Association between peripheral IFN-gamma producing CD8+ T-cells and disability score in relapsing-remitting multiple sclerosis

A large body of evidence supports the involvement of the immune system in the pathogenesis of multiple sclerosis (MS). Nevertheless, how the peripheral T-cells phenotypes are associated with factors such as the disability score, the effects of immunomodulatory treatments, or the activation period is poorly understood. In this study, we have centered our attention on the presence of IFN-gamma and IL-4 producing CD4+ and CD8+ T-cells in the peripheral blood of 58 relapsing-remitting MS (RRMS) patients, 48 that were stable and 10 who were in relapse period, and 30 healthy controls (HC). Our results support the existence of an independent association between the percentage of IFN-gamma producing CD8+ lymphocytes and the increased levels of disability score. Furthermore, the number of IFN-gamma producing CD8+ lymphocytes and the disability score were not correlated in patients treated with interferon-beta, evidence of its possible benefits in combating a pro-inflammatory profile. Finally, we compared the T-cell populations in RRMS patients in the stable or active period, and we found a significant decrease of IFN-gamma producing CD4+ lymphocytes in active patients. In conclusion our study supports the hypothesis that different peripheral blood T-cell phenotypes are associated with disability score or active period of the disease.     

Persistence, immune specificity, and functional ability of murine mutant ras epitope-specific CD4(+) and CD8(+) T lymphocytes following in vivo adoptive transfer.

Adoptive T-cell transfer has been shown to be a potentially effective strategy for cellular immunotherapy in some murine models of disease. However, several issues remain unresolved regarding some of the basic features involved in effective adoptive transfer, such as the influence of specific peptide antigen (Ag) boost after T-cell transfer, the addition of IL-2 post-T-cell transfer, the trafficking of transferred T cells to lymphoid and nonlymphoid tissues, and the functional stability of recoverable CD4(+) and CD8(+) T cells. We investigated several of these parameters, particularly as they relate to the persistence and maintenance of effector functions of murine CD4(+) and/or CD8(+) T lymphocytes after adoptive cellular transfer into partially gamma-irradiated syngeneic hosts. Our laboratory previously identified murine (H-2(d)) immunogenic CD4(+) and CD8(+) T-cell peptide epitopes reflecting codon 12 ras mutations as tumor-specific Ag. Therefore, the model system chosen here employed epitope-specific MHC class II-restricted CD4(+) T cells and MHC class I-restricted CD8(+) T cells produced from previously immunized BALB/c mice. Between 2 and 7 days after T-cell transfer, recipient mice received various combinations of peptide boosts and/or IL-2 treatments. At different times after the T-cell transfer, spleen and lung tissues were analyzed phenotypically to monitor the persistence of the immune T cells and functionally (via proliferation or cytotoxicity assays) to assess the maintenance of peptide specificity. The results showed that immune donor T lymphocytes (uncultured immune T cells or cloned T cells) were recoverable from the spleens and lungs of recipient mice after transfer. The recovery of Ag-specific T-cell responses was greatest from recipient mice that received peptide boosts and IL-2 treatment. However, mice that received a peptide boost without IL-2 treatment responded nearly as well, which suggested that including a peptide boost after T-cell transfer was more obligatory than exogenous IL-2 treatment to sustain adoptively transferred T cells in vivo. Ag-specific T-cell responses were weak in mice that either received IL-2 alone or did not receive the cognate peptide boost after T-cell transfer. The T-cell clones were also monitored by flow cytometry or RT-PCR based on expression of the T-cell receptor Vbeta-chain, which was previously characterized. Ag-specific T cells were recovered from both spleens and lungs of recipient mice, demonstrating that the T-cell clones could localize to both lymphoid and nonlymphoid tissues. This study demonstrates that both uncultured and in vitro-cloned T lymphocytes can migrate to lymphoid tissues and nonlymphoid (e.g., lung) tissues in recipient hosts and that their functional activities can be maintained at these sites after transfer, if they are exposed to peptide Ag in vivo.     

