Respiratory Dendritic Cell Subsets Differ in Their Capacity to Support the Induction of Virus-Specific Cytotoxic CD8+ T Cell Responses

Dendritic cells located at the body surfaces, e.g. skin, respiratory and gastrointestinal tract, play an essential role in the induction of adaptive immune responses to pathogens and inert antigens present at these surfaces. In the respiratory tract, multiple subsets of dendritic cells (RDC) have been identified in both the normal and inflamed lungs. While the importance of RDC in antigen transport from the inflamed or infected respiratory tract to the lymph nodes draining this site is well recognized, the contribution of individual RDC subsets to this process and the precise role of migrant RDC within the lymph nodes in antigen presentation to T cells is not clear. In this report, we demonstrate that two distinct subsets of migrant RDC - exhibiting the CD103⁺ and CD11b^hi phenotype, respectively - are the primary DC presenting antigen to naïve CD4⁺ and CD8⁺ T lymphocytes in the draining nodes in response to respiratory influenza virus infection. Furthermore, the migrant CD103⁺ RDC subset preferentially drives efficient proliferation and differentiation of naive CD8⁺ T cells responding to infection into effector cells, and only the CD103⁺ RDC subset can present to naïve CD8⁺ T cells non-infectious viral vaccine introduced into the respiratory tract. These results identify CD103⁺ and CD11b^hi RDC as critical regulators of the adaptive immune response to respiratory tract infection and potential targets in the design of mucosal vaccines.     

Negative Immune Regulator Tim-3 Is Overexpressed on T Cells in Hepatitis C Virus Infection and Its Blockade Rescues Dysfunctional CD4+ and CD8+ T Cells

A number of emerging molecules and pathways have been implicated in mediating the T-cell exhaustion characteristic of chronic viral infection. Not all dysfunctional T cells express PD-1, nor are they all rescued by blockade of the PD-1/PD-1 ligand pathway. In this study, we characterize the expression of T-cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) in chronic hepatitis C infection. For the first time, we found that Tim-3 expression is increased on CD4⁺ and CD8⁺ T cells in chronic hepatitis C virus (HCV) infection. The proportion of dually PD-1/Tim-3-expressing cells is greatest in liver-resident T cells, significantly more so in HCV-specific than in cytomegalovirus-specific cytotoxic T lymphocytes. Tim-3 expression correlates with a dysfunctional and senescent phenotype (CD127^low CD57^high), a central rather than effector memory profile (CD45RA^negative CCR7^high), and reduced Th1/Tc1 cytokine production. We also demonstrate the ability to enhance T-cell proliferation and gamma interferon production in response to HCV-specific antigens by blocking the Tim-3-Tim-3 ligand interaction. These findings have implications for the development of novel immunotherapeutic approaches to this common viral infection.Hepatitis C virus (HCV) is a major causative agent of chronic hepatitis, affecting approximately 200 million people throughout the world; the majority of individuals exposed to HCV become persistently infected (19). A broad array of functional impairments of virus-specific T cells from early to chronic stages of infection, including exhaustion (decreased antiviral cytokine production, cytotoxicity, and proliferative capacity) (8, 24) and arrested stages of differentiation (1, 13), is supported by considerable evidence. Recently, upregulation of programmed death 1 (PD-1) and downmodulation of CD127 (interleukin-7 [IL-7] receptor) have been linked to functional exhaustion of T cells in chronic HCV infection (5-7, 15, 21, 22). However, not all exhausted T cells express these phenotypic changes, and blockade of the PD-1/PD-L1 signaling pathway does not always reconstitute Th1/Tc1 cytokine production (4, 5), indicating that other molecules may contribute to the exhaustion typically associated with chronic viral infections (9). One such molecule is Tim-3 (T-cell immunoglobulin and mucin domain-containing molecule 3), a membrane protein initially identified on terminally differentiated Th1 but not Th2 cells in mice (9). A recent analysis of human immunodeficiency virus (HIV) infection demonstrates that Tim-3 is upregulated on both CD4⁺ and CD8⁺ T cells from patients with chronic infection relative to uninfected individuals and that virus-specific cells expressing high levels of Tim-3 secrete less IFN-γ than do Tim-3-negative cells (10). In light of these findings, for the first time, this study assessed the expression of Tim-3 in chronic HCV infection. We found a higher frequency of Tim3-expressing CD4⁺ and CD8⁺ T cells in chronic HCV infection, with the highest on HCV-specific cytotoxic T lymphocytes (CTLs). Tim-3 expression correlates with a dysfunctional phenotype and reduced Th1/Tc1 cytokine production but not viral load. We also demonstrated the ability to enhance T-cell proliferation in response to HCV-specific antigens by blocking the Tim-3-Tim-3 ligand interaction. These findings have implications for the development of novel immunotherapeutic approaches to this common disease.     

