Mycobacterium tuberculosis Specific CD8+ T Cells Rapidly Decline with Antituberculosis Treatment

Rationale

Biomarkers associated with response to therapy in tuberculosis could have broad clinical utility. We postulated that the frequency of Mycobacterium tuberculosis (Mtb) specific CD8⁺ T cells, by virtue of detecting intracellular infection, could be a surrogate marker of response to therapy and would decrease during effective antituberculosis treatment.Objectives: We sought to determine the relationship of Mtb specific CD4⁺ T cells and CD8⁺ T cells with duration of antituberculosis treatment.

Materials and Methods

We performed a prospective cohort study, enrolling between June 2008 and August 2010, of HIV-uninfected Ugandan adults (n = 50) with acid-fast bacillus smear-positive, culture confirmed pulmonary TB at the onset of antituberculosis treatment and the Mtb specific CD4⁺ and CD8⁺ T cell responses to ESAT-6 and CFP-10 were measured by IFN-γ ELISPOT at enrollment, week 8 and 24.

Results

There was a significant difference in the Mtb specific CD8⁺ T response, but not the CD4⁺ T cell response, over 24 weeks of antituberculosis treatment (p<0.0001), with an early difference observed at 8 weeks of therapy (p = 0.023). At 24 weeks, the estimated Mtb specific CD8⁺ T cell response decreased by 58%. In contrast, there was no significant difference in the Mtb specific CD4⁺ T cell during the treatment. The Mtb specific CD4⁺ T cell response, but not the CD8⁺ response, was negatively impacted by the body mass index.

Conclusions

Our data provide evidence that the Mtb specific CD8⁺ T cell response declines with antituberculosis treatment and could be a surrogate marker of response to therapy. Additional research is needed to determine if the Mtb specific CD8⁺ T cell response can detect early treatment failure, relapse, or to predict disease progression.     

Dynamic Imaging of CD8+ T Cells and Dendritic Cells during Infection with Toxoplasma gondii

To better understand the initiation of CD8⁺ T cell responses during infection, the primary response to the intracellular parasite Toxoplasma gondii was characterized using 2-photon microscopy combined with an experimental system that allowed visualization of dendritic cells (DCs) and parasite specific CD8⁺ T cells. Infection with T. gondii induced localization of both these populations to the sub-capsular/interfollicular region of the draining lymph node and DCs were required for the expansion of the T cells. Consistent with current models, in the presence of cognate antigen, the average velocity of CD8⁺ T cells decreased. Unexpectedly, infection also resulted in modulation of the behavior of non-parasite specific T cells. This TCR-independent process correlated with the re-modeling of the lymph node micro-architecture and changes in expression of CCL21 and CCL3. Infection also resulted in sustained interactions between the DCs and CD8⁺ T cells that were visualized only in the presence of cognate antigen and were limited to an early phase in the response. Infected DCs were rare within the lymph node during this time frame; however, DCs presenting the cognate antigen were detected. Together, these data provide novel insights into the earliest interaction between DCs and CD8⁺ T cells and suggest that cross presentation by bystander DCs rather than infected DCs is an important route of antigen presentation during toxoplasmosis.     

IL-4 Induced Innate CD8+ T Cells Control Persistent Viral Infection

Memory-like CD8⁺ T cells expressing eomesodermin are a subset of innate T cells initially identified in a number of genetically modified mice, and also exist in wild mice and human. The acquisition of memory phenotype and function by these T cells is dependent on IL–4 produced by PLZF⁺ innate T cells; however, their physiologic function is still not known. Here we found that these IL-4-induced innate CD8⁺ T cells are critical for accelerating the control of chronic virus infection. In CIITA-transgenic mice, which have a substantial population of IL-4-induced innate CD8⁺ T cells, this population facilitated rapid control of viremia and induction of functional anti-viral T-cell responses during infection with chronic form of lymphocytic choriomeningitis virus. Characteristically, anti-viral innate CD8⁺ T cells accumulated sufficiently during early phase of infection. They produced a robust amount of IFN-γ and TNF-α with enhanced expression of a degranulation marker. Furthermore, this finding was confirmed in wild-type mice. Taken together, the results from our study show that innate CD8⁺ T cells works as an early defense mechanism against chronic viral infection.     

