Functional capacity of Mycobacterium tuberculosis-specific T cell responses in humans is associated with mycobacterial load

High Ag load in chronic viral infections has been associated with impairment of Ag-specific T cell responses; however, the relationship between Ag load in chronic Mycobacterium tuberculosis infection and functional capacity of M. tuberculosis-specific T cells in humans is not clear. We compared M. tuberculosis-specific T cell-associated cytokine production and proliferative capacity in peripheral blood from adults with progressively higher mycobacterial loads-that is, persons with latent M. tuberculosis infection (LTBI), with smear-negative pulmonary tuberculosis (TB), and smear-positive TB. Patients with smear-positive TB had decreased polyfunctional IFN-γ(+)IL-2(+)TNF-α(+) and IL-2-producing specific CD4 T cells and increased TNF-α single-positive cells, when compared with smear-negative TB and LTBI. TB patients also had increased frequencies of M. tuberculosis-specific CD8 T cells, compared with LTBI. M. tuberculosis-specific CD4 and CD8 T cell proliferative capacity was profoundly impaired in individuals with smear-positive TB, and correlated positively with ex vivo IFN-γ(+)IL-2(+)TNF-α(+) CD4 T cells, and inversely with TNF-α single-positive CD4 T cells. During 6 mo of anti-TB treatment, specific IFN-γ(+)IL-2(+)TNF-α(+) CD4 and CD8 T cells increased, whereas TNF-α and IFN-γ single-positive T cells decreased. These results suggest progressive impairment of M. tuberculosis-specific T cell responses with increasing mycobacterial load and recovery of responses during therapy. Furthermore, these data provide a link between specific cytokine-producing subsets and functional capacity of M. tuberculosis-specific T cells, and between the presence of specific CD8 T cells ex vivo and active TB disease. These data have potentially significant applications for the diagnosis of TB and for the identification of T cell correlates of TB disease progression.     

The duration of antigen-stimulation significantly alters the diversity of multifunctional CD4 T cells measured by intracellular cytokine staining

The assessment of antigen-specific T cell responses by intracellular cytokine staining (ICS) has become a routine technique in studies of vaccination and immunity. Here, we highlight how the duration of in vitro antigen pre-stimulation, combined with the cytokine accumulation period, are critical parameters of these methods. The effect of varying these parameters upon the diversity and frequency of multifunctional CD4 T cell subsets has been investigated using a murine model of TB vaccination and in cattle naturally infected with Mycobacterium bovis. We demonstrate a substantial influence of the duration of the antigen pre-stimulation period on the repertoire of the antigen-specific CD4 T cell responses. Increasing pre-stimulation from 2 to 6 hours amplified the diversity of the seven potential multifunctional CD4 T cell subsets that secreted any combination of IFN-γ, IL-2 and TNF-α. However, increasing pre-stimulation from 6 to 16 hours markedly altered the multifunctional CD4 T cell repertoire to a dominant IFN-γ(+) only response. This was observed in both murine and cattle models.Whilst these data are of particular relevance to the measurement of vaccine and infection induced immunity in TB, more generally, they demonstrate the importance of the empirical determination of the optimum duration of the individual culture steps of ICS assays for any model. We highlight the potential significance of variations in these parameters, particularly when comparing data between studies and/or models including clinical trials.     

Identification of Mycobacterium tuberculosis-specific Th1, Th17 and Th22 cells using the expression of CD40L in tuberculous pleurisy