Combining Regulatory T Cell Depletion and Inhibitory Receptor Blockade Improves Reactivation of Exhausted Virus-Specific CD8+ T Cells and Efficiently Reduces Chronic Retroviral Loads

Chronic infections with human viruses, such as HIV and HCV, or mouse viruses, such as LCMV or Friend Virus (FV), result in functional exhaustion of CD8⁺ T cells. Two main mechanisms have been described that mediate this exhaustion: expression of inhibitory receptors on CD8⁺ T cells and expansion of regulatory T cells (Tregs) that suppress CD8⁺ T cell activity. Several studies show that blockage of one of these pathways results in reactivation of CD8⁺ T cells and partial reduction in chronic viral loads. Using blocking antibodies against PD-1 ligand and Tim-3 and transgenic mice in which Tregs can be selectively ablated, we compared these two treatment strategies and combined them for the first time in a model of chronic retrovirus infection. Blocking inhibitory receptors was more efficient than transient depletion of Tregs in reactivating exhausted CD8⁺ T cells and reducing viral set points. However, a combination therapy was superior to any single treatment and further augmented CD8⁺ T cell responses and resulted in a sustained reduction in chronic viral loads. These results demonstrate that Tregs and inhibitory receptors are non-overlapping factors in the maintenance of chronic viral infections and that immunotherapies targeting both pathways may be a promising strategy to treat chronic infectious diseases.     

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.     

Inhibition of Donor-Reactive CD8+ T Cell Responses by Selective CD28 Blockade Is Independent of Reduced ICOS Expression

Programmed T cell differentiation is critically influenced by the complement of costimulatory and coinhibitory signals transmitted during initial antigen encounter. We previously showed that selective CD28 blockade with novel domain antibodies that leave CTLA-4-mediated coinhibitory signaling intact resulted in more profound attenuation of donor-reactive T cell responses and improved graft survival in a murine transplant model. Selective CD28 blockade was also associated with decreased ICOS expression on donor-reactive CD8⁺ T cell responses as compared to CTLA-4 Ig, but the functional importance of this reduced ICOS expression was not known. In this study, we created retrogenic donor-reactive CD8⁺ T cells that overexpress ICOS in order to determine whether reduced ICOS expression mechanistically underlies the increased efficacy of selective CD28 blockade in controlling graft-specific T cell responses as compared to conventional costimulation blockade with CTLA-4 Ig. Results indicated that the ability of selective CD28 blockade to blunt donor-reactive CD8⁺ T cell expansion following transplantation was independent of its ability to inhibit ICOS expression. Furthermore, we have previously published that 2B4 coinhibitory signals are functionally important for controlling graft-specific CD8⁺ T cell responses in mice treated with CD28 blockade. Here we used a co-adoptive transfer approach to determine that 2B4 coinhibitory signals on antigen-specific CD8⁺ T cells function in a cell-intrinsic manner to limit ICOS expression in the setting of selective CD28 blockade.     

CD8+ T Cell Priming by Dendritic Cell Vaccines Requires Antigen Transfer to Endogenous Antigen Presenting Cells

Background

Immunotherapeutic strategies to stimulate anti-tumor immunity are promising approaches for cancer treatment. A major barrier to their success is the immunosuppressive microenvironment of tumors, which inhibits the functions of endogenous dendritic cells (DCs) that are necessary for the generation of anti-tumor CD8+ T cells. To overcome this problem, autologous DCs are generated ex vivo, loaded with tumor antigens, and activated in this non-suppressive environment before administration to patients. However, DC-based vaccines rarely induce tumor regression.

