CD8-mediated Responses Research Articles

Classical Hodgkin Lymphoma: Tumor Structure and Prognostic Value of the Immune Microenvironment

Classical Hodgkin lymphoma (cHL) is a unique malignant lymphoid neoplasm characterized by tumor (Hodgkin and Reed-Sternberg) cells in the inflammatory and immunosuppressive microenvironment. The cHL microenvironment is a complex dynamic environment with immune cells, stromal elements, and extracellular matrix components, all of them interacting with each other and with tumor cells. This interaction basically underlies both disease progression and response to therapy. Currently, there is a growing interest in studying the structure and functions of cHL microenvironment, its prognostic value, and the potential of its components to be used as new therapeutic targets. During the last decade, the outcomes of refractory cHL treatment have considerably improved, in particular due to the administration of such PD-1 inhibitors as nivolumab and pembrolizumab. High cHL sensitivity to anti-PD-1 therapy can be accounted for by the PD-1/PD-L1-associated niche being formed in the tumor tissue as a result of intensive PD-L1 expression by tumor cells and macrophages as well as the expression of its PD-1 receptor by T-cells and M2-macrophages. More and more information becomes available about the possible mechanisms of antitumor response in anti-PD-1 treated cHL patients which seems to contradict the traditional understanding of CD8-mediated response in solid tumors. Cytotoxic effects of anti-PD-1 therapy in cHL tissues are likely to result from the interaction between tumor cells, macrophages, and CD4-positive Т-lymphocytes. This review discusses structural and regulatory relationships between tumor cells and microenvironment components, deals with new therapy approaches using various microenvironment components as targets, and summarizes currently available knowledge on prognosis based on the study of cHL microenvironment.

Vaccination with post-translational modified, homocitrullinated peptides induces CD8 T-cell responses that mediate antitumor immunity

BackgroundPost-translational modification of proteins has the potential to alter the ability of T cells to recognize major histocompatibility complex (MHC) class -I and class-II restricted antigens, thereby resulting in altered...

The localization of a heterologous displayed antigen in the baculovirus-budded virion determines the type and strength of induced adaptive immune response.

In the search of strategies of presentation of heterologous antigens to elicit humoral or cellular immune responses that modulate and properly potentiate each type of response, researchers have been studying baculovirus (BV) as vaccine vectors with promising results. For some years, several research groups explored different antigen presentation approaches using the BV AcNPV by expressing polypeptides on the surface of budded virions or by de novo synthesis of heterologous antigens by transduction of mammalian cells. In the case of expression on the surface of budded virions, for example, researchers have expressed polypeptides in peplomers as GP64 glycoprotein fusions or distributed throughout the entire surface by fusions to portions of the G protein of vesicular stomatitis virus, VSV. Recently, our group developed the strategy of cross-presentation of antigens by fusions of GP64 to the capsid protein VP39 (capsid display) for the generation of cytotoxic responses. While the different strategies showed to be effective in raising immune responses, the individuality of each analysis makes difficult the comparison of the results. Here, by comparing the different strategies, we show that localization of the model antigen ovalbumin (OVA) strongly determined the quality and intensity of the adaptive response to the heterologous antigen. Furthermore, surface display favored humoral responses, whereas capsid display favored cytotoxic responses. Finally, capsid display showed a much more efficient strategy to activate CD8-mediated responses than transduction. The incorporation of adjuvants in baculovirus formulations dramatically diminished the immunostimulatory properties of baculovirus.

