Enhanced Virus-Specific CD8+ T Cell Responses by Listeria monocytogenes-Infected Dendritic Cells in the Context of Tim-3 Blockade

In this study, we engineered Listeria monocytogens (Lm) by deleting the LmΔactA/ΔinlB virulence determinants and inserting HCV-NS5B consensus antigens to develop a therapeutic vaccine against hepatitis C virus (HCV) infection. We tested this recombinant Lm-HCV vaccine in triggering of innate and adaptive immune responses in vitro using immune cells from HCV-infected and uninfected individuals. This live-attenuated Lm-HCV vaccine could naturally infect human dendritic cells (DC), thereby driving DC maturation and antigen presentation, producing Th1 cytokines, and triggering CTL responses in uninfected individuals. However, vaccine responses were diminished when using DC and T cells derived from chronically HCV-infected individuals, who express higher levels of inhibitory molecule Tim-3 on immune cells. Notably, blocking Tim-3 signaling significantly improved the innate and adaptive immune responses in chronically HCV-infected patients, indicating that novel strategies to enhance the potential of antigen presentation and cellular responses are essential for developing an effective therapeutic vaccine against HCV infection.     

PD-1 and Tim-3 Pathways Regulate CD8+ T Cells Function in Atherosclerosis

T cell-mediated immunity plays a significant role in the development of atherosclerosis (AS). There is increasing evidence that CD8⁺ T cells are also involved in AS but their exact roles remain unclear. The inhibitory receptors programmed cell death-1 (PD-1) and T cell immunoglobulin and mucin domain 3 (Tim-3) are well known inhibitory molecules that play a crucial role in regulating CD8⁺ T cell activation or tolerance. Here, we demonstrate that the co-expression of PD-1 and Tim-3 on CD8⁺ T cells is up-regulated in AS patients. PD-1⁺ Tim-3⁺ CD8⁺ T cells are enriched for within the central T (T_CM) cell subset, with high proliferative activity and CD127 expression. Co-expression of PD-1 and Tim-3 on CD8⁺ T cells is associated with increased anti-atherogenic cytokine production as well as decreased pro-atherogenic cytokine production. Blockade of PD-1 and Tim-3 results in a decrease of anti-atherogenic cytokine production by PD-1⁺ Tim-3⁺ CD8⁺ T cells and in an augmentation of TNF-α and IFN-γ production. These findings highlight the important role of the PD-1 and Tim-3 pathways in regulating CD8⁺ T cells function in human AS.     

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.     

Infection with Feline Immunodeficiency Virus Directly Activates CD4+ CD25+ T Regulatory Cells

Lentivirus infection activates CD4⁺ CD25⁺ T regulatory (Treg) cells. Activation of Treg cells may be due to direct virus infection or chronic antigenic stimulation. Herein we demonstrate that in vitro feline immunodeficiency virus (FIV) infection, but not UV-inactivated virus, activates Treg cells as measured by immunosuppressive function and upregulation of GARP, FoxP3, and membrane-bound transforming growth factor β (TGF-β). These data demonstrate for the first time that AIDS lentiviruses infect and activate Treg cells, potentially contributing to immune dysfunction.     

Dynamics of Cytokine/Chemokine Responses in Intestinal CD4+ and CD8+ T Cells during Acute Simian Immunodeficiency Virus Infection

Loss of intestinal CD4⁺ T cells was associated with decreased production of several T-helper 1 (T_H1) and T_H2 cytokines and increased production of interleukin 17 (IL-17), gamma interferon (IFN-γ), CCL4, and granulocyte-macrophage colony-stimulating factor (GM-CSF) by CD8⁺ T cells 21 days after simian immunodeficiency virus (SIV) infection in rhesus macaques. Shifting of mucosal T_H1 to T_H2 or T-cytotoxic 1 (T_C1) to T_C2 cytokine profiles was not evident. Additionally, both CD4⁺ and CD8⁺ T cells showed upregulation of macrophage migration inhibition factor (MIF) and basic fibroblast growth factor (FGF-basic) cytokines that have been linked to HIV disease progression.     

