatment is monitored by sputum culture conversion, which takes several weeks for results. Here, we sought to identify blood-based host biomarkers associated with ATB and hypothesized that immune activation markers on Mtb-specific CD4+ T cells would be associated with Mtb load in vivo and could thus provide a gauge of Mtb infection. METHODS: Using polychromatic flow cytometry, we evaluated the expression of immune activation markers on Mtb-specific CD4+ T cells from individuals with asymptomatic latent Mtb infection (LTBI) and ATB as well as from ATB patients undergoing anti-TB treatment. RESULTS: Frequencies of Mtb-specific IFN-γ+CD4+ T cells that expressed immune activation markers CD38 and HLA-DR as well as intracellular proliferation marker Ki-67 were substantially higher in subjects with ATB compared with those with LTBI. These markers accurately classified ATB and LTBI status, with cutoff values of 18%, 60%, and 5% for CD38+IFN-γ+, HLA-DR+IFN-γ+, and Ki-67+IFN-γ+, respectively, with 100% specificity and greater than 96% sensitivity. These markers also distinguished individuals with untreated ATB from those who had successfully completed anti-TB treatment and correlated with decreasing mycobacterial loads during treatment. CONCLUSION: We have identified host blood-based biomarkers on Mtb-specific CD4+ T cells that discriminate between ATB and LTBI and provide a set of tools for monitoring treatment response and cure. TRIAL REGISTRATION: Registration is not required for observational studies. FUNDING: This study was funded by Emory University, the NIH, and the Yerkes National Primate Center.

Systems biological analyses reveal the hepatitis C virus (HCV)-specific regulation of hematopoietic development.

UNLABELLED: Chronic liver disease is characterized by the liver enrichment of myeloid dendritic cells (DCs). To assess the role of disease on myelopoiesis, we utilized a systems biology approach to study development in liver-resident cells expressing stem cell marker CD34. In patients with endstage liver disease, liver CD34+ cells were comprised of two subsets, designated CD34+CD146+ and CD34+CD146-, and hematopoietic function was restricted to CD34+CD146- cells. Liver CD34 frequencies were reduced during nonalcoholic steatohepatitis (NASH) and chronic hepatitis C virus (HCV) compared to alcohol liver disease (ALD), and this reduction correlated with viral load in the HCV cohort. To better understand the relationship between liver CD34+CD146+ and CD34+CD146- subsets and any effects of disease on CD34 development, we used gene expression profiling and computational modeling to compare each subset during ALD and HCV. For CD34+CD146+ cells, increased expression of endothelial cell genes including von Willebrand factor, VE-cadherin, and eNOS were observed when compared to CD34+CD146- cells, and minimal effects of ALD and HCV diseases on gene expression were observed. Importantly for CD34+CD146- cells, chronic HCV was associated with a distinct "imprint" of programs related to cell cycle, DNA repair, chemotaxis, development, and activation, with an emphasis on myeloid and B lymphocyte lineages. This HCV signature was further translated in side-by-side analyses, where HCV CD34+CD146- cells demonstrated superior hematopoietic growth, colony formation, and diversification compared to ALD and NASH when cultured identically. Disease-associated effects on hematopoiesis were also evident by phenotypic alterations in the expression of CD14, HLA-DR, and CD16 by myeloid progeny cells. CONCLUSION: Etiology drives progenitor fate within diseased tissues. The liver may be a useful source of hematopoietic cells for therapy, or as therapeutic targets.

Impaired degranulation and proliferative capacity of Mycobacterium tuberculosis-specific CD8+ T cells in HIV-infected individuals with latent tuberculosis.

Human immunodeficiency virus (HIV)-infected individuals with latent Mycobacterium tuberculosis infection have substantially higher rates of progression to active tuberculosis than HIV-uninfected individuals with latent tuberculosis. To explore HIV-induced deficits in M. tuberculosis-specific CD8+ T-cell functions, we compared interferon γ production, degranulation, and proliferation of CD8+ T cells in response to M. tuberculosis peptides (ESAT-6/CFP-10) between HIV-infected (median CD4+ T-cell count, 522 cells/µL; interquartile range, 318-585 cells/µL) and HIV-uninfected individuals with latent tuberculosis from South Africa. We found that M. tuberculosis-specific degranulation and proliferative capacities were impaired in the HIV-infected group. Thus, our results suggest that HIV coinfection compromises CD8+ T-cell functions in latent tuberculosis.