Methodology/Principal Findings

We examined the fate and function of these DCs following their injection using murine models, in order to better understand their interaction with the host immune system. Contrary to previous assumptions, we show that DC vaccines have an insignificant role in directly priming CD8+ T cells, but instead function primarily as vehicles for transferring antigens to endogenous antigen presenting cells, which are responsible for the subsequent activation of T cells.

Conclusions/Significance

This reliance on endogenous immune cells may explain the limited success of current DC vaccines to treat cancer and offers new insight into how these therapies can be improved. Future approaches should focus on creating DC vaccines that are more effective at directly priming T cells, or abrogating the tumor induced suppression of endogenous DCs.     

Development of Virus-Specific CD4+ and CD8+ Regulatory T Cells Induced by Human Herpesvirus 6 Infection

Human herpesvirus 6 (HHV-6) is an important immunosuppressive and immunomodulatory virus. The mechanisms by which HHV-6 establishes latency and immunosuppression in its host are not well understood. Here we characterized HHV-6-specific T cells in peripheral blood mononuclear cells (PBMCs) from HHV-6-infected donors. Our results showed that HHV-6 infection could induce both CD4⁺ and CD8⁺ HHV-6-specific regulatory T (Treg) cells. These HHV-6-specific Treg cells had potent suppressive activity and expressed high levels of Treg-associated molecules CD25, FoxP3, and GITR. Both CD4⁺ and CD8⁺ Treg cells secreted gamma interferon (IFN-γ) and interleukin-10 (IL-10) but little or no IL-2, IL-4, or transforming growth factor β (TGF-β). Furthermore, HHV-6-specifc Treg cells not only could suppress naive and HHV-6-specific CD4⁺ effector T cell immune responses but also could impair dendritic cell (DC) maturation and functions. In addition, the suppressive effects mediated by HHV-6-specific Treg cells were mainly through a cell-to-cell contact-dependent mechanism but not through the identified cytokines. These results suggest that HHV-6 may utilize the induction of Treg cells as a strategy to escape antivirus immune responses and maintain the latency and immunosuppression in infected hosts.     

Priming of Salmonella enterica Serovar Typhi-Specific CD8+ T Cells by Suicide Dendritic Cell Cross-Presentation in Humans

Background

The emergence of antibiotic-resistant strains of Salmonella enterica serovar Typhi (S. Typhi), the etiologic agent of typhoid fever, has aggravated an already important public health problem and added new urgency to the development of more effective typhoid vaccines. To this end it is critical to better understand the induction of immunity to S. Typhi. CD8⁺ T cells are likely to play an important role in host defense against S. Typhi by several effector mechanisms, including killing of infected cells and IFN-γ secretion. However, how S. Typhi regulates the development of specific CD8⁺ responses in humans remains unclear. Recent studies in mice have shown that dendritic cells (DC) can either directly (upon uptake and processing of Salmonella) or indirectly (by bystander mechanisms) elicit Salmonella-specific CD8⁺ T cells.

Methodology/Principal Findings

We report here that upon infection with live S. Typhi, human DC produced high levels of pro-inflammatory cytokines IL-6, IL-8 and TNF-α, but low levels of IL-12 p70 and IFN-γ. In contrast, DC co-cultured with S. Typhi-infected cells, through suicide cross-presentation, uptake S. Typhi-infected human cells and release high levels of IFN-γ and IL-12p70, leading to the subsequent presentation of bacterial antigens and triggering the induction of memory T cells, mostly CD3⁺CD8⁺CD45RA⁻CD62L⁻ effector/memory T cells.

Conclusions/Significance

This study is the first to demonstrate the effect of S. Typhi on human DC maturation and on their ability to prime CD8⁺ cells and highlights the significance of these phenomena in eliciting adaptive immunity to S. Typhi.     