Trafficking of IL-17-producing CD4+, but not CD8+, T cells to the skin requires CCR6 expression

Abstract Contact hypersensitivity (CHS) is a CD8-mediated response to hapten sensitization and challenge of skin. During elicitation of CHS, hapten-primed CD8 T cells producing IFN-g and IL-17 are recruited to the challenge site by hapten-presenting endothelial cell (EC)-derived chemokines. Our previous results have shown that CXCL9 directs localization of IFN-g producing CD8 T cells to the challenge site, but chemokines directing localization of IL-17-producing CD8 T cells to the site remain unknown. DNFB hapten application to the skin of sensitized mice or to EC in culture induced TNFa-dependent expression of CCL20 within 1 h. Hapten-stimulated ELISPOT assay of flow-sorted CD8 T cells from draining lymph nodes of sensitized mice indicated many CCR6+CXCR3+ and CCR6+CXCR3− producing IL-17, but low numbers of CCR6−CXCR3+ cells producing IL-17. Hapten-induced IFN-g production was restricted to CXCR3+ CD8 T cells. Anti-CCL20 mAb did not inhibit CHS responses when given to sensitized WT mice during challenge and normal CHS responses were observed in sensitized and challenged CCR6−/− mice. Sensitized CCR6−/− mice had increased hapten-specific IFN-g and IL-17 producing CD8 T cells in the skin-draining lymph nodes when compared to sensitized WT mice. In contrast to sensitized WT mice, sensitization of CCR6−/− mice also induced high numbers of hapten-primed CD4+T cells producing IFN-g and IL-17. However, depletion of CD8, but not CD4, T cells from sensitized CCR6−/− mice abrogated the CHS response to hapten challenge. These results indicate that the CD8 T cell mediated CHS response can occur independently of CCR6 expression whereas trafficking of hapten-primed CD4 T cells to the skin to mediate the response requires CCR6.

Intrathymic injection of lentiviral vector curtails the immune response in the periphery of normal mice

Gene transfer in the thymus, based on HIV-derived lentiviral vectors, is a promising avenue for modulation of T cell selection and autoimmunity. However, the impact of intrathymic (IT) injections on an antigen-specific immune response elicited in the periphery of normal mice has not been investigated yet. Highly concentrated stocks of lentiviral vectors expressing the soluble form of hemaglutinin of the influenza virus (LvHA) were injected in the thymus of normal BALB/c mice. The CD4 and CD8-mediated immune responses to HA after peripheral immunization were measured by various parameters. We first show that a lentiviral vector expressing the luciferase was detected for at least 2 months after IT-injections. We then show that the LvHA vector could elicit a functional CD4- and CD8-T cell-mediated immune responses in the peripheral lymphoid organs of BALB/c mice. IT-injection of the LvHA vector significantly curbed this response: lower numbers of transferred HA-specific CD4(+) T cells were found in LvHA-injected compared to control animals. Furthermore, lower frequencies of HA-specific CD8(+) T cells, interferon γ-producing cells and cytotoxic cells were detected from 3 weeks to 3 months in LvHA-injected mice compared to controls. However, these reduced CD8-mediated responses were not increased after depletion of CD25(+) cells in vitro or in vivo. The results obtained in the present study show that injection of the LvHA lentiviral vector significantly curtailed the immune response to the same antigen in the periphery. Increased selection of HA-specific regulatory T cells and negative selection of HA-specific CD8(+) T cell precursors may explain the results. Our work establish the feasibility of IT-injections of lentiviral vectors to manipulate T cell tolerance in the thymus of normal mice, for basic and pre-clinical research.

Vital Role for CD8+Cells in Controlling Retroviral Infections

Antiviral adaptive immune defenses consist of humoral and cell-mediated responses, which together eliminate extracellular and intracellular virus. As most retrovirus-infected individuals do not raise efficient protective antivirus immune responses, the relative importance of humoral and cell-mediated responses in restraining retroviral infection is not well understood. We utilized retrovirus-resistant I/LnJ mice, which control infection with mouse mammary tumor virus (MMTV) and murine leukemia virus (MuLV) via an adaptive immune mechanism, to assess the contribution of cellular responses and virus-neutralizing antibodies (Abs) to the control of retroviral infection. We found that in retrovirus-infected CD8-deficient I/LnJ mice, viral titers exceed the neutralizing capability of antiviral Abs, resulting in augmented virus spread and disease induction. Thus, even in the presence of robust neutralizing Ab responses, CD8-mediated responses are essential for full protection against retroviral infection.