Rapid accumulation of adoptively transferred CD8+ T cells at the tumor site is associated with long-term control of SV40 T antigen-induced tumors

We previously established a model to study CD8⁺ T cell (T_CD8)-based adoptive immunotherapy of cancer using line SV11 mice that develop choroid plexus tumors in the brain due to transgenic expression of Simian Virus 40 large T antigen (Tag). These mice are tolerant to the three dominant T_CD8-recognized Tag epitopes I, II/III and IV. However, adoptive transfer of spleen cells from naïve C57BL/6 (B6) mice prolongs SV11 survival following T_CD8 priming against the endogenous Tag epitope IV. In addition, survival of SV11 mice is dramatically increased following transfer of lymphocytes from Tag-immune B6 mice. In the current study, we compared the kinetics and magnitude of Tag-specific T_CD8 accumulation at the tumor site following adoptive transfer with a high dose of either Tag-immune or naïve donor cells or decreasing doses of Tag-immune lymphocytes. Following adoptive transfer of Tag-immune cells, epitope I- and IV-specific T_CD8 accumulated to high levels in the brain of SV11 mice, peaking at 5–7 days, while epitope IV-specific T_CD8 derived from naïve donors required three weeks to achieve peak levels. A similar delay in the peak of epitope IV-specific T_CD8 accumulation was observed when tenfold fewer Tag-immune donor cells were administered, reducing control of tumor progression. These results suggest that efficient and prolonged control of established autochthonous tumors is associated with high-level early accumulation of adoptively transferred T cells. We also provide evidence that although multiple specificities are represented in the Tag immune donor lymphocytes, epitope IV-specific donor T_CD8 play a predominant role in control of tumor growth.     

Highly protective in vivo function of cytomegalovirus IE1 epitope-specific memory CD8 T cells purified by T-cell receptor-based cell sorting

Reconstitution of antiviral CD8 T cells is essential for controlling cytomegalovirus (CMV) infection after bone marrow transplantation. Accordingly, polyclonal CD8 T cells derived from BALB/c mice infected with murine CMV protect immunocompromised adoptive transfer recipients against CMV disease. The protective population comprises CD8 T cells with T-cell receptors (TCRs) specific for defined and for as-yet-unknown viral epitopes, as well as a majority of nonprotective cells with unrelated specificities. Defined epitopes include IE1/m123 and m164, which are immunodominant in terms of the magnitude of the CD8 T-cell response, and a panel of subordinate epitopes (m04, m18, M45, M83, and M84). While cytolytic T-lymphocyte lines (CTLLs) were shown to be protective regardless of the immunodominance of the respective epitope, the individual contributions of in vivo resident epitope-specific CD8 T cells to the antiviral control awaited investigation. The IE1 peptide 168-YPHFMPTNL-176 is generated from the immediate-early protein 1 (IE1) (pp89/76) of murine CMV and is presented by the major histocompatibility complex class I (MHC-I) molecule Ld. To quantitate its contribution to the protective potential of a CD8-T memory (CD8-TM) cell population, IE1-TCR+ and IE1-TCR- CD8-TM cells were purified by epitope-specific cell sorting with IE1 peptide-loaded MHC-immunoglobulin G1 dimers as ligands of cognate TCRs. Of relevance for clinical approaches to an adoptive cellular immunotherapy, sorted IE1 epitope-specific CD8-TM cells were found to be exceedingly protective upon adoptive transfer. Compared with CTLLs specific for the same epitope and of comparable avidity and TCR beta-chain variable region (Vbeta)-defined polyclonality, sorted CD8-TM cells proved to be superior by more than 2 orders of magnitude.     

Functional CD4(+) and CD8(+) T-cell responses induced by autologous mitomycin C treated Epstein-Barr virus transformed lymphoblastoid cell lines

Epstein-Barr virus (EBV) gene expression in tumor cells of posttransplant lymphoproliferative disorder (PTLD) patients resembles that of EBV transformed B-cell lines (LCL). EBV-specific cytotoxic T-lymphocytes can be generated by stimulating peripheral blood lymphocytes with autologous LCL. We describe a standardized method for the growth inactivation and cryopreservation of LCL for optimal T-cell stimulation and analyzed the function and phenotype of responding T-cells. LCL growth was completely blocked by mitomycin C treatment (McLCL) and McLCL could be cryopreserved while retaining excellent APC function. McLCL stimulated both CD4(+) and CD8(+) T-cells as measured by HLA-DR and CD25 expression using FACS analysis. EBV-specific CTL activity and T-cell proliferation were induced and immunocytochemical staining showed CD4(+) and (granzyme B positive) CD8(+) T-cells rosetting with McLCL. Granzymes A and B, IFN-gamma, and IL-6 were detected at significant levels in the supernatant. Thus, ex vivo T-cell activation with cryopreserved McLCL results in activation of both CD4(+) and CD8(+) T-cells producing a Th1-like cytokine profile, making this a suitable protocol for adoptive therapy of PTLD.     