Important advances have been made in the immunodiagnosis of tuberculosis (TB) based on the detection of Mycobacterium tuberculosis (MTB)-specific T cells. However, the sensitivity and specificity of the immunological approach are relatively low because there are no specific markers for antigen-specific Th cells, and some of the Th cells that do not produce cytokines can be overlooked using this approach. In this study, we found that MTB-specific peptides of ESAT-6/CFP-10 can stimulate the expression of CD40L specifically in CD4(+) T cells but not other cells from pleural fluid cells (PFCs) in patients with tuberculous pleurisy (TBP). CD4(+)CD40L(+) but not CD4(+)CD40L(-) T cells express IFN-γ, IL-2, TNF-α, IL-17 or IL-22 after stimulation with MTB-specific peptides. In addition, CD4(+)CD40L(+) T cells were found to be mostly polyfunctional T cells that simultaneously produce IFN-γ, IL-2 and TNF-α and display an effector or effector memory phenotype (CD45RA(-)CD45RO(+)CCR7(-)CD62L(-)ICOS(-)). To determine the specificity of CD4(+)CD40L(+) T cells, we incubated PFCs with ESTA-6/CFP-10 peptides and sorted live CD4(+)CD40L(+) and CD4(+)CD40L(-) T cells by flow cytometry. We further demonstrated that sorted CD4(+)CD40L(+), but not CD4(+)CD40L(-) fractions, principally produced IFN-γ, IL-2, TNF-α, IL-17 and IL-22 following restimulation with ESTA-6/CFP-10 peptides. Taken together, our data indicate that the expression of CD40L on MTB-specific CD4(+) T cells could be a good marker for the evaluation and isolation of MTB-specific Th cells and might also be useful in the diagnosis of TB.     

Identification of M. tuberculosis-specific Th1 cells expressing CD69 generated in vivo in pleural fluid cells from patients with tuberculous pleurisy

Th1 cell-mediated immune responses at the site of active infection are important to restrict the growth of M. tuberculosis (MTB) and for the spontaneous resolution of patients with tuberculous pleurisy (TBP). In the present study, we found that without any stimulation, CD4(+) T cells in pleural fluid cells (PFCs) from patients with TBP expressed significantly higher levels of CD69 than PBMCs from patients with tuberculosis (TB) or healthy donors. CD4(+)CD69(+) T cells expressed T-bet and IL-12Rβ2. After stimulation with MTB-specific antigens, CD4(+)CD69(+) T cells expressed significantly higher levels of IFN-γ, IL-2 and TNF-α than CD4(+)CD69(-) T cells, demonstrating that CD4(+)CD69(+) T cells were MTB-specific Th1 cells. In addition, CD4(+)CD69(+) T cells were mostly polyfunctional Th1 cells that simultaneously produced IFN-γ, IL-2, TNF-α and displayed an effector or effector memory phenotype (CD45RA(-)CCR7(-)CD62L(-)CD27(-)). Moreover, the percentages of CD4(+)CD69(+) T cells were significantly and positively correlated with polyfunctional T cells. Interestingly, sorted CD4(+)CD69(+) but not CD4(+)CD69(-) fractions by flow cytometry produced IFN-γ, IL-2 and TNF-α that were significantly regulated by CD4(+)CD25(+) Treg cells. Taken together, based on the expression of CD69, we found a direct quantitative and qualitative method to detect and evaluate the in vivo generated MTB-specific polyfunctional CD4(+) T cells in PFCs from patients with TBP. This method can be used for the potential diagnosis and enrichment or isolation of MTB-specific Th1 cells in the investigations.     

Blockade of CTLA-4 decreases the generation of multifunctional memory CD4+ T cells in vivo

CTLA-4 is known as a central inhibitor of T cell responses. It terminates T cell activation and proliferation and induces resistance against activation induced cell death. However, its impact on memory formation of adaptive immune responses is still unknown. In this study, we demonstrate that although anti-CTLA-4 mAb treatment during primary immunization of mice initially enhances the number of IFN-γ-producing CD4(+) T cells, it does not affect the size of the memory pool. Interestingly, we find that the CTLA-4 blockade modulates the quality of the memory pool: it decreases the amount of specialized "multifunctional" memory CD4(+) T cells coproducing IFN-γ, TNF-α, and IL-2 in response to Ag. The reduction of these cells causes an immense decrease of IFN-γ-producing T cells after in vivo antigenic rechallenge. Chimeric mice expressing CTLA-4-competent and -deficient cells unmask, which these CTLA-4-driven mechanisms are mediated CD4(+) T cell nonautonomously. In addition, the depletion of CD25(+) T cells prior to the generation of Ag-specific memory cells reveals that the constitutively CTLA-4-expressing natural regulatory T cells determine the quality of memory CD4(+) T cells. Taken together, these results indicate that although the inhibitory molecule CTLA-4 damps the primary immune response, its engagement positively regulates the formation of a high-quality memory pool equipped with multifunctional CD4(+) T cells capable of mounting a robust response to Ag rechallenge.     