Emergence of simian immunodeficiency virus-specific cytotoxic CD4+ T cells and increased humoral responses correlate with control of rebounding viremia in CD8-depleted macaques infected with Rev-independent live-attenuated simian immunodeficiency virus.

Indian rhesus macaques infected with the Rev-independent live-attenuated SIVmac239 strains control viremia to undetectable levels, have persistent but low cellular and humoral anti-SIV responses, and show no signs of immune deficiency. To analyze the immune mechanisms responsible for viral control, five macaques infected at day 1 after birth were subjected to CD8(+) cell depletion at 6.7 y postinfection. This resulted in viremia increases to 3.7-5.5 log(10) RNA copies, supporting a role of CD8-mediated responses in the control of viral replication. The rebounding viremia was rapidly controlled to levels below the threshold of detection, and occurred in the absence of SIV-specific CD8(+) T cells and significant CD8(+) T cell recovery in four of the five animals, suggesting that other mechanisms are involved in the immunological control of viremia. Monitoring immune responses at the time of viral control demonstrated a burst of circulating SIV-specific CD4(+) T cells characterized as CD45RA(-)CD28(+)CD95(+)CCR7(-) and also granzyme B(+), suggesting cytotoxic ability. Control of viremia was also concomitant with increases in humoral responses to Gag and Env, including a transient increase in neutralizing Abs against the neutralization-resistant SIVmac239 in four of five animals. These data demonstrate that a combination of cellular responses mediated by CD4(+) T cells and humoral responses was associated with the rapid control of the rebounding viremia in macaques infected by the Rev-independent live-attenuated SIV, even in the absence of measurable SIV-specific CD8(+) T cells in the blood, emphasizing the importance of different components of the immune response for full control of SIV infection.

Neo-Lymphoid Aggregates in the Adult Liver Can Initiate Potent Cell-Mediated Immunity

Author SummaryLymph nodes (LNs) are believed to be the most important tissues initiating immune responses by facilitating the activation of T and B lymphocytes. Mice lacking such LNs (called alymphoplastic) are severely immune compromised and resistant to immunizations. We discovered that the immune-deficiency of such alymphoplastic mice is actually not caused by the loss of LNs, but rather by the underlying genetic lesion. Surprisingly, mice lacking all lymph nodes can still mount potent T cell-mediated immune responses. We also discovered that T and B cells have completely different structural requirements for their activation/maturation. Whereas B cells rely on LNs to become efficient antibody-producing cells, T cells can be activated successfully outside of such dedicated tissues. So—in the absence of LNs—antigens delivered by immunization are actively transported into the liver where cellular immunity is initiated. The mammalian fetal liver is responsible for the early formation of blood and immune cells, and we propose that the adult liver can still provide a niche for T cell–antigen encounters. During evolution, T and B cells emerged simultaneously, allowing cold-blooded vertebrates (which lack LNs) to launch adaptive immune responses. The development of LNs in mammals coincided with a drastic improvement in antibody affinity maturation, whereas T cells remain LN-independent to this day.

Early kinetics of innate and adaptive immune responses during hepatitis B virus infection

Background and Aim:Innate immunity appears to be silent in acutely heptitis B virus (HBV)-infected chimpanzees, as shown by microarray analysis of intrahepatic gene expression. Whether this observation also applies to...