Early immunization induces persistent tumor-infiltrating CD8+ T cells against an immunodominant epitope and promotes lifelong control of pancreatic tumor progression in SV40 tumor antigen transgenic mice

The ability to recruit the host's CD8+ T lymphocytes (T(CD8)) against cancer is often limited by the development of peripheral tolerance toward the dominant tumor-associated Ags. Because multiple epitopes derived from a given tumor Ag (T Ag) can be targeted by T(CD8), vaccine approaches should be directed toward those T(CD8) that are more likely to survive under conditions of persistent Ag expression. In this study, we investigated the effect of peripheral tolerance on the endogenous T(CD8) response toward two epitopes, designated epitopes I and IV, from the SV40 large T Ag. Using rat insulin promoter (RIP) 1-Tag4 transgenic mice that express T Ag from the RIP and develop pancreatic insulinomas, we demonstrate that epitope IV- but not epitope I-specific T(CD8) are maintained long term in tumor-bearing RIP1-Tag4 mice. Even large numbers of TCR-transgenic T cells specific for epitope I were rapidly eliminated from RIP1-Tag4 mice after adoptive transfer and recognition of the endogenous T Ag. Importantly, immunization of RIP1-Tag4 mice at 5 wk of age against epitope IV resulted in complete protection from tumor progression over a 2-year period despite continued expression of T Ag in the pancreas. This extensive control of tumor progression was associated with the persistence of functional epitope IV-specific T(CD8) within the pancreas for the lifetime of the mice without the development of diabetes. This study indicates that an equilibrium is reached in which immune surveillance for spontaneous cancer can be achieved for the lifespan of the host while maintaining normal organ function.     

Contribution of CD8 T lymphocytes to the immuno-pathogenesis of multiple sclerosis and its animal models

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) characterized by multi-focal demyelination, axonal loss, and immune cell infiltration. Numerous immune mediators are detected within MS lesions, including CD4⁺ and CD8⁺ T lymphocytes suggesting that they participate in the related pathogenesis. Although CD4⁺ T lymphocytes are traditionally considered the main actors in MS immunopathology, multiple lines of evidence suggest that CD8⁺ T lymphocytes are also implicated in the pathogenesis. In this review, we outline the recent literature pertaining to the potential roles of CD8⁺ T lymphocytes both in MS and its animal models. The CD8⁺ T lymphocytes detected in MS lesions demonstrate characteristics of activated and clonally expanded cells supporting the notion that these cells actively contribute to the observed injury. Moreover, several experimental in vivo models mediated by CD8⁺ T lymphocytes recapitulate important features of the human disease. Whether the CD8⁺ T cells can induce or aggravate tissue destruction in the CNS needs to be fully explored. Strengthening our understanding of the pathogenic potential of CD8⁺ T cells in MS should provide promising new avenues for the treatment of this disabling inflammatory disease.     

Pathogenic potential of borna disease virus lacking the immunodominant CD8 T-cell epitope

Borna disease virus (BDV) is a highly neurotropic, noncytolytic virus. Experimentally infected B10.BR mice remain healthy unless specific antiviral T cells that infiltrate the infected brain are triggered by immunization. In contrast, infected MRL mice spontaneously mount an antiviral T-cell response that can result in meningoencephalitis and neurological disease. The antiviral T cells may, alternatively, eliminate the virus without inducing disease if they are present in sufficient numbers before the virus replicates to high titers. Since the immune response of H-2(k) mice is directed mainly against the epitope TELEISSI located in the viral nucleoprotein N, we generated BDV mutants that feature TQLEISSI in place of TELEISSI. We show that adoptive transfer of BDV N-specific CD8 T cells induced neurological disease in B10.BR mice persistently infected with wild-type BDV but not with the mutant virus expressing TQLEISSI. Surprisingly, the mutant virus replicated less well in adult MRL wild-type mice than in mutant mice lacking mature CD8 T cells. Furthermore, when MRL mice were infected with the TQLEISSI-expressing BDV mutant as newborns, neurological disease was observed, although at a lower rate and with slower kinetics than in mice infected with wild-type virus. These results confirm that TELEISSI is the major CD8 T-cell epitope in H-2(k) mice and suggest that unidentified minor epitopes are present in the BDV proteome which are recognized rather efficiently by antiviral T cells if the dominant epitope is absent.     