Knockdown of HMGB1 in tumor cells attenuates their ability to induce regulatory T cells and uncovers naturally acquired CD8 T cell-dependent antitumor immunity

Although high mobility group box 1 (HMGB1) in tumor cells is involved in many aspects of tumor progression, its role in tumor immune suppression remains elusive. Host cell-derived IL-10 suppressed a naturally acquired CD8 T cell-dependent antitumor response. The suppressive activity of tumor-associated Foxp3(+)CD4(+)CD25(+) regulatory T cells (Treg) was IL-10 dependent. Neutralizing HMGB1 impaired tumor cell-promoted IL-10 production by Treg. Short hairpin RNA-mediated knockdown of HMGB1 (HMGB1 KD) in tumor cells did not affect tumor cell growth but uncovered naturally acquired long-lasting tumor-specific IFN-γ- or TNF-α-producing CD8 T cell responses and attenuated their ability to induce Treg, leading to naturally acquired CD8 T cell- or IFN-γ-dependent tumor rejection. The data suggest that tumor cell-derived HMGB1 may suppress naturally acquired CD8 T cell-dependent antitumor immunity via enhancing Treg to produce IL-10, which is necessary for Treg-mediated immune suppression.     

Protective immunity induced with the RTS,S/AS vaccine is associated with IL-2 and TNF-α producing effector and central memory CD4 T cells

A phase 2a RTS,S/AS malaria vaccine trial, conducted previously at the Walter Reed Army Institute of Research, conferred sterile immunity against a primary challenge with infectious sporozoites in 40% of the 80 subjects enrolled in the study. The frequency of Plasmodium falciparum circumsporozoite protein (CSP)-specific CD4(+) T cells was significantly higher in protected subjects as compared to non-protected subjects. Intrigued by these unique vaccine-related correlates of protection, in the present study we asked whether RTS,S also induced effector/effector memory (T(E/EM)) and/or central memory (T(CM)) CD4(+) T cells and whether one or both of these sub-populations is the primary source of cytokine production. We showed for the first time that PBMC from malaria-non-exposed RTS,S-immunized subjects contain both T(E/EM) and T(CM) cells that generate strong IL-2 responses following re-stimulation in vitro with CSP peptides. Moreover, both the frequencies and the total numbers of IL-2-producing CD4(+) T(E/EM) cells and of CD4(+) T(CM) cells from protected subjects were significantly higher than those from non-protected subjects. We also demonstrated for the first time that there is a strong association between the frequency of CSP peptide-reactive CD4(+) T cells producing IL-2 and the titers of CSP-specific antibodies in the same individual, suggesting that IL-2 may be acting as a growth factor for follicular Th cells and/or B cells. The frequencies of CSP peptide-reactive, TNF-α-producing CD4(+) T(E/EM) cells and of CD4(+) T(E/EM) cells secreting both IL-2 and TNF-α were also shown to be higher in protected vs. non-protected individuals. We have, therefore, demonstrated that in addition to TNF-α, IL-2 is also a significant contributing factor to RTS,S/AS vaccine induced immunity and that both T(E/EM) and T(CM) cells are major producers of IL-2.     

