The Role of Interleukin-2 in Memory CD8 Cell Differentiation

The current literature on the role of interleukin (IL)-2 in memory CD8(+) T-cell differentiation indicates a significant contribution of IL-2 during primary and also secondary expansion of CD8(+) T cells. IL-2 seems to be responsible for optimal expansion and generation of effector functions following primary antigenic challenge. As the magnitude of T-cell expansion determines the numbers of memory CD8(+) T cells surviving after pathogen elimination, these events influence memory cell generation. Moreover, during the contraction phase of an immune response where most antigen-specific CD8(+) T cells disappear by apoptosis, IL-2 signals are able to rescue CD8(+) T cells from cell death and provide a durable increase in memory CD8(+) T-cell counts. At the memory stage, CD8(+) T-cell frequencies can be boosted by administration of exogenous IL-2. Significantly, only CD8(+) T cells that have received IL-2 signals during initial priming are able to mediate efficient secondary expansion following renewed antigenic challenge. Thus, IL-2 signals during different phases of an immune response are key in optimizing CD8(+) T-cell functions, thereby affecting both primary and secondary responses of these T cells.

Expression hierarchy of T cell epitopes from melanoma differentiation antigens: unexpected high level presentation of tyrosinase-HLA-A2 Complexes revealed by peptide-specific, MHC-restricted, TCR-like antibodies.

Peptide Ags presented by class I MHC molecules on human melanomas and that are recognized by CD8(+) T cells are the subjects of many studies of antitumor immunity and represent attractive candidates for therapeutic approaches. However, no direct quantitative measurements exist to reveal their expression hierarchy on the cell surface. Using novel recombinant Abs which bind these Ags with a peptide-specific, MHC-restricted manner, we demonstrate a defined pattern of expression hierarchy of peptide-HLA-A2 complexes derived from three major differentiation Ags: gp100, Melan-A/Mart-1, and tyrosinase. Studying melanoma cell lines derived from multiple patients, we reveal a surprisingly high level of presentation of tyrosinase-derived complexes and moderate to very low expression of complexes derived from other Ags. No correlation between Ag presentation and mRNA expression was found; however, protein stability may play a major role. These results provide new insights into the characteristics of Ag presentation and are particularly important when such targets are being considered for immunotherapy. These results may shed new light on relationships between Ag presentation and immune response to cancer Ags.

Peptide vaccine induces enhanced tumor growth associated with apoptosis induction in CD8+ T cells.

CD8(+) CTLs play a critical role in antitumor immunity. However, vaccination with synthetic peptide containing CTL epitopes has not been generally effective in inducing protective antitumor immunity. In this study, we addressed the detailed mechanism(s) involved in this failure using a new tumor model of BALB/c transplanted tumors expressing NY-ESO-1, an extensively studied human cancer/testis Ag. Whereas peptide immunization with an H2-D(d)-restricted CTL epitope derived from NY-ESO-1 (NY-ESO-1 p81-88) induced NY-ESO-1(81-88)-specific CD8(+) T cells in draining lymph nodes and spleens, tumor growth was significantly enhanced. Single-cell analysis of specific CD8(+) T cells revealed that peptide immunization caused apoptosis of >80% of NY-ESO-1(81-88)-specific CD8(+) T cells at tumor sites and repetitive immunization further diminished the number of specific CD8(+) T cells. This phenomenon was associated with elevated surface expression of Fas and programmed death-1. When peptide vaccination was combined with an adjuvant, a TLR9 ligand CpG, the elevated Fas and programmed death-1 expression and apoptosis induction were not observed, and vaccine with peptide and CpG was associated with strong tumor growth inhibition. Selection of appropriate adjuvants is essential for development of effective cancer vaccines, with protection of effector T cells from peptide vaccine-induced apoptosis being a prime objective.

Dendritic cells exposed to MVA-based HIV-1 vaccine induce highly functional HIV-1-specific CD8+ T cell responses in HIV-1-infected individuals

Currently, MVA virus vectors carrying HIV-1 genes are being developed as HIV-1/AIDS prophylactic/therapeutic vaccines. Nevertheless, little is known about the impact of these vectors on human dendritic cells (DC) and their capacity to present HIV-1 antigens to human HIV-specific T cells. This study aimed to characterize the interaction of MVA and MVA expressing the HIV-1 genes Env-Gag-Pol-Nef of clade B (referred to as MVA-B) in human monocyte-derived dendritic cells (MDDC) and the subsequent processes of HIV-1 antigen presentation and activation of memory HIV-1-specific T lymphocytes. For these purposes, we performed ex vivo assays with MDDC and autologous lymphocytes from asymptomatic HIV-infected patients. Infection of MDDC with MVA-B or MVA, at the optimal dose of 0.3 PFU/MDDC, induced by itself a moderate degree of maturation of MDDC, involving secretion of cytokines and chemokines (IL1-ra, IL-7, TNF-α, IL-6, IL-12, IL-15, IL-8, MCP-1, MIP-1α, MIP-1β, RANTES, IP-10, MIG, and IFN-α). MDDC infected with MVA or MVA-B and following a period of 48 h or 72 h of maturation were able to migrate toward CCL19 or CCL21 chemokine gradients. MVA-B infection induced apoptosis of the infected cells and the resulting apoptotic bodies were engulfed by the uninfected MDDC, which cross-presented HIV-1 antigens to autologous CD8+ T lymphocytes. MVA-B-infected MDDC co-cultured with autologous T lymphocytes induced a highly functional HIV-specific CD8+ T cell response including proliferation, secretion of IFN-γ, IL-2, TNF-α, MIP-1β, MIP-1α, RANTES and IL-6, and strong cytotoxic activity against autologous HIV-1-infected CD4+ T lymphocytes. These results evidence the adjuvant role of the vector itself (MVA) and support the clinical development of prophylactic and therapeutic anti-HIV vaccines based on MVA-B.