Functional CD8+ T cell responses in lethal Ebola virus infection

Ebola virus (EBOV) causes highly lethal hemorrhagic fever that leads to death in up to 90% of infected humans. Like many other infections, EBOV induces massive lymphocyte apoptosis, which is thought to prevent the development of a functional adaptive immune response. In a lethal mouse model of EBOV infection, we show that there is an increase in expression of the activation/maturation marker CD44 in CD4(+) and CD8(+) T cells late in infection, preceding a dramatic rebound of lymphocyte numbers in the blood. Furthermore, we observed both lymphoblasts and apoptotic lymphocytes in spleen late in infection, suggesting that there is lymphocyte activation despite substantial bystander apoptosis. To test whether these activated lymphocytes were functional, we performed adoptive transfer studies. Whole splenocytes from moribund day 7 EBOV-infected animals protected naive animals from EBOV, but not Marburgvirus, challenge. In addition, we observed EBOV-specific CD8(+) T cell IFN-gamma responses in moribund day 7 EBOV-infected mice, and adoptive transfer of CD8(+) T cells alone from day 7 mice could confer protection to EBOV-challenged naive mice. Furthermore, CD8(+) cells from day 7, but not day 0, mice proliferated after transfer to infected recipients. Therefore, despite significant lymphocyte apoptosis, a functional and specific, albeit insufficient, adaptive immune response is made in lethal EBOV infection and is protective upon transfer to naive infected recipients. These findings should cause a change in the current view of the 'impaired' immune response to EBOV challenge and may help spark new therapeutic strategies to control lethal filovirus disease.     

Sorting and pooling strategy: a novel tool to map a virus proteome for CD8 T-cell epitopes

Human cytomegalovirus (CMV) is a major cause of morbidity in immunocompromised individuals. However, no efficient vaccine has been developed to date. Identification of T-cell target proteins and epitopes is crucial not only for developing a successful immunization strategy, but also for new approaches using adoptive transfer of antigen-specific T-cells. The CMV genome has more than 200 open reading frames potentially coding for as many proteins. Here, we describe a robust, fast, and simple SPOT synthesis strategy, which allowed us to micro-synthesize every possible CD8 T-cell epitope in the entire potential CMV proteome. So far, 9069 of these peptides have been tested in an ex vivo T-cell stimulation assay. As well as confirming a number of previously known epitopes, we identified several new ones.     

Comparison of influenza and SIV specific CD8 T cell responses in macaques

Macaques are a potentially useful non-human primate model to compare memory T-cell immunity to acute virus pathogens such as influenza virus and effector T-cell responses to chronic viral pathogens such as SIV. However, immunological reagents to study influenza CD8(+) T-cell responses in the macaque model are limited. We recently developed an influenza-SIV vaccination model of pigtail macaques (Macaca nemestrina) and used this to study both influenza-specific and SIV-specific CD8(+) T-cells in 39 pigtail macaques expressing the common Mane-A*10(+) (Mane-A01*084) MHC-I allele. To perform comparative studies between influenza and SIV responses a common influenza nucleoprotein-specific CD8(+) T-cell response was mapped to a minimal epitope (termed RA9), MHC-restricted to Mane-A*10 and an MHC tetramer developed to study this response. Influenza-specific memory CD8(+) T-cell response maintained a highly functional profile in terms of multitude of effector molecule expression (CD107a, IFN-γ, TNF-α, MIP-1β and IL-2) and showed high avidity even in the setting of SIV infection. In contrast, within weeks following active SIV infection, SIV-specific CD8(+) effector T-cells expressed fewer cytokines/degranulation markers and had a lower avidity compared to influenza specific CD8(+) T-cells. Further, the influenza specific memory CD8 T-cell response retained stable expression of the exhaustion marker programmed death-marker-1 (PD-1) and co-stimulatory molecule CD28 following infection with SIV. This contrasted with the effector SIV-specific CD8(+) T-cells following SIV infection which expressed significantly higher amounts of PD-1 and lower amounts of CD28. Our results suggest that strategies to maintain a more functional CD8(+) T-cell response, profile may assist in controlling HIV disease.     