Impact of Soluble CD26 on Treatment Outcome and Hepatitis C Virus-Specific T Cells in Chronic Hepatitis C Virus Genotype 1 Infection

Background

Interferon and ribavirin therapy for chronic hepatitis C virus (HCV) infection yields sustained virological response (SVR) rates of 50–80%. Several factors such as non-1 genotype, beneficial IL28B genetic variants, low baseline IP-10, and the functionality of HCV-specific T cells predict SVR. With the pending introduction of new therapies for HCV entailing very rapid clearance of plasma HCV RNA, the importance of baseline biomarkers likely will increase in order to tailor therapy. CD26 (DPPIV) truncates the chemokine IP-10 into a shorter antagonistic form, and this truncation of IP-10 has been suggested to influence treatment outcome in patients with chronic HCV infection patients. In addition, previous reports have shown CD26 to be a co-stimulator for T cells. The aim of the present study was to assess the utility of CD26 as a biomarker for treatment outcome in chronic hepatitis C and to define its association with HCV-specific T cells.

Methods

Baseline plasma from 153 genotype 1 and 58 genotype 2/3 infected patients enrolled in an international multicenter phase III trial (DITTO-HCV) and 36 genotype 1 infected patients participating in a Swedish trial (TTG1) were evaluated regarding baseline soluble CD26 (sCD26) and the functionality of HCV-specific CD8⁺ T cells.

Results

Genotype 1 infected patients achieving SVR in the DITTO (P = 0.002) and the TTG1 (P = 0.02) studies had lower pretreatment sCD26 concentrations compared with non-SVR patients. Sixty-five percent of patients with sCD26 concentrations below 600 ng/mL achieved SVR compared with 39% of the patients with sCD26 exceeding 600 ng/mL (P = 0.01). Patients with sCD26 concentrations below 600 ng/mL had significantly higher frequencies of HCV-specific CD8⁺ T cells (P = 0.02).

Conclusions

Low baseline systemic concentrations of sCD26 predict favorable treatment outcome in chronic HCV infection and may be associated with higher blood counts of HCV-specific CD8⁺ T cells.     

TGF-β and IL-10 Production by HIV-Specific CD8+ T Cells Is Regulated by CTLA-4 Signaling on CD4+ T Cells

Immune dysregulation in HIV-1 infection is associated with increased expression of inhibitory molecules such as CTLA-4, TGF-β, and IL-10. In this study we examined one potential mechanism for regulating TGF-β and IL-10 expression by HIV-specific suppressor CD8+ T cells. No overlap between TGF-β, IL-10, and IFN-γ cytokine production by HIV-specific CD8+ T cells was observed. TGF-β positive and IL-10 positive cells were FOXP3 negative, CD25 negative, and displayed a heterogeneous surface expression of CD127. TGF-β and IL-10 positive CD8+ T cells did not express CTLA-4. Nevertheless, CTLA-4 blockade resulted in a significant decrease in HIV-specific TGF-β positive and IL-10 positive CD8+ T cell responses, and a concomitant increase in HIV-specific IFN-γ positive CD8+ T cell responses. Depletion of CD4+ T cells abrogated the impact of CTLA-4 on HIV-specific TGF-β positive and IL-10 positive CD8+ T cells. Our study suggests that CTLA-4 Signaling on CD4+ T cells regulates the inhibitory functions of the HIV-specific suppressor CD8+ T cells.     

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.     

Differential Targeting of Viral Components by CD4+ versus CD8+ T Lymphocytes in Dengue Virus Infection