Human and Murine Clonal CD8+ T Cell Expansions Arise during Tuberculosis Because of TCR Selection

The immune system can recognize virtually any antigen, yet T cell responses against several pathogens, including Mycobacterium tuberculosis, are restricted to a limited number of immunodominant epitopes. The host factors that affect immunodominance are incompletely understood. Whether immunodominant epitopes elicit protective CD8+ T cell responses or instead act as decoys to subvert immunity and allow pathogens to establish chronic infection is unknown. Here we show that anatomically distinct human granulomas contain clonally expanded CD8+ T cells with overlapping T cell receptor (TCR) repertoires. Similarly, the murine CD8+ T cell response against M. tuberculosis is dominated by TB10.4_4-11-specific T cells with extreme TCRβ bias. Using a retrogenic model of TB10.4_4-11-specific CD8+ T cells, we show that TCR dominance can arise because of competition between clonotypes driven by differences in affinity. Finally, we demonstrate that TB10.4-specific CD8+ T cells mediate protection against tuberculosis, which requires interferon-γ production and TAP1-dependent antigen presentation in vivo. Our study of how immunodominance, biased TCR repertoires, and protection are inter-related, provides a new way to measure the quality of T cell immunity, which if applied to vaccine evaluation, could enhance our understanding of how to elicit protective T cell immunity.     

Clonal expansion of CD8+ effector T cells in childhood tuberculosis

The role of CD8(+) T cells in human tuberculosis (TB) remains elusive. We analyzed the T cell repertoire and phenotype in 1) children with active TB (< or =4 years), 2) healthy latently Mycobacterium tuberculosis-infected children, and 3) noninfected age-matched (tuberculin skin test-negative) controls. Ex vivo phenotyping of T cell subpopulations by flow cytometry revealed a significant increase in the proportion of CD8(+)CD45RO(-)CD62L(-)CD28(-)CD27(-) effector T cells (T(EF)) in the peripheral blood of children with active TB (22.1 vs 9.5% in latently M. tuberculosis-infected children, vs 8.5% in tuberculin skin test-negative controls). Analyses of TCR variable beta-chains revealed markedly skewed repertoires in CD8(+) T(EF) and effector memory T cells. Expansions were restricted to single TCR variable beta-chains in individual donors indicating clonal growth. CDR3 spectratyping and DNA sequencing verified clonal expansion as the cause for CD8(+) effector T cell enrichment in individual TB patients. The most prominent enrichment of highly similar T(EF) clones (>70% of CD8(+) T(EF)) was found in two children with active severe TB. Therefore, clonal expansion of CD8(+) T(EF) occurs in childhood TB with potential impact on course and severity of disease.     

Co-Infection with Mycobacterium tuberculosis Impairs HIV-Specific CD8+ and CD4+ T Cell Functionality

The ability of antigen-specific T cells to simultaneously produce multiple cytokines is thought to correlate with the functional capacity and efficacy of T cells. These ‘polyfunctional’ T cells have been associated with control of HIV. We aimed to assess the impact of co-infection with Mycobacterium tuberculosis (MTB) on HIV-specific CD8+ and CD4+ T cell function. We assessed T cell functionality in 34 South African adults by investigating the IFN-y, IL-2, TNF-α, IL-21 and IL-17 cytokine secretion capacity, using polychromatic flow cytometry, following HIV Gag-specific stimulation of peripheral blood mononuclear cells. We show that MTB is associated with lower HIV-specific T cell function in co-infected as compared to HIV mono-infected individuals. This decline in function was greatest in co-infection with active Tuberculosis (TB) compared to co-infection with latent MTB (LTBI), suggesting that mycobacterial load may contribute to this loss of function. The described impact of MTB on HIV-specific T cell function may be a mechanism for increased HIV disease progression in co-infected subjects as functionally impaired T cells may be less able to control HIV.     