Reinforcement of cancer immunotherapy by adoptive transfer of cblb-deficient CD8+ T cells combined with a DC vaccine

The success of cancer immunotherapy is limited by potent endogenous immune-evasion mechanisms, which are at least in part mediated by transforming growth factor-β (TGF-β). The E3 ubiquitin ligase Cbl-b is a key regulator of T cell activation and is established to regulate TGF-β sensitivity. cblb-deficient animals reject tumors via CD8(+) T cells, which make Cbl-b an ideal target for improvement of adoptive T-cell transfer (ATC) therapy. In this study, we show that cblb-deficient CD8(+) T cells are hyper-responsive to T-cell receptor (TCR)/CD28-stimulation and are in part protected against the negative cues induced by TGF-β in vitro. Notably, adoptive transfer of polyclonal, non-TCR transgenic cblb-deficient CD8(+) T cells is not sufficient to reject B16-ova or EG7 tumors in vivo. Thus, cblb-deficient ATC requires proper in vivo re-activation by a dendritic cell (DC) vaccine. In strict contrast to ATC monotherapy, this approach delayed tumor outgrowth and significantly increased survival rates, which is paralleled by increased CD8(+) T-cells infiltration to the tumor site and enrichment of ova-specific and interferon-γ (IFN-γ)-secreting CD8(+) T cell in the draining lymph node (LN). Moreover, CD8(+) T cells from cblb-deficient mice vaccinated with the DC vaccine show increased cytolytic activity in vivo. In summary, our data using cblb-deficient polyclonal, non-TCR-transgenic adoptively transferred CD8(+) T cells into immuno-competent non-lymphodepleted recipients suggest that targeting Cbl-b might serve as a novel 'adjuvant approach', suitable to augment the effectiveness of established anti-cancer immunotherapies.     

Trafficking of antigen-specific CD8+ T lymphocytes to mucosal surfaces following intramuscular vaccination

A critical goal of vaccine development for a wide variety of pathogens is the induction of potent and durable mucosal immunity. However, it has been assumed that this goal would be difficult to achieve by systemic vaccination due to the anatomic and functional distinctness of the systemic and mucosal immune systems and the resultant compartmentalization of immune responses. In this study, we show that Ag-specific CD8(+) T lymphocytes traffic efficiently to mucosal surfaces following systemic vaccination. Intramuscular immunization with recombinant adenovirus (rAd) vector-based vaccines expressing SIV Gag resulted in potent, durable, and functional CD8(+) T lymphocyte responses at multiple mucosal effector sites in both mice and rhesus monkeys. In adoptive transfer studies in mice, vaccine-elicited systemic CD8(+) T lymphocytes exhibited phenotypic plasticity, up-regulated mucosal homing integrins and chemokine receptors, and trafficked rapidly to mucosal surfaces. Moreover, the migration of systemic CD8(+) T lymphocytes to mucosal compartments accounted for the vast majority of Ag-specific mucosal CD8(+) T lymphocytes induced by systemic vaccination. Thus, i.m. vaccination can overcome immune compartmentalization and generate robust mucosal CD8(+) T lymphocyte memory. These data demonstrate that the systemic and mucosal immune systems are highly coordinated following vaccination.     