Role of intestinal mucosal integrity in HIV transmission to infants through breast-feeding: the BAN study.

BACKGROUND: Increased intestinal permeability may be one of the mechanisms of transmission of human immunodeficiency virus (HIV) to infants through breast-feeding. Intestinal permeability correlates with microbial translocation, which can be measured through quantification of bacterial lipopolysaccharide (LPS). METHODS: We evaluated levels of plasma LPS (by the Limulus amebocyte lysate assay) and immune activation markers in serial specimens from infants exposed to but uninfected with HIV and infants infected with HIV from the Breastfeeding, Antiretrovirals, and Nutrition (BAN) study. RESULTS: Plasma LPS levels increased after infants in the BAN study were weaned from the breast, at 24 weeks of age. Cotrimoxazole prophylaxis was associated with higher plasma LPS levels (P = .004). Infants with HIV infection had higher LPS levels, compared with uninfected infants (P = .004). Higher preinfection plasma LPS levels were a significant predictor of infant HIV infection through breast-feeding (hazard ratio = 1.60 for every unit increase in plasma LPS level; P = .01) and of lower infant length-for-age z scores (P = .02). CONCLUSIONS: These findings suggest that disruption in intestinal integrity is a mechanism of HIV transmission to infants through breast-feeding. Weaning from breast milk and use of antibiotic prophylaxis was associated with increased levels of microbial translocation, which could facilitate HIV entry through the intestine. Complementary approaches to enhance intestinal mucosal integrity in the infant may further reduce breast-feeding transmission of HIV.

A three-dose intramuscular injection schedule of anthrax vaccine adsorbed generates sustained humoral and cellular immune responses to protective antigen and provides long-term protection against inhalation anthrax in rhesus macaques.

A 3-dose (0, 1, and 6 months) intramuscular (3-IM) priming series of a human dose (HuAVA) and dilutions of up to 1:10 of anthrax vaccine adsorbed (AVA) provided statistically significant levels of protection (60 to 100%) against inhalation anthrax for up to 4 years in rhesus macaques. Serum anti-protective antigen (anti-PA) IgG and lethal toxin neutralization activity (TNA) were detectable following a single injection of HuAVA or 1:5 AVA or following two injections of diluted vaccine (1:10, 1:20, or 1:40 AVA). Anti-PA and TNA were highly correlated (overall r(2) = 0.89 for log(10)-transformed data). Peak responses were seen at 6.5 months. In general, with the exception of animals receiving 1:40 AVA, serum anti-PA and TNA responses remained significantly above control levels at 28.5 months (the last time point measured for 1:20 AVA), and through 50.5 months for the HuAVA and 1:5 and 1:10 AVA groups (P < 0.05). PA-specific gamma interferon (IFN-γ) and interleukin-4 (IL-4) CD4(+) cell frequencies and T cell stimulation indices were sustained through 50.5 months (the last time point measured). PA-specific memory B cell frequencies were highly variable but, in general, were detectable in peripheral blood mononuclear cells (PBMC) by 2 months, were significantly above control levels by 7 months, and remained detectable in the HuAVA and 1:5 and 1:20 AVA groups through 42 months (the last time point measured). HuAVA and diluted AVA elicited a combined Th1/Th2 response and robust immunological priming, with sustained production of high-avidity PA-specific functional antibody, long-term immune cell competence, and immunological memory (30 months for 1:20 AVA and 52 months for 1:10 AVA). Vaccinated animals surviving inhalation anthrax developed high-magnitude anamnestic anti-PA IgG and TNA responses.

Distinct effector memory CD4+ T cell signatures in latent Mycobacterium tuberculosis infection, BCG vaccination and clinically resolved tuberculosis.