Identification of a Novel Immunogenic HLA-A*0201-Binding Epitope of HER-2/neu with Potent Antitumor Properties

HER-2/neu oncoprotein is overexpressed in a variety of human tumors and is associated with aggressive disease. Immunogenic HER-2/neu CTL epitopes have been used as vaccines for the treatment of HER-2/neu positive malignancies with limited success. By applying prediction algorithms for MHC class I ligands and proteosomal cleavages, in this study, we describe the identification of HER-2/neu decamer LIAHNQVRQV spanning residues 85-94 (HER-2(10(85))). HER-2(10(85)) proved to bind with high affinity to HLA-A2.1 and was stable for 4 h in an off-kinetics assay. This peptide was immunogenic in HLA-A2.1 transgenic (HHD) mice inducing peptide-specific CTL, which responded to tumor cell lines of various origin coexpressing human HER-2/neu and HLA-A2.1. This demonstrates that HER-2(10(85)) is naturally processed from endogenous HER-2/neu. Five of sixteen HER-2/neu+ HLA-A2.1+ breast cancer patients analyzed had HER-2(10(85))-reactive T cells ranging from 0.35-0.70% of CD8+ T cells. Depletion of T regulatory cells from PBMC enabled the rapid expansion of HLA-A2.1/HER-2(10(85))pentamer+/CD8+ cells (PENT+/CD8+), whereas significantly lower numbers of CTL could be generated from unfractionated PBMC. HER-2(10(85))-specific human CTL recognized the HER-2/neu+ HLA-A2.1+ tumor cell line SKBR3.A2, as determined by IFN-gamma intracellular staining and in the high sensitivity CD107alpha degranulation assay. Finally, HER-2(10(85)) significantly prolonged the survival of HHD mice inoculated with the transplantable ALC.A2.1.HER tumor both in prophylactic and therapeutic settings. These data demonstrate that HER-2(10(85)) is an immunogenic peptide, capable of eliciting CD8-mediated responses in vitro and in vivo, providing the platform for further exploitation of HER-2(10(85)) as a possible target for anticancer immunotherapy.

Gliadin Activates HLA Class I-Restricted CD8+ T Cells in Celiac Disease Intestinal Mucosa and Induces the Enterocyte Apoptosis

The extensive infiltration of CD8(+) T cells in the intestinal mucosa of celiac disease (CD) patients is a hallmark of the disease. We identified a gliadin peptide (pA2) that is selectively recognized by CD8(+) T cells infiltrating intestinal mucosa of HLA-A2(+) CD patients. Herein, we investigated the phenotype, the tissue localization, and the effector mechanism of cells responsive to pA2 by using the organ culture of CD intestinal mucosa. The target of pA2-mediated cytotoxicity was also investigated by using the intestinal epithelial cell lines Caco2 and HT29, A2(+) and A2(-), respectively, as target cells. Jejunal biopsy specimens from CD patients were cultured in vitro with pA2, and cellular activation was evaluated by immunohistochemistry and cytofluorimetric analysis. Cytotoxicity of pA2-specific, intestinal CD8(+) T cells was assayed by granzyme-B and interferon-gamma release and by apoptosis of target cells. pA2 challenge of A2(+) CD mucosa increased the percentage of CD8(+)CD25(+) and of CD80(+) cells in the lamina propria, the former mainly localized beneath the epithelium, as well as the number of terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling-positive cells (TUNEL(+)) in the epithelium. Intraepithelial CD3(+) cells and enterocyte expression of Fas were also increased. CD8(+)CD25(+) and CD8(+)FASL(+) T cells were significantly increased in cell preparations from biopsy specimens cultured with pA2. CD8(+) T-cell lines released both granzyme-B and interferon-gamma following recognition of pA2 when presented by Caco2 and not by HT29. These data indicate that gliadins contain peptides able to activate, through a TCR/HLA class I interaction, CD8-mediated response in intestinal CD mucosa and to induce the enterocyte apoptosis.