Functional CD169 on Macrophages Mediates Interaction with Dendritic Cells for CD8+ T Cell Cross-Priming

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Neuroantigen-Specific Autoregulatory CD8+ T Cells Inhibit Autoimmune Demyelination through Modulation of Dendritic Cell Function

Experimental autoimmune encephalomyelitis (EAE) is a well-established murine model of multiple sclerosis, an immune-mediated demyelinating disorder of the central nervous system (CNS). We have previously shown that CNS-specific CD8+ T cells (CNS-CD8+) ameliorate EAE, at least in part through modulation of CNS-specific CD4+ T cell responses. In this study, we show that CNS-CD8+ also modulate the function of CD11c+ dendritic cells (DC), but not other APCs such as CD11b+ monocytes or B220+ B cells. DC from mice receiving either myelin oligodendrocyte glycoprotein-specific CD8+ (MOG-CD8+) or proteolipid protein-specific CD8+ (PLP-CD8+) T cells were rendered inefficient in priming T cell responses from naïve CD4+ T cells (OT-II) or supporting recall responses from CNS-specific CD4+ T cells. CNS-CD8+ did not alter DC subset distribution or MHC class II and CD86 expression, suggesting that DC maturation was not affected. However, the cytokine profile of DC from CNS-CD8+ recipients showed lower IL-12 and higher IL-10 production. These functions were not modulated in the absence of immunization with CD8-cognate antigen, suggesting an antigen-specific mechanism likely requiring CNS-CD8-DC interaction. Interestingly, blockade of IL-10 in vitro rescued CD4+ proliferation and in vivo expression of IL-10 was necessary for the suppression of EAE by MOG-CD8+. These studies demonstrate a complex interplay between CNS-specific CD8+ T cells, DC and pathogenic CD4+ T cells, with important implications for therapeutic interventions in this disease.     

A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ

The numbers and locations of virus-specific CD8+ T cells relative to the numbers and locations of their infected cell targets is thought to be critical in determining outcomes that range from clearance to chronic persistent infections. We describe here a method for assessing the spatial and quantitative relationships between immune effector (E) virus-specific CD8+ T cells and infected targets (T) that combines in situ tetramer (IST) staining to detect virus-specific CD8+ T cells and in situ hybridization (ISH) to detect viral-RNA+ cells in the tissues where the battle between immune defenses and infection takes place. The combination of IST staining and ISH, abbreviated ISTH, enables visualization and mapping of the locations of immune effector cells and targets, and facile determination of E:T ratios. These parameters in turn can then be used to determine the relationships between spatial proximity, and the timing and magnitude of the immune response that predict outcomes in early infection.Download video file.^{(240M, mp4)}     

Identification of Varicella-Zoster Virus-Specific CD8 T Cells in Patients after T-Cell-Depleted Allogeneic Stem Cell Transplantation