Two billion people worldwide are estimated to be latently infected with Mycobacterium tuberculosis (Mtb) and are at risk for developing active tuberculosis since Mtb can reactivate to cause TB disease in immune-compromised hosts. Individuals with latent Mtb infection (LTBI) and BCG-vaccinated individuals who are uninfected with Mtb, harbor antigen-specific memory CD4(+) T cells. However, the differences between long-lived memory CD4(+) T cells induced by latent Mtb infection (LTBI) versus BCG vaccination are unclear. In this study, we characterized the immune phenotype and functionality of antigen-specific memory CD4(+) T cells in healthy BCG-vaccinated individuals who were either infected (LTBI) or uninfected (BCG) with Mtb. Individuals were classified into LTBI and BCG groups based on IFN-γ ELISPOT using cell wall antigens and ESAT-6/CFP-10 peptides. We show that LTBI individuals harbored high frequencies of late-stage differentiated (CD45RA(-)CD27(-)) antigen-specific effector memory CD4(+) T cells that expressed PD-1. In contrast, BCG individuals had primarily early-stage (CD45RA(-)CD27(+)) cells with low PD-1 expression. CD27(+) and CD27(-) as well as PD-1(+) and PD-1(-) antigen-specific subsets were polyfunctional, suggesting that loss of CD27 expression and up-regulation of PD-1 did not compromise their capacity to produce IFN-γ, TNF-α and IL-2. PD-1 was preferentially expressed on CD27(-) antigen-specific CD4(+) T cells, indicating that PD-1 is associated with the stage of differentiation. Using statistical models, we determined that CD27 and PD-1 predicted LTBI versus BCG status in healthy individuals and distinguished LTBI individuals from those who had clinically resolved Mtb infection after anti-tuberculosis treatment. This study shows that CD4(+) memory responses induced by latent Mtb infection, BCG vaccination and clinically resolved Mtb infection are immunologically distinct. Our data suggest that differentiation into CD27(-)PD-1(+) subsets in LTBI is driven by Mtb antigenic stimulation in vivo and that CD27 and PD-1 have the potential to improve our ability to evaluate true LTBI status.

Use of replication restricted recombinant vesicular stomatitis virus vectors for detection of antigen-specific T cells.

Detection of antigen-specific T cells at the single-cell level by ELISpot or flow cytometry techniques employing intracellular cytokine staining (ICS) is now an indispensable tool in many areas of immunology. When precisely mapped, optimal MHC-binding peptide epitopes are unknown, these assays use antigen in a variety of forms, including recombinant proteins, overlapping peptide sets representing one or more target protein sequences, microbial lysates, lysates of microbially-infected cells, or gene delivery vectors such as DNA expression plasmids or recombinant vaccinia or adenoviruses expressing a target protein of interest. Here we introduce replication-restricted, recombinant vesicular stomatitis virus (VSV) vectors as a safe, easy to produce, simple to use, and highly effective vector for genetic antigen delivery for the detection of human antigen-specific helper and cytotoxic T cells. To demonstrate the broad applicability of this approach, we have used these vectors to detect human T cell responses to the immunodominant pp65 antigen of human cytomegalovirus, individual segments of the yellow fever virus polyprotein, and to various influenza proteins.

Phenotype, function, and gene expression profiles of programmed death-1(hi) CD8 T cells in healthy human adults.

T cell dysfunction is an important feature of many chronic viral infections. In particular, it was shown that programmed death-1 (PD-1) regulates T cell dysfunction during chronic lymphocytic choriomeningitis virus infection in mice, and PD-1(hi) cells exhibit an intense exhausted gene signature. These findings were extended to human chronic infections such as HIV, hepatitis C virus, and hepatitis B virus. However, it is not known if PD-1(hi) cells of healthy humans have the traits of exhausted cells. In this study, we provide a comprehensive description of phenotype, function, and gene expression profiles of PD-1(hi) versus PD-1(lo) CD8 T cells in the peripheral blood of healthy human adults as follows: 1) the percentage of naive and memory CD8 T cells varied widely in the peripheral blood cells of healthy humans, and PD-1 was expressed by the memory CD8 T cells; 2) PD-1(hi) CD8 T cells in healthy humans did not significantly correlate with the PD-1(hi) exhausted gene signature of HIV-specific human CD8 T cells or chronic lymphocytic choriomeningitis virus-specific CD8 T cells from mice; 3) PD-1 expression did not directly affect the ability of CD8 T cells to secrete cytokines in healthy adults; 4) PD-1 was expressed by the effector memory compared with terminally differentiated effector CD8 T cells; and 5) finally, an interesting inverse relationship between CD45RA and PD-1 expression was observed. In conclusion, our study shows that most PD-1(hi) CD8 T cells in healthy adult humans are effector memory cells rather than exhausted cells.