Multifunctional CD4 cells expressing gamma interferon and perforin mediate protection against lethal influenza virus infection

CD4 effectors generated in vitro can promote survival against a highly pathogenic influenza virus via an antibody-independent mechanism involving class II-restricted, perforin-mediated cytotoxicity. However, it is not known whether CD4 cells activated during influenza virus infection can acquire cytolytic activity that contributes to protection against lethal challenge. CD4 cells isolated from the lungs of infected mice were able to confer protection against a lethal dose of H1N1 influenza virus A/Puerto Rico 8/34 (PR8). Infection of BALB/c mice with PR8 induced a multifunctional CD4 population with proliferative capacity and ability to secrete interleukin-2 (IL-2) and tumor necrosis factor alpha (TNF-α) in the draining lymph node (DLN) and gamma interferon (IFN-γ) and IL-10 in the lung. IFN-γ-deficient CD4 cells produced larger amounts of IL-17 and similar levels of TNF-α, IL-10, and IL-2 compared to wild-type (WT) CD4 cells. Both WT and IFN-γ(-/-) CD4 cells exhibit influenza virus-specific cytotoxicity; however, IFN-γ-deficient CD4 cells did not promote recovery after lethal infection as effectively as WT CD4 cells. PR8 infection induced a population of cytolytic CD4 effectors that resided in the lung but not the DLN. These cells expressed granzyme B (GrB) and required perforin to lyse peptide-pulsed targets. Lethally infected mice given influenza virus-specific CD4 cells deficient in perforin showed greater weight loss and a slower time to recovery than mice given WT influenza virus-specific CD4 cells. Taken together, these data strengthen the concept that CD4 T cell effectors are broadly multifunctional with direct roles in promoting protection against lethal influenza virus infection.     

Long-term control of simian immunodeficiency virus mac251 viremia to undetectable levels in half of infected female rhesus macaques nasally vaccinated with simian immunodeficiency virus DNA/recombinant modified vaccinia virus Ankara

The efficacy of two SIV DNA plus recombinant modified vaccinia virus Ankara nasal vaccine regimens, one combined with plasmids expressing IL-2 and IL-15, the other with plasmids expressing GM-CSF, IL-12, and TNF-α, which may better stimulate humoral responses, was evaluated in two female rhesus macaque groups. Vaccination stimulated significant SIV-specific mucosal and systemic cell-mediated immunity in both groups, whereas SIV-specific IgA titers were sporadic and IgG titers negative. All vaccinated animals, except one, became infected after intravaginal SIV(mac251) low-dose challenge. Half of the vaccinated, infected animals (7/13) promptly controlled virus replication to undetectable viremia for the duration of the trial (130 wk) and displayed virological and immunological phenotypes similar to those of exposed, uninfected individuals. When all vaccinated animals were considered, a 3-log viremia reduction was observed, compared with controls. The excellent viral replication containment achieved in vaccinated animals translated into significant preservation of circulating α4β7(high+)/CD4(+) T cells and of circulating and mucosal CD4(+)/C(M) T cells and in reduced immune activation. A more significant long-term survival was also observed in these animals. Median survival was 72 wk for the control group, whereas >50% of the vaccinated animals were still disease free 130 wk postchallenge, when the trial was closed. There was a statistically significant correlation between levels of CD4(+)/IFN-γ(+) and CD8(+)/IFN-γ(+) T cell percentages on the day of challenge and the control of viremia at week 60 postchallenge or survival. Postchallenge immunological correlates of protection were systemic anti-SIV Gag + Env CD4(+)/IL-2(+), CD4(+)/IFN-γ(+), and CD8(+)/TNF-α(+) T cells and vaginal anti-SIV Gag + Env CD8(+) T cell total monofunctional responses.     

Extracellular purine metabolism and signaling of CD73-derived adenosine in murine Treg and Teff cells