Mycobacterium tuberculosis Multidrug Resistant Strain M Induces an Altered Activation of Cytotoxic CD8+ T Cells

In human tuberculosis (TB), CD8⁺ T cells contribute to host defense by the release of Th1 cytokines and the direct killing of Mycobacterium tuberculosis (Mtb)-infected macrophages via granule exocytosis pathway or the engagement of receptors on target cells. Previously we demonstrated that strain M, the most prevalent multidrug-resistant (MDR) Mtb strain in Argentine, is a weak inducer of IFN-γ and elicits a remarkably low CD8-dependent cytotoxic T cell activity (CTL). In contrast, the closely related strain 410, which caused a unique case of MDR-TB, elicits a CTL response similar to H37Rv. In this work we extend our previous study investigating some parameters that can account for this discrepancy. We evaluated the expressions of the lytic molecules perforin, granzyme B and granulysin and the chemokine CCL5 in CD8⁺ T cells as well as activation markers CD69 and CD25 and IL-2 expression in CD4⁺ and CD8⁺ T cells stimulated with strains H37Rv, M and 410. Our results demonstrate that M-stimulated CD8⁺ T cells from purified protein derivative positive healthy donors show low intracellular expression of perforin, granzyme B, granulysin and CCL5 together with an impaired ability to form conjugates with autologous M-pulsed macrophages. Besides, M induces low CD69 and IL-2 expression in CD4⁺ and CD8⁺ T cells, being CD69 and IL-2 expression closely associated. Furthermore, IL-2 addition enhanced perforin and granulysin expression as well as the degranulation marker CD107 in M-stimulated CD8⁺ T cells, making no differences with cells stimulated with strains H37Rv or 410. Thus, our results highlight the role of IL-2 in M-induced CTL activity that drives the proper activation of CD8⁺ T cells as well as CD4⁺ T cells collaboration.     

HIV-specific IL-10-positive CD8+ T cells suppress cytolysis and IL-2 production by CD8+ T cells

IL-10 producing T cells inhibit Ag-specific CD8+ T cell responses and may play a role in the immune dysregulation observed in HIV infection. We have previously observed the presence of HIV-specific IL-10-positive CD8+ T cells in advanced HIV disease. In this study, we examined the suppressive function of the Gag-specific IL-10-positive CD8+ T cells. Removal of these IL-10-positive CD8+ T cells resulted in increased cytolysis and IL-2, but not IFN-gamma, production by both HIV- and human CMV-specific CD8+ T cells. In addition, these IL-10-positive CD8+ T cells mediated suppression through direct cell-cell contact, and had a distinct immunophenotypic profile compared with other regulatory T cells. We describe a new suppressor CD8+ T cell population in advanced HIV infection that may contribute to the immune dysfunction observed in HIV infection.     

CD4+ Primary T Cells Expressing HCV-Core Protein Upregulate Foxp3 and IL-10, Suppressing CD4 and CD8 T Cells

Adaptive T cell responses are critical for controlling HCV infection. While there is clinical evidence of a relevant role for regulatory T cells in chronic HCV-infected patients, based on their increased number and function; mechanisms underlying such a phenomena are still poorly understood. Accumulating evidence suggests that proteins from Hepatitis C virus can suppress host immune responses. We and others have shown that HCV is present in CD4+ lymphocytes from chronically infected patients and that HCV-core protein induces a state of unresponsiveness in the CD4+ tumor cell line Jurkat. Here we show that CD4+ primary T cells lentivirally transduced with HCV-core, not only acquire an anergic phenotype but also inhibit IL-2 production and proliferation of bystander CD4+ or CD8+ T cells in response to anti-CD3 plus anti-CD28 stimulation. Core-transduced CD4+ T cells show a phenotype characterized by an increased basal secretion of the regulatory cytokine IL-10, a decreased IFN-γ production upon stimulation, as well as expression of regulatory T cell markers, CTLA-4, and Foxp3. A significant induction of CD4+CD25+CD127^lowPD-1^highTIM-3^high regulatory T cells with an exhausted phenotype was also observed. Moreover, CCR7 expression decreased in HCV-core expressing CD4+ T cells explaining their sequestration in inflamed tissues such as the infected liver. This work provides a new perspective on de novo generation of regulatory CD4+ T cells in the periphery, induced by the expression of a single viral protein.     