To study the role of CD8 T cells in the control of varicella-zoster virus (VZV) reactivation, we developed multimeric major histocompatibility complexes to identify VZV-specific CD8 T cells. Potential HLA-A2 binding peptides from the putative immediate-early 62 protein (IE62) of VZV were tested for binding, and peptides with sufficient binding capacity were used to generate pentamers. Patients with VZV reactivation following stem cell transplantation were screened with these pentamers, leading to the identification of the first validated class I-restricted epitope of VZV. In 42% of HLA-A2 patients following VZV reactivation, these IE62-ALW-A2 T cells could be detected ex vivo.Varicella-zoster virus (VZV) infects about 95% of the population, persists throughout life, and may lead to herpes zoster when the virus reactivates. After T-cell-depleted allogeneic stem cell transplantation (TCD alloSCT), reactivation of the virus leads to considerable morbidity (10). Primary infection elicits both humoral and cellular responses, but cellular immunity is essential for preventing herpes zoster. The VZV genome comprises more than 70 unique open reading frames that encode proteins that are coordinately expressed during replication. The product of open reading frame 62, the immediate-early 62 (IE62) protein, is required for the initiation of VZV replication (9) and is expressed at high levels before viral replication has occurred (8). Previous research has demonstrated that IE62-specific T cells were detected after primary VZV infection and in immune subjects (2, 4). In addition, T cells recognizing various other IE proteins and glycoproteins of VZV, as demonstrated by gamma interferon (IFN-γ) production upon stimulation with peptides or lysate derived from these proteins, have been described (1, 6, 13). The VZV-specific memory T cells found in these studies were predominantly CD4 T cells, while no VZV-specific CD8 T cells were demonstrated without prior in vitro expansion, possibly due to the low frequency of VZV-specific CD8 T cells or to the low sensitivity of the screening methods used to detect CD8 T cells by IFN-γ production upon stimulation. Frey et al. described CD8 epitopes of IE62 detected following in vitro restimulation. However, the HLA restriction and specificity of these T cells were not confirmed (4). Due to the lack of validated VZV-derived immunodominant peptides for major histocompatibility complex (MHC) class I, the analysis of VZV-specific CD8 T-cell responses is hampered (14). To be able to analyze the role of CD8 T cells in VZV reactivation, we therefore set out to identify epitopes for VZV by using VZV-IE62-specific MHC class I peptide complexes.The predictive algorithms BIMAS (11) and SYFPEITHI (12) were used to select potential HLA-A2 binding peptides from the IE62 protein. Peptides with a score of ≥3 (BIMAS) or ≥20 (SYFPEITHI) were considered to have potentially significant binding affinity. The 81 resulting 9-mer peptides were synthesized and tested for binding affinity with the REVEAL MHC-peptide binding assay (ProImmune, Oxford, United Kingdom). HLA-A2 binding affinity was determined by the ability of the peptides to stabilize the HLA-peptide complex. Based on the binding affinity measurements, 34 high- to medium-affinity HLA-A2 binding peptides were selected and used to generate ProVE MHC pentamers (ProImmune, Oxford, United Kingdom). To enable screening of this large number of pentamers, the pentamers were divided into five pools, each containing six or seven pentamers. In the initial screening with pooled pentamers, four HLA-A2-positive patients were screened after a clinical diagnosis of VZV reactivation after TCD alloSCT. The presence of viral DNA in plasma at the time of clinical observations of VZV reactivation was confirmed by real-time PCR on plasma samples as previously described (7). After informed consent was obtained, peripheral blood mononuclear cells (PBMCs) were cryopreserved and thawed and 0.5 × 10⁶ cells were incubated with pentamers at a concentration of 0.03 mg/ml for 10 min at room temperature in RPMI medium supplemented with 2% fetal bovine serum. After the cells were washed twice, 8 μl of FluoroTag-phycoerythrin (PE) was added for 20 min of incubation at 4°C and the cells were counterstained with CD4, CD40, and CD19-fluorescein isothiocyanate (FITC). Flow cytometric analysis was performed on a FACScalibur fluorescence-activated cell sorter (FACS; Becton-Dickinson [BD], San Jose, CA). In one of four patients, pentamer pool 6, containing pentamers 61, 62, 64, 65, 66, and 67, was positive (0.06% of CD8 T cells); no other positive signals were observed. Staining with the individual pentamers revealed that pentamer 66, containing the epitope ALWALPHAA derived from the IE62 protein of VZV (IE62-ALW-A2) was responsible for the positive signal (0.06% of CD8 T cells, Fig. ​Fig.1B1B).Open in a separate windowFIG. 