Dengue virus (DENV) is the principal arthropod-borne viral pathogen afflicting human populations. While repertoires of antibodies to DENV have been linked to protection or enhanced infection, the role of T lymphocytes in these processes remains poorly defined. This study provides a comprehensive overview of CD4⁺ and CD8⁺ T cell epitope reactivities against the DENV 2 proteome in adult patients experiencing secondary DENV infection. Dengue virus-specific T cell responses directed against an overlapping 15mer peptide library spanning the DENV 2 proteome were analyzed ex vivo by enzyme-linked immunosorbent spot assay, and recognition of individual peptides was further characterized in specific T cell lines. Thirty novel T cell epitopes were identified, 9 of which are CD4⁺ and 21 are CD8⁺ T cell epitopes. We observe that whereas CD8⁺ T cell epitopes preferentially target nonstructural proteins (NS3 and NS5), CD4⁺ epitopes are skewed toward recognition of viral components that are also targeted by B lymphocytes (envelope, capsid, and NS1). Consistently, a large proportion of dengue virus-specific CD4⁺ T cells have phenotypic characteristics of circulating follicular helper T cells (CXCR5 expression and production of interleukin-21 or gamma interferon), suggesting that they are interacting with B cells in vivo. This study shows that during a dengue virus infection, the protein targets of human CD4⁺ and CD8⁺ T cells are largely distinct, thus highlighting key differences in the immunodominance of DENV proteins for these two cell types. This has important implications for our understanding of how the two arms of the human adaptive immune system are differentially targeted and employed as part of our response to DENV infection.     

B7-H1 Blockade Increases Survival of Dysfunctional CD8+ T Cells and Confers Protection against Leishmania donovani Infections

Experimental visceral leishmaniasis (VL) represents an exquisite model to study CD8⁺ T cell responses in a context of chronic inflammation and antigen persistence, since it is characterized by chronic infection in the spleen and CD8⁺ T cells are required for the development of protective immunity. However, antigen-specific CD8⁺ T cell responses in VL have so far not been studied, due to the absence of any defined Leishmania-specific CD8⁺ T cell epitopes. In this study, transgenic Leishmania donovani parasites expressing ovalbumin were used to characterize the development, function, and fate of Leishmania-specific CD8⁺ T cell responses. Here we show that L. donovani parasites evade CD8⁺ T cell responses by limiting their expansion and inducing functional exhaustion and cell death. Dysfunctional CD8⁺ T cells could be partially rescued by in vivo B7-H1 blockade, which increased CD8⁺ T cell survival but failed to restore cytokine production. Nevertheless, B7-H1 blockade significantly reduced the splenic parasite burden. These findings could be exploited for the design of new strategies for immunotherapeutic interventions against VL.     

Tim-3-Expressing CD4+ and CD8+ T Cells in Human Tuberculosis (TB) Exhibit Polarized Effector Memory Phenotypes and Stronger Anti-TB Effector Functions

T-cell immune responses modulated by T-cell immunoglobulin and mucin domain-containing molecule 3 (Tim-3) during Mycobacterium tuberculosis (Mtb) infection in humans remain poorly understood. Here, we found that active TB patients exhibited increases in numbers of Tim-3-expressing CD4⁺ and CD8⁺ T cells, which preferentially displayed polarized effector memory phenotypes. Consistent with effector phenotypes, Tim-3⁺CD4⁺ and Tim-3⁺CD8⁺ T-cell subsets showed greater effector functions for producing Th1/Th22 cytokines and CTL effector molecules than Tim-3⁻ counterparts, and Tim-3-expressing T cells more apparently limited intracellular Mtb replication in macrophages. The increased effector functions for Tim-3-expressing T cells consisted with cellular activation signaling as Tim-3⁺CD4⁺ and Tim-3⁺CD8⁺ T-cell subsets expressed much higher levels of phosphorylated signaling molecules p38, stat3, stat5, and Erk1/2 than Tim-3- controls. Mechanistic experiments showed that siRNA silencing of Tim-3 or soluble Tim-3 treatment interfering with membrane Tim-3-ligand interaction reduced de novo production of IFN-γ and TNF-α by Tim-3-expressing T cells. Furthermore, stimulation of Tim-3 signaling pathways by antibody cross-linking of membrane Tim-3 augmented effector function of IFN-γ production by CD4⁺ and CD8⁺ T cells, suggesting that Tim-3 signaling helped to drive stronger effector functions in active TB patients. This study therefore uncovered a previously unknown mechanism for T-cell immune responses regulated by Tim-3, and findings may have implications for potential immune intervention in TB.     