Antigen-specific CD8+ T cells and the development of central memory during Mycobacterium tuberculosis infection

Whether true memory T cells develop in the face of chronic infection such as tuberculosis remains controversial. To address this question, we studied CD8+ T cells specific for the Mycobacterium tuberculosis ESAT6-related Ags TB10.3 and TB10.4. The shared epitope TB10.3/10.4(20-28) is presented by H-2 K(d), and 20-30% of the CD8+ T cells in the lungs of chronically infected mice are specific for this Ag following respiratory infection with M. tuberculosis. These TB10.3/10.4(20-28)-specific CD8+ T cells produce IFN-gamma and TNF and express CD107 on their cell surface, which indicates their likely role as CTL in vivo. Nearly all of the Ag-specific CD8+ T cells in the lungs of chronically infected mice had a T effector cell phenotype based on their low expression of CD62L and CD45RB. In contrast, a population of TB10.3/10.4(20-28)-specific CD8+ T cells was identified in the lymphoid organs that express high levels of CD62L and CD45RB. Antibiotic treatment to resolve the infection led to a contraction of the Ag-specific CD8+ T cell population and was accompanied by an increase in the proportion of CD8+ T cells with a central memory phenotype. Finally, challenge of memory-immune mice with M. tuberculosis was accompanied by significant expansion of TB10.3/10.4(20-28)-specific CD8+ T cells, which suggests that these cells are in fact functional memory T cells.     

Contribution of CD8+ T cells to control of Mycobacterium tuberculosis infection

Tuberculosis is the number one cause of death due to infectious disease in the world today. Understanding the dynamics of the immune response is crucial to elaborating differences between individuals who contain infection vs those who suffer active disease. Key cells in an adaptive immune response to intracellular pathogens include CD8(+) T cells. Once stimulated, these cells provide a number of different effector functions, each aimed at clearing or containing the pathogen. To explore the role of CD8(+) T cells in an integrative way, we synthesize both published and unpublished data to build and test a mathematical model of the immune response to Mycobacterium tuberculosis in the lung. The model is then used to perform a series of simulations mimicking experimental situations. Selective deletion of CD8(+) T cell subsets suggests a differential contribution for CD8(+) T cell effectors that are cytotoxic as compared with those that produce IFN-gamma. We also determined the minimum levels of effector memory cells of each T cell subset (CD4(+) and CD8(+)) in providing effective protection following vaccination.     

Mycobacterium tuberculosis directs immunofocusing of CD8+ T cell responses despite vaccination

Vaccines that elicit T cell responses try to mimic protective memory T cell immunity after infection by increasing the frequency of Ag-specific T cells in the immune repertoire. However, the factors that determine immunodominance during infection and after vaccination and the relation between immunodominance and protection are incompletely understood. We previously identified TB10.4(20-28) as an immunodominant epitope recognized by H2-K(d)-restricted CD8(+) T cells after M. tuberculosis infection. Here we report a second epitope, EspA(150-158), that is recognized by a substantial number of pulmonary CD8(+) T cells. The relative abundance of these T cells in the naive repertoire only partially predicts their relative frequency after M. tuberculosis infection. Furthermore, although vaccination with recombinant vaccinia virus expressing these epitopes changes their relative immunodominance in the preinfection T cell repertoire, this change is transient after challenge with M. tuberculosis. We speculate that factors intrinsic to the chronic nature of M. tuberculosis infection establishes the hierarchy of immunodominance and may explain the failure of some vaccines to provide protection.     