Identification and Characterization of ErbB-3-Binding Protein-1 as a Target for Immunotherapy

Based on immune reactivity in response to a whole-cell colon tumor vaccine and using serological identification of Ags by recombinant cDNA expression cloning, we here describe the molecular and functional identification of a novel human tumor Ag. By screening a cDNA expression library derived from the coloncarcinoma cell line HT-29 with pooled colorectal cancer patients' sera, 26 clones reactive with IgG Abs could be identified. Characterization of these cDNA clones by sequence analysis and alignment, and detailed serological analysis revealed cancer-related immunoreactivity for the ErbB-3-binding protein-1 (Ebp1). Immunohistochemical staining of colorectal tumors and neighboring normal colon tissue indicated the observed cancer-related immunogenicity of Ebp1 to be related to overexpression. Via reverse immunology, five potential HLA-A2-restricted T cell epitopes were identified, of which two (Ebp1(45-54) and Ebp1(59-67)) bound HLA-A2 with intermediate and high affinity, respectively. Analysis of their immunogenicity in vitro indicated that only the high-affinity Ebp1(59) epitope gave rise to CD8(+) T cells capable of recognizing both exogenously loaded Ebp1 peptide and endogenously expressed Ebp1 on target cells. In addition, in vivo CD8(+) T cell responsiveness against the Ebp1(59) epitope could be detected in two of nine and three of six cancer patients PBMC and tumor draining lymph nodes, respectively, but not in nine of nine healthy donors tested. These data confirm that Ebp1 is an immunogenic protein, capable of eliciting CD8-mediated responses in vivo and in vitro, providing a rationale for further exploration of Ebp1 as a possible target for anticancer immunotherapy.

Modified anthrax fusion proteins deliver HIV antigens through MHC Class I and II pathways

T cell-based HIV vaccine candidates have focused on eliciting both CD4- and CD8-mediated responses. One challenge in vaccine development is the successful introduction and presentation of exogenous antigen to elicit an immune response. Modified bacterial toxins have been studied extensively as intracellular delivery agents because of their unique capability to translocate antigen across the cell membrane without affecting cell viability. Modified anthrax toxin lethal factor (LFn) fusion protein is able to effectively induce anti-HIV cytotoxic T lymphocytes in the absence of protective antigen (PA) and is being evaluated as a vaccine candidate. Here we describe, for the first time, the processing and presentation of LFn fusion proteins by the MHC Class II pathway. The ability of LFn–HIV to induce both CD8- and CD4-mediated responses may have relevance in current approaches to vaccine design. Furthermore, the translocation and presentation of antigens occurs in the absence of PA, which proposes a modified molecular mechanism of antigen presentation by the anthrax toxin model. Additionally, we found that LFn–HIV is specific and sensitive in detecting HIV-specific CD4 + and CD8 + T cell responses in T cell assays, further broadening the value of this antigen delivery system as a useful immunologic tool.

Antiviral CD8-mediated responses in chronic HCV carriers with HBV superinfection.

Hepatitis B virus (HBV) superinfection in chronic hepatitis C represents a natural model to investigate whether or not hepatitis C virus (HCV) can influence priming and maturation of antiviral T cells; whether or not HBV superinfection, which is known to determine control of HCV replication, can restore HCV-specific T cell responsiveness; and whether or not cytokines stimulated by HBV infection can contribute to HCV control. To address these issues, the function of CD8 cells specific for HBV and HCV was studied longitudinally in two chronic HCV patients superinfected with HBV. Patients with acute hepatitis B were also examined. Frequency and function of HBV tetramer+ CD8 cells were comparable in patients acutely infected with HBV with or without chronic HCV infection. HBV-specific CD8 cell function was efficiently expressed irrespective of serum HCV-RNA levels. Moreover, fluctuations of HCV viremia at the time of HBV superinfection were not associated with evident changes of CD8 responsiveness to HCV. Finally, no correlation was found between serum levels of interferon alpha, interleukin (IL)-12, IL-10, or IL-18 and control of HCV replication. In conclusion, HCV did not affect the induction of primary and memory HBV-specific CD8 responses. HCV-specific CD8 responses were undetectable when HCV-RNA was negative, showing that inhibition of HCV replication in the setting of a HBV superinfection was not sufficient to induce a restoration of CD8 reactivity against HCV.