Prior exposure to an attenuated Listeria vaccine does not reduce immunogenicity: pre-clinical assessment of the efficacy of a Listeria vaccine in the induction of immune responses against HIV.

BACKGROUND: We have evaluated an attenuated Listeria monocytogenes (Lm) candidate vaccine vector in nonhuman primates using a delivery regimen relying solely on oral vaccination. We sought to determine the impact of prior Lm vector exposure on the development of new immune responses against HIV antigens. FINDINGS: Two groups of rhesus macaques one Lm naive, the other having documented prior Lm vector exposures, were evaluated in response to oral inoculations of the same vector expressing recombinant HIV-1 Gag protein. The efficacy of the Lm vector was determined by ELISA to assess the generation of anti-Listerial antibodies; cellular responses were measured by HIV-Gag specific ELISpot assay. Our results show that prior Lm exposures did not diminish the generation of de novo cellular responses against HIV, as compared to Listeria-naïve monkeys. Moreover, empty vector exposures did not elicit potent antibody responses, consistent with the intracellular nature of Lm. CONCLUSIONS: The present study demonstrates in a pre-clinical vaccine model, that prior oral immunization with an empty Lm vector does not diminish immunogenicity to Lm-expressed HIV genes. This work underscores the need for the continued development of attenuated Lm as an orally deliverable vaccine.

Transient CD86 expression on hepatitis C virus-specific CD8+ T cells in acute infection is linked to sufficient IL-2 signaling.

Costimulatory signals via B7/CD28 family molecules (signal 2) are critical for effective adaptive CD8(+) T cell immune responses. In addition to costimulatory signals, B7/CD28 family coinhibitory receptor/ligands that modulate immune responses have been identified. In acute hepatitis C virus (HCV) infection, programmed death receptor 1, an inhibitory receptor in the CD28 family, is highly expressed on virus-specific CD8(+) T cells, yet vigorous immune responses often develop. We hypothesized that other costimulatory signals present during the acute phase of HCV infection would be important to counter this negative signaling. In this study, we found that CD86 was highly expressed on HCV-specific CD8(+) T cells early in acute HCV infection and was lost on transition to chronic HCV infection; the expression of CD86 was different from other activation markers, because expression was delayed after in vitro TCR stimulation and required sufficient IL-2 signaling; and HCV-specific CD8(+) T cells in the liver of patients with chronic HCV infection were highly activated (CD69, CD38, and HLA-DR expression), but only a minority expressed CD86 or showed evidence of recent IL-2 signaling (low basal phosphorylated STAT5), despite persistent viremia. Our study identified B7 ligand expression on HCV-specific CD8(+) T cells as a distinct marker of effective T cell stimulation with IL-2 signaling in acute HCV infection. Expression of costimulatory molecules, such as CD86, early in HCV infection may be essential in overcoming inhibitory signals from the high level of programmed death receptor 1 expression also seen at this phase of infection.

Differential expression of CD26 on virus-specific CD8(+) T cells during active, latent and resolved infection.

The hallmark of effective establishment of immune memory is the long-term memory cell that persists in the absence of antigen. To explore its characteristics, we investigated the differences between a resolved successful immune response, such as after influenza (flu) vaccination, and the state of chronic infection with persistent antigen, such as with cytomegalovirus (CMV), Epstein-Barr virus (EBV) or human immunodeficiency virus (HIV), which leads to defective T-cell memory. Immunophenotypic analyses using multi-parameter flow cytometry and tetramer technology identified a unique pattern of CD26(high) expression among influenza-specific CD8(+) T cells, but not among CD8(+) T cells specific for CMV, EBV (three different epitopes) or HIV. The median percentage of CD8(+) T cells expressing CD26 was 95.5% for influenza, but for cells specific for CMV, EBV and HIV it was 10.5%, 12%-19%, and 13.2%, respectively. These findings suggest that expression of CD26(high) may be a characteristic of a memory cell. CD26(high) expression correlates with expression of CD127, a marker of memory cells. Furthermore, CD26(high) cells can produce interleukin-2. These findings offer insight into the dynamics of T-cell differentiation, and they may offer a specific marker of a successfully developed memory CD8(+) T cell, that of CD26(high). This marker has the potential to be useful in studies of immune responses to infectious agents, and to new vaccine candidates.