CD73-derived adenosine acts as potent inhibitor of inflammation, and regulatory T cells (Treg) have been shown to express CD73 as a novel marker. This study explored the role of endogenously formed adenosine in modulating NF-κB activity and cytokine/chemokine release from murine Treg and effector T cells (Teff) including key enzymes/purinergic receptors of extracellular ATP catabolism. Stimulating murine splenocytes and CD4(+) T cells with anti-CD3/anti-CD28 significantly upregulated activated NF-κB in CD73(-/-) T cells (wild type: 4.36 ± 0.21; CD73(-/-): 6.58 ± 0.75; n = 4; P = 0.029). This was associated with an augmented release of proinflammatory cytokines IL-2, TNF-α, and IFN-γ. Similar changes were observed with the CD73 inhibitor APCP (50 μM) on NF-κB and IFN-γ in wild-type CD4(+) T-cells. Treatment of stimulated CD4(+) T-cells with adenosine (25 μM) potently reduced IFN-γ release which is mediated by adenosine A2a receptors (A2aR). AMP (50 μM) also reduced cytokine release which was not inhibited by APCP. In Teff, A2aR activation (CGS21680) potently inhibited the release of IL-1, IL-2, IL-3, IL-4, IL-12, IL-13, IFN-γ, TNF-α, granulocyte-macrophage colony-stimulating factor (GM-CSF), CCL3, and CCL4. However, in Treg, CGS21680 did not alter cytokine/chemokine release. In summary, CD73-derived adenosine tonically inhibits active NF-κB in CD4(+) T-cells, thereby modulating the release of a broad spectrum of proinflammatory cytokines and chemokines. Downregulation of P2X7 and upregulation of CD73 in Treg after antigenic stimulation may be an important mechanism to maintain the ability of Treg to generate immunosuppressive adenosine.     

Interleukin 24 as a novel potential cytokine immunotherapy for the treatment of Mycobacterium tuberculosis infection

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) remains a major global health problem. Interleukin 24 (IL-24) is a novel tumor suppressor and a unique member of the IL-10 family of cytokines. However, the in vivo immunological consequences of this cytokine's activity during Mtb infection are still unknown. We found that IL-24 concentration was significantly decreased in the sera of TB patients, and Mtb infection suppressed IL-24 expression of human peripheral blood mononuclear cells (PBMCs) in vitro. Furthermore, we used a mouse infection model utilizing the virulent Mtb H37Rv strain to demonstrate that the administration of exogenous IL-24 had a protective effect against the bacteria. We found that IL-24 could activate human CD8(+) T cells, driving CD8(+) T cells to produce interferon-γ (IFN-γ) and counteract TB. This activity was found to be dependent on early involvement of neutrophils in the mouse model. IL-24 strongly stimulated IFN-γ production mainly by signaling through the IL-24 receptors of human CD8(+) T cells. IL-12 secretion from neutrophils in response to IL-24 treatment might be a minor factor in activating human CD8(+) T cells to secrete IFN-γ. Suppression of IL-24 expression by Mtb infection might enhance susceptibility to infection and promote the development of chronic TB. This new information could potentially stimulate the development of a new cytokine-based immunotherapeutic approach using IL-24 to stimulate immunity against TB.     

The relationship of cytomegalovirus (CMV) infection with circulatory IFN-α levels and IL-7 receptor α expression on CD8+ T cells in human aging

The IL-7 receptor alpha (IL-7Rα) is the high affinity receptor for IL-7 which is essential for T cell homeostasis. We recently reported an age-associated expansion of human effector memory (EM) CD8(+) T cells expressing IL-7Rα low (IL-7Rα(low)), which could be detrimental to hosts by occupying "immunological space". We investigated the potential mechanisms for this phenomenon, focusing on cytomegalovirus (CMV) infection and INF-α. In the elderly (age ≥ 65), CMV infection was associated with a decreased frequency of na?ve CD8(+) T cells as well as with an increased frequency of total EM and IL-7Rα(low) EM CD8(+) T cells. However, in the young (age ≤ 40), this viral infection was associated only with an increased frequency of IL-7Rα(low) EM CD8(+) T cells. There was no association found between CMV immune status and plasma levels of IFN-α. In CMV-infected young and elderly people, INF-α levels had no correlation with the frequency of IL-7Rα(low) EM CD8(+) T cells although this cytokine levels correlated with the frequency of IL-7Rα(low) CD45RA(+) EM CD8(+) T cells in CMV-uninfected elderly people. Our findings suggest that the effect of CMV infection on the frequency of CD8(+) T cell subsets may begin with IL-7Rα(low) EM CD8(+) T cells and spread to other subsets with aging. Also, IFN-α could be associated with the expansion of IL-7Rα(low) CD45RA(+) EM CD8(+) T cells in the CMV-uninfected elderly.     