PD-1 blockage reverses immune dysfunction and hepatitis B viral persistence in a mouse animal model

Persistent hepatitis B viral (HBV) infection results in chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC). Recent studies in animal models of viral infection indicate that the interaction between the inhibitory receptor, programmed death (PD)-1, on lymphocytes and its ligand (PD-L1) play a critical role in T-cell exhaustion by inducing T-cell inactivation. High PD-1 expression levels by peripheral T-lymphocytes and the possibility of improving T-cell function by blocking PD-1-mediated signaling confirm the importance of this inhibitory pathway in inducing T-cell exhaustion. We studied T-cell exhaustion and the effects of PD-1 and PD-L1 blockade on intrahepatic infiltrating T-cells in our recently developed mouse model of HBV persistence. In this mouse animal model, we demonstrated that there were increased intrahepatic PD-1-expressing CD8+ and CD4+ T cells in mice with HBV persistence, but PD-1 upregulation was resolved in mice which had cleared HBV. The Intrahepatic CD8+ T-cells expressed higher levels of PD-1 and lower levels of CD127 in mice with HBV persistence. Blockade of PD-1/PD-L1 interactions increased HBcAg-specific interferon (IFN)-γ production in intrahepatic T lymphocytes. Furthermore, blocking the interaction of PD-1 with PD-L1 by an anti-PD-1 monoclonal antibody (mAb) reversed the exhausted phenotype in intrahepatic T lymphocytes and viral persistence to clearance of HBV in vivo. Our results indicated that PD-1 blockage reverses immune dysfunction and viral persistence of HBV infection in a mouse animal model, suggesting that the anti-PD-1 mAb might be a good therapeutic candidate for chronic HBV infection.     

CD4+ T-cell-epitope escape mutant virus selected in vivo.

Mutations in viral genomes that affect T-cell-receptor recognition by CD8+ cytotoxic T lymphocytes have been shown to allow viral evasion from immune surveillance during persistent viral infections. Although CD4+ T-helper cells are crucially involved in the maintenance of effective cytotoxic T-lymphocyte and neutralizing-antibody responses, their role in viral clearance and therefore in imposing similar selective pressures on the virus is unclear. We show here that transgenic virus-specific CD4+ Tcells, transferred into mice persistently infected with lymphocytic choriomeningitis virus, select for T-helper epitope mutant viruses that are not recognized. Together with the observed antigenic variation of the same T-helper epitope during polyclonal CD4+ T-cell responses in infected pore-forming protein-deficient C57BL/6 mice, this finding indicates that viral escape from CD4+ T lymphocytes is a possible mechanism of virus persistence.     

Fate and function of anti-CD3/CD28-activated T cells following adoptive transfer: IL-2 promotes development of anti-tumor memory T cells in vivo

BACKGROUND: Adoptive immunotherapy with T cells activated through CD3 alone requires exogenous IL-2 for T-cell function and survival after transfer, but the in vivo cytokine requirement of T cells activated through CD3 and CD28 is unknown. We hypothesized that CD3/CD28-activated T cells, unlike those activated through CD3 alone, might develop into long-lived memory T cells, either with or without systemic IL-2. METHODS: We used MHC class I-restricted TCR transgenic T cells from the OT-1 mouse, specific for the surrogate tumor Ag ovalbumin (OVA), to assess the trafficking kinetics, antigenic responsiveness and anti-tumor efficacy of dual-activated T cells in vivo as a function of IL-2 administration. At days 7, 14, and 28 after transfer, lymph node cells and splenocytes were examined for donor cell persistence and antigenic responsiveness by FACS and ELISA, respectively. RESULTS: In IL-2-treated mice, donor CD8+ T cells persisted and developed a memory phenotype, based on CD44 and Ly6c expression at day 28, while mice given no IL-2 had fewer donor cells at all time points. OVA-specific release of IFN-gamma was higher from lymphocytes of IL-2-treated mice compared with no-IL-2 mice (P<0.02 at all time points). In mice challenged with an OVA-bearing subline of the AML leukemia model C1498, IL-2 did not confer added protection from tumor challenge at 1 or 2 weeks after adoptive transfer, but gave improved survival at 4 weeks post-transfer. DISCUSSION: We conclude that exogenous IL-2 is not required for anti-tumor activity of CD3/CD28-activated CD8+ cells early after adoptive transfer, but promotes T-cell persistence that confers disease protection at more remote times.     