1.Screening with pentamers containing VZV-derived immunogenic epitopes. PBMCs of a patient after VZV reactivation following TCD alloSCT were incubated with pentamers and then stained with FluoroTag-PE to detect the pentamer-positive cells (A and B) and counterstained with CD4-, CD40-, and CD19-FITC. Pentamer staining of the CD4-, CD40-, and CD19-negative cells is shown. (A) PBMCs stained with pentamer 67 containing the epitope ALPHAAAAV, showing no specific staining. (B) PBMCs stained with pentamer 66 containing the epitope ALWALPHAA, showing specific staining. IE62-ALW-A2-specific T-cell clones were sorted into a single cell per well and expanded nonspecifically. The clones were stained with an irrelevant tetramer (C) and the IE62-ALW-A2 tetramer (D) in combination with CD8-FITC. Clones 1 and 2 were stained with a Vβ kit (BD) to demonstrate that clone 1 (E) and clone 2 (F) express different T-cell receptors. The results demonstrate that we isolated different T-cell clones that specifically stain with the IE62-ALW-A2 tetramer.To confirm the specificity of the IE62-ALW-A2-specific T cells, the pentamer-positive T cells were sorted into a single cell per well with a FACSDiva (BD) and expanded as previously described (5). The expanded T-cell clones were labeled specifically with the IE62-ALW-A2 PE-conjugated tetramer that was constructed as previously described (3) (Fig. ​(Fig.1D),1D), and Vβ analysis with the T-cell receptor Vβ repertoire kit (BD) showed that at least two different T-cell clones were isolated, demonstrating the oligoclonal origin of IE62-ALW-A2-positive T cells (Fig. 1E and F). To assess the cytolytic capacity of IE62-ALW-A2 T cells, chromium release assays were performed as described earlier (5). ⁵¹Cr-labeled Epstein-Barr virus (EBV) lymphoblastoid cell lines (LCLs) loaded with the IE62-ALW peptide were incubated with IE62-ALW-A2 T cells for 4 h. As demonstrated in Fig. ​Fig.2A,2A, HLA-A2-positive EBV LCLs loaded with the IE62-ALW-A2 peptide were lysed by both T-cell clones, whereas unloaded EBV LCLs were not lysed. To determine the avidity of the T-cell clones, the IE62-ALW-A2 peptide was titrated on EBV LCLs, and after 24 h of coculture, supernatants were harvested and used to determine the IFN-γ production of the stimulated T cells by standard enzyme-linked immunosorbent assay. Half-maximum IFN-γ production of the T-cell clones was observed when the stimulator cells were loaded with 10 ng/ml peptide, indicative of high-avidity T-cell clones (Fig. ​(Fig.2B).2B). To determine whether the T cells recognized cells endogenously expressing the IE-62-encoding gene, COS-A2 cells were transfected with Lipofectamine (Invitrogen, Carlsbad, CA) by using pcDNA vectors coding for different VZV genes, which were kindly provided by E. Wiertz (Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands). The transfected COS-A2 cells were used 24 h after transfection as stimulator cells in this assay. After 24 h of coculture, supernatants were harvested and used to determine the IFN-γ production of the stimulated T cells. IE62-ALW-A2 T-cell clones produced IFN-γ in response to COS-A2 cells endogenously expressing the IE62 protein, as well as COS-A2 cells pulsed with the IE62-ALW-A2 peptide. No IFN-γ was produced when the COS-A2 cells were transfected with the IE63-encoding gene of VZV or pulsed with an irrelevant peptide (Fig. ​(Fig.2C2C).Open in a separate windowFIG. 2.IE62-ALW-A2 T cells recognize IE62-ALW-A2 peptide-loaded target cells and target cells endogenously expressing IE62. (A) The cytolytic activity of IE62-ALW-A2-positive T-cell clones 1 and 2 was analyzed with the ⁵¹Cr release assay. T cells were incubated for 4 h with IE62-ALW-A2 peptide (pep)-loaded or unloaded, HLA-A2-positive EBV LCLs at an effector-to-target ratio of 10:1. (B) IE62-ALW-A2 T-cell clone 1 was stimulated with HLA-A2-positive EBV LCLs loaded with different concentrations of the IE62-ALW-A2 peptide. Release of IFN-γ (pg/ml) after 24 h of stimulation is shown. (C) IE62-ALW-A2 T-cell clones 1 and 2 were stimulated with HLA-A2-positive COS-A2 cells, left untreated, or loaded with the IE62-ALW-A2 peptide or with the IE4-ALR-B8 peptide as an irrelevant peptide or transfected with the IE63-encoding gene (COS-A2-IE63) or the IE62-encoding gene (COS-A2-IE62). Release of IFN-γ (picograms per milliliter) after 24 h of stimulation is shown.To determine whether IE62-ALW-A2-specific T cells were present in healthy individuals, cryopreserved PBMCs from 18 healthy, VZV-seropositive, HLA-A2-positive individuals were screened with the PE-conjugated VZV tetramer. PBMCs were labeled with tetramers for 15 min at 37°C in RPMI medium without phenol supplemented with 2% fetal bovine serum, washed, and analyzed with a FACScalibur. In 3 of these 18 serologically VZV-positive individuals, IE62-ALW-A2 tetramer-positive T cells could be detected (range, 0.01 to 0.02% of CD8 T cells). These data demonstrate that IE62-ALW-A2-specific T cells can be observed and that the frequency of these T cells is low under steady-state conditions in immunocompetent persons.To assess the frequency of IE62-ALW-A2-specific T cells in a cohort of patient who suffered from VZV reactivation following TCD alloSCT, 19 HLA-A2-positive patients after VZV reactivation following TCD alloSCT were screened by using the IE62-ALW-A2 tetramer. We screened these patients at a median of 47 days after the clinical diagnosis of VZV reactivation. In 8 of these 19 patients, IE62-ALW-A2-specific T cells could be directly detected ex vivo (mean, 0.04% [range, 0.01 to 0.11%] of CD8 T cells), indicating that this epitope is recognized in 42% of the HLA-A2-positive patients during VZV reactivation (Table ​(Table1).1). In VZV-seronegative patients (six screened), no IE62-ALW-A2 tetramer-positive cells could be detected.