Immunization with live and dead Chlamydia muridarum induces different levels of protective immunity in a murine genital tract model: correlation with MHC class II peptide presentation and multifunctional Th1 cells

Mice that were intranasally vaccinated with live or dead Chlamydia muridarum with or without CpG-containing oligodeoxynucleotide 1862 elicited widely disparate levels of protective immunity to genital tract challenge. We found that the frequency of multifunctional T cells coexpressing IFN-γ and TNF-α with or without IL-2 induced by live C. muridarum most accurately correlated with the pattern of protection against C. muridarum genital tract infection, suggesting that IFN-γ(+)-producing CD4(+) T cells that highly coexpress TNF-α may be the optimal effector cells for protective immunity. We also used an immunoproteomic approach to analyze MHC class II-bound peptides eluted from dendritic cells (DCs) that were pulsed with live or dead C. muridarum elementary bodies (EBs). We found that DCs pulsed with live EBs presented 45 MHC class II C. muridarum peptides mapping to 13 proteins. In contrast, DCs pulsed with dead EBs presented only six MHC class II C. muridarum peptides mapping to three proteins. Only two epitopes were shared in common between the live and dead EB-pulsed groups. This study provides insights into the role of Ag presentation and cytokine secretion patterns of CD4(+) T effector cells that correlate with protective immunity elicited by live and dead C. muridarum. These insights should prove useful for improving vaccine design for Chlamydia trachomatis.     

Simian immunodeficiency virus-induced changes in T cell cytokine responses in cynomolgus macaques with latent Mycobacterium tuberculosis infection are associated with timing of reactivation

Understanding the early immunologic events accompanying reactivated tuberculosis (TB) in HIV-infected individuals may yield insight into causes of reactivation and improve treatment modalities. We used the cynomolgus macaque (Macaca fascicularis) model of HIV-Mycobacterium tuberculosis coinfection to investigate the dynamics of multifunctional T cell responses and granuloma T cell phenotypes in reactivated TB. CD4(+) and CD8(+) T cells expressing Th1 cytokines (IFN-γ, IL-2, TNF) and Th2 cytokines (IL-4 and IL-10) were followed from latent M. tuberculosis infection to reactivation after coinfection with a pathogenic SIV. Coinfected animals experienced increased Th1 cytokine responses to M. tuberculosis Ags above the latent-response baseline 3-5 wk post-SIV infection that corresponded with peak plasma viremia. Th2 cytokine expression was not Ag specific, but strong, transient IL-4 expression was noted 4-7 wk post-SIV infection. Animals reactivating <17 wk post-SIV infection had significantly more multifunctional CD4(+) T cells 3-5 wk post-SIV infection and more Th2-polarized and fewer Th0-, Th1-polarized CD8(+) T cells during weeks 1-10 post-SIV infection than animals reactivating >26 wk post-SIV infection. Granuloma T cells included Th0-, Th1-, and Th2-polarized phenotypes but were particularly rich in cytolytic (CD107(+)) T cells. When combined with the changes in peripheral blood T cells, these factors indicate that events during acute HIV infection are likely to include distortions in proinflammatory and anti-inflammatory T cell responses within the granuloma that have significant effects on reactivation of latent TB. Moreover, it appears that mycobacteria-specific multifunctional T cells are better correlates of Ag load (i.e., disease status) than of protection.     