Protection against vaccinia virus challenge by CD8 memory T cells resolved by molecular mimicry

Live vaccinia virus (VV) vaccination has been highly successful in eradicating smallpox. However, the mechanisms of immunity involved in mediating this protective effect are still poorly understood, and the roles of CD8 T-cell responses in primary and secondary VV infections are not clearly identified. By applying the concept of molecular mimicry to identify potential CD8 T-cell epitopes that stimulate cross-reactive T cells specific to lymphocytic choriomeningitis virus (LCMV) and VV, we identified after screening only 115 peptides two VV-specific immunogenic epitopes that mediated protective immunity against VV. An immunodominant epitope, VV-e7r130, did not generate cross-reactive T-cell responses to LCMV, and a subdominant epitope, VV-a11r198, did generate cross-reactive responses to LCMV. Infection with VV induced strong epitope-specific responses which were stable into long-term memory and peaked at the time virus was cleared, consistent with CD8 T cells assisting in the control of VV. Two different approaches, direct adoptive transfer of VV-e7r-specific CD8 T cells and prior immunization with a VV-e7r-expressing ubiquitinated minigene, demonstrated that memory CD8 T cells alone could play a significant role in protective immunity against VV. These studies suggest that exploiting cross-reactive responses between viruses may be a useful tool to complement existing technology in predicting immunogenic epitopes to large viruses, such as VV, leading to a better understanding of the role CD8 T cells play during these viral infections.     

Identification of hexon-specific CD4 and CD8 T-cell epitopes for vaccine and immunotherapy

Adenoviral infections in the immunocompromised host are associated with significant morbidity and mortality. Although the adoptive transfer of adenovirus-specific T cells may prevent and treat such infections, the T-cell immune response to the multiplicity of adenovirus serotypes and subspecies that infect humans has not been well characterized, impeding the development of such approaches. We have, therefore, analyzed the specificities of T-cell responses to the viral capsid hexon antigen, since this structure is highly conserved in human pathogens. We screened 25 human cytotoxic T-cell lines with adenovirus specificity to extensively characterize their responses to adenoviral hexon and to identify a panel of novel CD4⁺ and CD8⁺ T-cell epitopes. Using a peptide library spanning the entire sequence of the hexon protein, we confirmed the responsiveness of these cytotoxic T-cell lines to seven peptides described previously and also identified 33 new CD4- or CD8-restricted hexon epitopes. Importantly, the majority of these epitopes were shared among different adenovirus subspecies, suggesting that T cells with such specificities could recognize and be protective against multiple serotypes, simplifying the task of effective adoptive transfer or vaccine-based immunotherapy for treating infection by this virus.     

Bacteria modulate the CD8+ T cell epitope repertoire of host cytosol-exposed proteins to manipulate the host immune response

The main adaptive immune response to bacteria is mediated by B cells and CD4+ T-cells. However, some bacterial proteins reach the cytosol of host cells and are exposed to the host CD8+ T-cells response. Both gram-negative and gram-positive bacteria can translocate proteins to the cytosol through type III and IV secretion and ESX-1 systems, respectively. The translocated proteins are often essential for the bacterium survival. Once injected, these proteins can be degraded and presented on MHC-I molecules to CD8+ T-cells. The CD8+ T-cells, in turn, can induce cell death and destroy the bacteria's habitat. In viruses, escape mutations arise to avoid this detection. The accumulation of escape mutations in bacteria has never been systematically studied. We show for the first time that such mutations are systematically present in most bacteria tested. We combine multiple bioinformatic algorithms to compute CD8+ T-cell epitope libraries of bacteria with secretion systems that translocate proteins to the host cytosol. In all bacteria tested, proteins not translocated to the cytosol show no escape mutations in their CD8+ T-cell epitopes. However, proteins translocated to the cytosol show clear escape mutations and have low epitope densities for most tested HLA alleles. The low epitope densities suggest that bacteria, like viruses, are evolutionarily selected to ensure their survival in the presence of CD8+ T-cells. In contrast with most other translocated proteins examined, Pseudomonas aeruginosa's ExoU, which ultimately induces host cell death, was found to have high epitope density. This finding suggests a novel mechanism for the manipulation of CD8+ T-cells by pathogens. The ExoU effector may have evolved to maintain high epitope density enabling it to efficiently induce CD8+ T-cell mediated cell death. These results were tested using multiple epitope prediction algorithms, and were found to be consistent for most proteins tested.