Immune Surveillance by Rhinovirus-Specific Circulating CD4+ and CD8+ T Lymphocytes

Background

It is difficult to experimentally infect volunteers with RV strains to which the subject demonstrates serological immunity. However, in RV challenges, viral clearance begins before de novo adaptive immune responses would develop. We speculated that adaptive immunity to RV reflects heterologous immunity by effector memory cells.

Methods

DCs were generated from monocytes using GM-CSF and IL-4 and RV39 loading accomplished with a dose of ∼350 TCID₅₀/10⁵ cells. RV-induced maturation was established as modulation of MHC class II, CD80, CD83, and CD86. Circulating RV targeting CD4 and CD8 T cells were investigated as induction of RV-specific proliferation (CFSE-dilution).

Results

Maturation of DC by RV was confirmed as upregulation of MHC Class II (83.3±5.0% to 87.8±4.1%), CD80 (39.4±7.2% to 47.6±7.7%) and CD86 (78.4±4.7% to 84.1±3.4%). Both CD4 and CD8 memory T cells were recognized in the circulation of healthy subjects.

Conclusions

RV drives DC maturation and results in their ability to present RV antigens to both T helper and cytotoxic lymphocytes. Both CD4 and CD8 cells capable of recognizing RV-associated antigens are present in the circulation of healthy subjects where they are presumably involved in immune surveillance and explain the rapid recruitment of an adaptive immune response during RV infection.     

Protective CD4+ and CD8+ T Cells against Influenza Virus Induced by Vaccination with Nucleoprotein DNA

DNA vaccination is an effective means of eliciting both humoral and cellular immunity, including cytotoxic T lymphocytes (CTL). Using an influenza virus model, we previously demonstrated that injection of DNA encoding influenza virus nucleoprotein (NP) induced major histocompatibility complex class I-restricted CTL and cross-strain protection from lethal virus challenge in mice (J. B. Ulmer et al., Science 259:1745–1749, 1993). In the present study, we have characterized in more detail the cellular immune responses induced by NP DNA, which included robust lymphoproliferation and Th1-type cytokine secretion (high levels of gamma interferon and interleukin-2 [IL-2], with little IL-4 or IL-10) in response to antigen-specific restimulation of splenocytes in vitro. These responses were mediated by CD4⁺ T cells, as shown by in vitro depletion of T-cell subsets. Taken together, these results indicate that immunization with NP DNA primes both cytolytic CD8⁺ T cells and cytokine-secreting CD4⁺ T cells. Further, we demonstrate by adoptive transfer and in vivo depletion of T-cell subsets that both of these types of T cells act as effectors in protective immunity against influenza virus challenge conferred by NP DNA.Cellular immune responses play an important role in protection from disease caused by infectious pathogens, such as viruses and certain bacteria (e.g., Mycobacterium tuberculosis). The specific T cells involved in conferring immunity can include both CD4⁺ and CD8⁺ T cells, often through the action of secreted cytokines and cytolytic activity, respectively. Certain types of vaccines, such as subunit proteins and whole or partially purified preparations of inactivated organisms, in general induce CD4⁺ T-cell responses but not CD8⁺ cytotoxic T lymphocytes (CTL). In contrast, live attenuated organisms and subunit proteins formulated with certain experimental adjuvants can induce both types of responses. Recently, a different approach consisting of direct immunization with plasmid DNA expression vectors (i.e., DNA vaccines) has shown promise as a viable means of inducing broad-spectrum T-cell responses. The effectiveness of DNA vaccines in animal models is likely due, at least in part, to expression of antigens in situ (35), leading to the induction of CTL (29), antibodies (3, 4, 10, 21, 22, 32), and cytokine-secreting lymphocyte responses (12, 36). During the past 5 years, many reports have been published on the immunogenicity of DNA vaccines encoding various antigens in several animal models, thereby illustrating the applicability of the technology to many pathogens (for a review, see reference 6). However, in only a few instances has the nature of the effector cells responsible for protective immunity been described (7, 16). In the present study, we have analyzed in detail the cellular immune responses induced by influenza virus nucleoprotein (NP) DNA and have established that both CD4⁺ T cells secreting Th1-type cytokines and CD8⁺ cytotoxic T cells play important effector roles in heterosubtypic protective immunity against lethal influenza virus challenge in mice.