CD8+ T cells induce thyroid epithelial cell hyperplasia and fibrosis

CD8(+) T cells can be important effector cells in autoimmune inflammation, generally because they can damage target cells by cytotoxicity. This study shows that activated CD8(+) T cells induce thyroid epithelial cell hyperplasia and proliferation and fibrosis in IFN-γ(-/-) NOD.H-2h4 SCID mice in the absence of CD4(+) T cells. Because CD8(+) T cells induce proliferation rather than cytotoxicity of target cells, these results describe a novel function for CD8(+) T cells in autoimmune disease. In contrast to the ability of purified CD8(+) T cells to induce thyrocyte proliferation, CD4(+) T cells or CD8 T cell-depleted splenocytes induced only mild thyroid lesions in SCID recipients. T cells in both spleens and thyroids highly produce TNF-α. TNF-α promotes proliferation of thyrocytes in vitro, and anti-TNF-α inhibits development of thyroid epithelial cell hyperplasia and proliferation in SCID recipients of IFN-γ(-/-) splenocytes. This suggests that targeting CD8(+) T cells and/or TNF-α may be effective for treating epithelial cell hyperplasia and fibrosis.     

Distinct kinetics of Gag-specific CD4+ and CD8+ T cell responses during acute HIV-1 infection

HIV infection is characterized by a gradual deterioration of immune function, mainly in the CD4 compartment. To better understand the dynamics of HIV-specific T cells, we analyzed the kinetics and polyfunctional profiles of Gag-specific CD4(+) and CD8(+) T cell responses in 12 subtype C-infected individuals with different disease-progression profiles, ranging from acute to chronic HIV infection. The frequencies of Gag-responsive CD4(+) and CD8(+) T cells showed distinct temporal kinetics. The peak frequency of Gag-responsive IFN-γ(+)CD4(+) T cells was observed at a median of 28 d (interquartile range: 21-81 d) post-Fiebig I/II staging, whereas Gag-specific IFN-γ(+)CD8(+) T cell responses peaked at a median of 253 d (interquartile range: 136-401 d) and showed a significant biphasic expansion. The proportion of TNF-α-expressing cells within the IFN-γ(+)CD4(+) T cell population increased (p = 0.001) over time, whereas TNF-α-expressing cells within IFN-γ(+)CD8(+) T cells declined (p = 0.005). Both Gag-responsive CD4(+) and CD8(+) T cells showed decreased Ki67 expression within the first 120 d post-Fiebig I/II staging. Prior to the disappearance of Gag-responsive Ki67(+)CD4(+) T cells, these cells positively correlated (p = 0.00038) with viremia, indicating that early Gag-responsive CD4 events are shaped by viral burden. No such associations were observed in the Gag-specific CD8(+) T cell compartment. Overall, these observations indicated that circulating Gag-responsive CD4(+) and CD8(+) T cell frequencies and functions are not synchronous, and properties change rapidly at different tempos during early HIV infection.     

Filarial infection suppresses malaria-specific multifunctional Th1 and Th17 responses in malaria and filarial coinfections

The mechanisms underlying the modulation of both the malaria-specific immune response and the course of clinical malaria in the context of concomitant helminth infection are poorly understood. We used multiparameter flow cytometry to characterize the quality and the magnitude of malaria-specific T cell responses in filaria-infected and -uninfected individuals with concomitant asymptomatic Plasmodium falciparum malaria in Mali. In comparison with filarial-uninfected subjects, filarial infection was associated with higher ex vivo frequencies of CD4(+) cells producing IL-4, IL-10, and IL-17A (p = 0.01, p = 0.001, and p = 0.03, respectively). In response to malaria Ag stimulation, however, filarial infection was associated with lower frequencies of CD4(+) T cells producing IFN-γ, TNF-α, and IL-17A (p < 0.001, p = 0.04, and p = 0.04, respectively) and with higher frequencies of CD4(+)IL10(+)T cells (p = 0.0005). Importantly, filarial infection was associated with markedly lower frequencies of malaria Ag-specific Th1 (p < 0.0001), Th17 (p = 0.012), and "TNF-α" (p = 0.0008) cells, and a complete absence of malaria-specific multifunctional Th1 cells. Filarial infection was also associated with a marked increase in the frequency of malaria-specific adaptive regulatory T/Tr1 cells (p = 0.024), and the addition of neutralizing anti-IL-10 Ab augmented the amount of Th1-associated cytokine produced per cell. Thus, among malaria-infected individuals, concomitant filarial infection diminishes dramatically the frequencies of malaria-specific Th1 and Th17 T cells, and alters the quality and magnitude of malaria-specific T cell responses.     