Role of CD8+ T lymphocytes in control of Mycobacterium tuberculosis infection

Tuberculosis remains a global health concern. Control of infection is dependent on cell-mediated immune responses, with CD4+ T lymphocytes playing a central role. In this article, data supporting the importance of CD8+ T lymphocytes is reviewed, with an emphasis on the unique functional roles that this lymphocyte subset may play.     

CD4+ T Cells Are Required for the Priming of CD8+ T Cells following Infection with Herpes Simplex Virus Type 1

The role of CD4⁺ helper T cells in modulating the acquired immune response to herpes simplex virus type 1 (HSV-1) remains ill defined; in particular, it is unclear whether CD4⁺ T cells are needed for the generation of the protective HSV-1-specific CD8⁺-T-cell response. This study examined the contribution of CD4⁺ T cells in the generation of the primary CD8⁺-T-cell responses following acute infection with HSV-1. The results demonstrate that the CD8⁺-T-cell response generated in the draining lymph nodes of CD4⁺-T-cell-depleted C57BL/6 mice and B6-MHC-II^−/− mice is quantitatively and qualitatively distinct from the CD8⁺ T cells generated in normal C57BL/6 mice. Phenotypic analyses show that virus-specific CD8⁺ T cells express comparable levels of the activation marker CD44 in mice lacking CD4⁺ T cells and normal mice. In contrast, CD8⁺ T cells generated in the absence of CD4⁺ T cells express the interleukin 2 receptor α-chain (CD25) at lower levels. Importantly, the CD8⁺ T cells in the CD4⁺-T-cell-deficient environment are functionally active with respect to the expression of cytolytic activity in vivo but exhibit a diminished capacity to produce gamma interferon and tumor necrosis factor alpha. Furthermore, the primary expansion of HSV-1-specific CD8⁺ T cells is diminished in the absence of CD4⁺-T-cell help. These results suggest that CD4⁺-T-cell help is essential for the generation of fully functional CD8⁺ T cells during the primary response to HSV-1 infection.Infection due to herpes simplex virus type 1 (HSV-1) results in a wide spectrum of clinical presentations depending on the host''s age, the host''s immune status, and the route of inoculation (47). HSV-1 typically causes mild and self-limited lesions on the orofacial areas or genital sites. However, the disease can be life-threatening, as in the case of neonatal and central nervous system infections (18). The host''s immune responses, particularly CD8⁺ T cells, play an important role in determining the outcome of HSV infections in both the natural human host (18, 19, 28) and experimental murine models (11, 43). Immunodepletion and adoptive transfer studies have demonstrated the role of CD8⁺ T cells in reducing viral replication, resolving cutaneous disease, and providing overall protection upon rechallenge (6, 25, 26). CD8⁺ T cells play a particularly important role in preventing infection of the peripheral nervous system (PNS) and the reactivation of latent virus from neurons in the sensory ganglia of infected mice (21, 24, 36). The mechanisms that CD8⁺ T cells employ include gamma interferon (IFN-γ) production and functions associated with cytolytic granule content at the sites of primary infection (23, 31, 38). In the PNS of infected mice, the mechanisms primarily involve IFN-γ secretion (16, 20, 29), particularly against infected neurons expressing surface Qa-1 (41). Histopathological evidence from HSV-1-infected human ganglion sections show a large CD8⁺-T-cell infiltrate and the presence of inflammatory cytokines, suggesting that the presence of activated, effector memory cells within the PNS is important for maintaining HSV-1 latency in the natural human host (10, 42).The generation of a robust CD8⁺-T-cell response is essential for the control of various infectious pathogens. Some studies suggest that a brief interaction with antigen-presenting cells (APCs) is sufficient for CD8⁺-T-cell activation and expansion into functional effectors (44). However, the magnitude and quality of the overall CD8⁺-T-cell response generated may be dependent on additional factors (49). Recent evidence suggests that CD4⁺ T cells facilitate the activation and development of CD8⁺-T-cell responses either directly