TABLE 1.

Presence of IE62-ALW-A2-specific T cells in HLA-A2 patients after VZV reactivation following TCD alloSCT

Persistent Enteric Murine Norovirus Infection Is Associated with Functionally Suboptimal Virus-Specific CD8 T Cell Responses

Norovirus (NV) gastroenteritis is a major contributor to global morbidity and mortality, yet little is known about immune mechanisms leading to NV control. Previous studies using the murine norovirus (MNV) model have established a key role for T cells in MNV clearance. Despite these advances, important questions remain regarding the magnitude, location, and dynamics of the MNV-specific T cell response. To address these questions, we identified MNV-specific major histocompatibility complex (MHC) class I immunodominant epitopes using an overlapping peptide screen. One of these epitopes (amino acids 519 to 527 of open reading frame 2 [ORF2^519-527]) was highly conserved among all NV genogroups. Using MHC class I peptide tetramers, we tracked MNV-specific CD8 T cells in lymphoid and mucosal sites during infection with two MNV strains with distinct biological behaviors, the acutely cleared strain CW3 and the persistent strain CR6. Here, we show that enteric MNV infection elicited robust T cell responses primarily in the intestinal mucosa and that MNV-specific CD8 T cells dynamically regulated the expression of surface molecules associated with activation, differentiation, and homing. Furthermore, compared to MNV-CW3 infection, chronic infection with MNV-CR6 resulted in fewer and less-functional CD8 T cells, and this difference was evident as early as day 8 postinfection. Finally, MNV-specific CD8 T cells were capable of reducing the viral load in persistently infected Rag1^−/− mice, suggesting that these cells are a crucial component of NV immunity. Collectively, these data provide fundamental new insights into the adaptive immune response to two closely related NV strains with distinct biological behaviors and bring us closer to understanding the correlates of protective antiviral immunity in the intestine.     

Enhanced Cross-Presentation and Improved CD8+ T Cell Responses after Mannosylation of Synthetic Long Peptides in Mice

The use of synthetic long peptides (SLP) has been proven to be a promising approach to induce adaptive immune responses in vaccination strategies. Here, we analyzed whether the efficiency to activate cytotoxic T cells by SLP-based vaccinations can be increased by conjugating SLPs to mannose residues. We could demonstrate that mannosylation of SLPs results in increased internalization by the mannose receptor (MR) on murine antigen-presenting cells. MR-mediated internalization targeted the mannosylated SLPs into early endosomes, from where they were cross-presented very efficiently compared to non-mannosylated SLPs. The influence of SLP mannosylation was specific for cross-presentation, as no influence on MHC II-restricted presentation was observed. Additionally, we showed that vaccination of mice with mannosylated SLPs containing epitopes from either ovalbumin or HPV E7 resulted in enhanced proliferation and activation of antigen-specific CD8⁺ T cells. These findings demonstrate that mannosylation of SLPs augments the induction of a cytotoxic T cell response in vitro and in vivo and might be a promising approach to induce cytotoxic T cell responses in e.g. cancer therapy and anti-viral immunity.