Heterogeneity of multifunctional IL-17A producing S. Typhi-specific CD8+ T cells in volunteers following Ty21a typhoid immunization

Salmonella enterica serovar Typhi (S. Typhi), the causative agent of typhoid fever, continues to cause significant morbidity and mortality world-wide. CD8+ T cells are an important component of the cell mediated immune (CMI) response against S. Typhi. Recently, interleukin (IL)-17A has been shown to contribute to mucosal immunity and protection against intracellular pathogens. To investigate multifunctional IL-17A responses against S. Typhi antigens in T memory subsets, we developed multiparametric flow cytometry methods to detect up to 6 cytokines/chemokines (IL-10, IL-17A, IL-2, interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and macrophage inflammatory protein-1β (MIP-1β)) simultaneously. Five volunteers were immunized with a 4 dose regimen of live-attenuated S. Typhi vaccine (Ty21a), peripheral blood mononuclear cells (PBMC) were isolated before and at 11 time points after immunization, and CMI responses were evaluated. Of the 5 immunized volunteers studied, 3 produced detectable CD8+ T cell responses following stimulation with S. Typhi-infected autologous B lymphoblastoid cell lines (B-LCL). Additionally, 2 volunteers had detectable levels of intracellular cytokines in response to stimulation with S. Typhi-infected HLA-E restricted cells. Although the kinetics of the responses differed among volunteers, all of the responses were bi- or tri-phasic and included multifunctional CD8+ T cells. Virtually all of the IL-17A detected was derived from multifunctional CD8+ T cells. The presence of these multifunctional IL-17A+ CD8+ T cells was confirmed using an unsupervised analysis program, flow cytometry clustering without K (FLOCK). This is the first report of IL-17A production in response to S. Typhi in humans, indicating the presence of a Tc17 response which may be important in protection. The presence of IL-17A in multifunctional cells co-producing Tc1 cytokines (IL-2, IFN-γ and TNF-α) may also indicate that the distinction between Tc17 and Tc1 responses in humans is not as clearly delineated as suggested by in vitro experiments and animal models.     

Whole-blood flow-cytometric analysis of antigen-specific CD4 T-cell cytokine profiles distinguishes active tuberculosis from non-active states

T-cell based IFN-γ release assays do not permit distinction of active tuberculosis (TB) from successfully treated disease or latent M. tuberculosis infection. We postulated that IFN-γ and IL-2 cytokine profiles of antigen-specific T cells measured by flow-cytometry ex vivo might correlate with TB disease activity in vivo. Tuberculin (PPD), ESAT-6 and CFP-10 were used as stimuli to determine antigen-specific cytokine profiles in CD4 T cells from 24 patients with active TB and 28 patients with successfully treated TB using flow-cytometry. Moreover, 25 individuals with immunity consistent with latent M. tuberculosis infection and BCG-vaccination, respectively, were recruited. Although the frequency of cytokine secreting PPD reactive CD4 T cells was higher in patients with active TB compared to patients with treated TB (median 0.81% vs. 0.39% of CD4 T cells, p?=?0.02), the overlap in frequencies precluded distinction between the groups on an individual basis. When assessing cytokine profiles, PPD specific CD4 T cells secreting both IFN-γ and IL-2 predominated in treated TB, latent infection and BCG-vaccination, whilst in active TB the cytokine profile was shifted towards cells secreting IFN-γ only (p<0.0001). Cytokine profiles of ESAT-6 or CFP-10 reactive CD4 T cells did not differ between the groups. Receiver operator characteristics (ROC) analysis revealed that frequencies of PPD specific IFN-γ/IL-2 dual-positive T cells below 56% were an accurate marker for active TB (specificity 100%, sensitivity 70%) enabling effective discrimination from non-active states. In conclusion, a frequency lower than 56% IFN-γ/IL-2 dual positive PPD-specific circulating CD4 T-cells is strongly indicative of active TB.