413 Functional features of HCV-specific CD8 mediated responses associated with different outcomes of HCV infection

The hepatitis C virus (HCV) production system consists of transfecting the human hepatoma cell line Huh7 with genomic HCV RNA (JFH1). To monitor HCV replication by fluorescence microscopy, we constructed a recombinant HCV clone expressing Azami-Green (mAG), a bright green fluorescent protein, by inserting the mAG gene into the nonstructural protein 5A (NS5A) gene; the resultant clone was designated JFH1-hmAG. The Huh-7.5.1 (a subclone of Huh7) cells transfected with JFH1-hmAG RNA were found to produce cytoplasmic NS5A-mAG, as readily visualized by fluorescence microscopy, and infectious virus, as assayed with the culture supernatant, indicating that JFH1-hmAG is infectious and replication-competent. Furthermore, the replication of this virus was inhibited by interferon alpha in a dose-dependent manner. These results suggest that JFH1-hmAG is useful for studying HCV life cycle and the mechanism of interferon’s anti-HCV action and for screening and testing new anti-HCV drugs.

Antiviral cell-mediated immune responses during hepatitis B and hepatitis C virus infections.

Cell-mediated immune responses to hepatitis B (HBV) and hepatitis C virus (HCV) antigens are vigorous and multispecific in acute, self-limited infections. Moreover, the prevalent cytokine pattern of circulating virus-specific T cells from patients who recover spontaneously from acute hepatitis is Th1-like. Longitudinal analysis of the T cell response to HCV antigens from the early stages of HCV infection in patients who recover from hepatitis and those who do not indicates that weaker responses and a prevalent Th2 pattern of cytokine production is associated with viral persistence and chronic evolution of disease. Although similar sequential studies are missing in hepatitis B, the observation that HBV-specific T cell responses are very weak or totally undetectable in the peripheral blood of patients with long-lasting chronic hepatitis B suggests that strength and quality of virus-specific T cell responses at the early stages of infection may influence the final outcome of both hepatitis B and C. While T cell hyporesponsiveness seems to be an important determinant for HBV persistence once chronic hepatitis has developed, this mechanism appears to be less critical in chronic HCV infection, because the vigor and quality of HCV-specific T cell responses seem to improve as a function of the duration of infection. This is shown by the finding that HCV-specific CD4- and CD8-mediated responses are easily detectable in the peripheral blood of patients with long-lasting chronic hepatitis C and that production of Th1 cytokines predominates within their livers. HCV therefore seems to be able to persist even in the face of an active T cell response and to acquire the capacity to survive within a host environment apparently unfavorable to its persistence. The high variability of HCV may explain its efficiency in escaping immune surveillance.

3 Immunology of Hodgkin's disease

Hodgkin's disease is characterized by an immune response in the involved tissues that is predominantly CD4 mediated. The CD4+ T-cells are CD45RO+ and CD45RBdim, they express several activation markers but lack CD26, and in vitro can be stimulated to produce gamma-interferon and IL-4, but not IL-2. This is not the usual immunophenotype and cytokine production pattern of Th1, Th2 or Th0 cells and may be a reflection of anergy. The cause of such an anergic reaction is not clear since RS cells express HLA class II as well as the co-stimulator molecules CD80 and CD86. It is possible that a (hypothetical) super antigen expressed on the RS cells may play a role. The absence of IL-2 production however explains the absence of a CD8 mediated response. In addition to that, RS cells generally do not express HLA class I, which allows them to escape CD8 mediated responses. The link between the ineffective immune response in the tissue and the generalized immune deficiency in Hodgkin's disease may consist of several components. These include the influx of mature T-cells into the affected tissues, the secretion of inhibitory molecules by the neoplastic cells and the spill-over of the anergic T-cell response into the general circulation by either the Hodgkin related antigen or also as a result of an IL-4 dominated response. The latter possibility may also be related to the hyper-gamma-globulinaemia and the frequently observed high IgE levels.