through the provision of cytokines or indirectly by the conditioning of dendritic cells (DC) (8, 48, 51). Those studies suggested that the latter mechanism is the dominant pathway, wherein CD4⁺ T cells assist CD8⁺-T-cell priming via the engagement of CD40 ligand (CD154) on CD4⁺ T cells and CD40 expressed on DC (4, 30, 33). This interaction results in the activation and maturation of DC, making them competent to stimulate antigen-specific CD8⁺-T-cell responses (35, 37).The requirement for CD4⁺-T-cell help in the generation of primary and secondary CD8⁺-T-cell responses to antigen varies. Primary CD8⁺-T-cell responses to infectious pathogens, such as Listeria monocytogenes, lymphocytic choriomeningitis virus (LCMV), influenza virus, and vaccinia virus, can be mounted effectively independently of CD4⁺-T-cell help (3, 12, 22, 34). In contrast, primary CD8⁺-T-cell responses to nonmicrobial antigens display an absolute dependence on CD4⁺-T-cell help (4, 5, 30, 33, 46). This observed difference in the requirement for CD4⁺-T-cell help may ultimately be a product of the initial inflammatory stimulus generated following immunization (49). Microbial antigens trigger an inflammatory response that can lead to the direct activation and priming of APCs, such as DC, thereby bypassing the need for CD4⁺-T-cell help. Nonmicrobial antigens, however, trigger an attenuated inflammatory response that does not directly activate and prime DCs. In the absence of this inflammation, CD4⁺ T cells are thought to condition and license DC functions through CD154/CD40 interactions, which leads to the subsequent activation of antigen-specific CD8⁺-T-cell responses (5, 49). Even in the case of pathogens where primary CD8⁺-T-cell responses were independent of CD4⁺-T-cell help, the secondary responses to these pathogens were found to be defective in the absence of CD4⁺-T-cell help (3, 12, 34, 40).The requirement for CD4⁺-T-cell help in priming CD8⁺-T-cell responses against HSV-1 infection is not well defined. Earlier studies with HSV-1 suggested that CD4⁺ T cells play an important role in the generation of primary CD8⁺-T-cell responses, detected in vitro, to acute infection with HSV-1 (14), principally through the provision of interleukin 2 (IL-2) for optimal CD8⁺-T-cell differentiation and proliferation. Subsequent studies, utilizing an in vivo approach, indicated that CD4⁺ T cells were not required for CD8⁺-T-cell-mediated cytolytic function (23). CD4⁺ T cells are thought to provide help by conditioning DC in a cognate, antigen-specific manner, thereby making them competent to stimulate HSV-1-specific CD8⁺-T-cell responses (37). By contrast, findings from other studies show that CD4⁺-T-cell-depleted mice were able to fully recover from acute infection with HSV-1 (38). These studies imply that the absence of CD4⁺ T cells does not prevent priming of CD8⁺ T cells in vivo.Studies from this laboratory have identified two distinct HSV-1-specific CD8⁺-T-cell subpopulations generated during the primary response, based upon the ability to synthesize IFN-γ following antigenic stimulation in vitro (1). To better understand the need for CD4⁺-T-cell help, we examined the functional characteristics and phenotypes of these CD8⁺-T-cell populations generated during a primary response to acute infection with HSV-1 in mice lacking CD4⁺ T cells. Our findings show that primary CD8⁺-T-cell responses to HSV-1 are compromised in the absence of CD4⁺-T-cell help. Specifically, the HSV-1 gB-specific CD8⁺ T cells produced in the absence of CD4⁺ T cells were found to be active with regard to cytolysis in vivo but were functionally impaired in the production of IFN-γ and TNF-α compared with intact C57BL/6 mice. Virus-specific CD8⁺ T cells were also reduced in number in CD4-depleted mice and in B6 mice lacking major histocompatibility complex (MHC) class II expression (B6-MHC-II^−/−) compared to wild-type (WT) mice. In addition, our data showed higher virus burdens in the infectious tissues obtained from mice lacking CD4⁺ T cells than in those from intact mice. Collectively, these findings demonstrate that CD4⁺-T-cell help is essential for the generation of primary CD8⁺-T-cell responses following acute cutaneous infection with HSV-1.