Lymphocyte populations in liver biopsy specimens from patients with chronic liver disease

The characterisation of lymphocytes from liver biopsies indicates that 'activated' T lymphocytes are present in the liver in alcohol induced hepatitis, chronic active hepatitis (HBS+ve and -ve), and in primary biliary cirrhosis but not in inactive cirrhosis, chronic persistent hepatitis, extrahepatic and drug induced cholestasis. A greater percentage of lymphocytes bear Fc-receptors in chronic active hepatitis than in alcohol induced hepatitis or cholestatic liver disease. The concentration of 'activated' T cells in the peripheral blood in all groups studied was within the normal range, suggesting that the 'activated' T cells found in the liver were reacting to either native or foreign antigens within the liver. The data on Fc-receptor bearing cells are consistent with the involvement of antibody assisted K cell mediated cytotoxicity in chronic active hepatitis.

The caspase 8 inhibitor c-FLIP(L) modulates T-cell receptor-induced proliferation but not activation-induced cell death of lymphocytes.

The caspase 8 inhibitor c-FLIP(L) can act in vitro as a molecular switch between cell death and growth signals transmitted by the death receptor Fas (CD95). To elucidate its function in vivo, transgenic mice were generated that overexpress c-FLIP(L) in the T-cell compartment (c-FLIP(L) Tg mice). As anticipated, FasL-induced apoptosis was inhibited in T cells from the c-FLIP(L) Tg mice. In contrast, activation-induced cell death of T cells in c-FLIP(L) Tg mice was unaffected, suggesting that this deletion process can proceed in the absence of active caspase 8. Accordingly, c-FLIP(L) Tg mice differed from Fas-deficient mice by showing no accumulation of B220(+) CD4(-) CD8(-) T cells. However, stimulation of T lymphocytes with suboptimal doses of anti-CD3 or antigen revealed increased proliferative responses in T cells from c-FLIP(L) Tg mice. Thus, a major role of c-FLIP(L) in vivo is the modulation of T-cell proliferation by decreasing the T-cell receptor signaling threshold.

Fas (CD95/APO-1) limits the expansion of T lymphocytes in an environment of limited T-cell antigen receptor/MHC contacts.

Fas-deficient mice (Fas(lpr/lpr)) and humans have profoundly dysregulated T lymphocyte homeostasis, which manifests as an accumulation of CD4(+) and CD8(+) T cells as well as an unusual population of CD4(-)CD8(-)TCRαβ(+) T cells. To date, no unifying model has explained both the increased T-cell numbers and the origin of the CD4(-)CD8(-)TCRαβ(+) T cells. As Fas(lpr/lpr) mice raised in a germ-free environment still manifest lymphadenopathy, we considered that this process is primarily driven by recurrent low-avidity TCR signaling in response to self-peptide/MHC as occurs during homeostatic proliferation. In these studies, we developed two independent systems to decrease the number of self-peptide/MHC contacts. First, expression of MHC class I was reduced in OT-I TCR transgenic mice. Although OT-I Fas(lpr/lpr) mice did not develop lymphadenopathy characteristic of Fas(lpr/lpr) mice, in the absence of MHC class I, OT-I Fas(lpr/lpr) T cells accumulated as both CD8(+) and CD4(-)CD8(-) T cells. In the second system, re-expression of β(2)m limited to thymic cortical epithelial cells of Fas(lpr/lpr) β(2)m-deficient mice yielded a model in which polyclonal CD8(+) thymocytes entered a peripheral environment devoid of MHC class I. These mice accumulated significantly greater numbers of CD4(-)CD8(-)TCRαβ(+) T cells than conventional Fas(lpr/lpr) mice. Thus, Fas shapes the peripheral T-cell repertoire by regulating the survival of a subset of T cells proliferating in response to limited self-peptide/MHC contacts.

Dendritic cells exposed to MVA-based HIV-1 vaccine induce highly functional HIV-1-specific CD8+ T cell responses in HIV-1-infected individuals

Currently, MVA virus vectors carrying HIV-1 genes are being developed as HIV-1/AIDS prophylactic/therapeutic vaccines. Nevertheless, little is known about the impact of these vectors on human dendritic cells (DC) and their capacity to present HIV-1 antigens to human HIV-specific T cells. This study aimed to characterize the interaction of MVA and MVA expressing the HIV-1 genes Env-Gag-Pol-Nef of clade B (referred to as MVA-B) in human monocyte-derived dendritic cells (MDDC) and the subsequent processes of HIV-1 antigen presentation and activation of memory HIV-1-specific T lymphocytes. For these purposes, we performed ex vivo assays with MDDC and autologous lymphocytes from asymptomatic HIV-infected patients. Infection of MDDC with MVA-B or MVA, at the optimal dose of 0.3 PFU/MDDC, induced by itself a moderate degree of maturation of MDDC, involving secretion of cytokines and chemokines (IL1-ra, IL-7, TNF-α, IL-6, IL-12, IL-15, IL-8, MCP-1, MIP-1α, MIP-1β, RANTES, IP-10, MIG, and IFN-α). MDDC infected with MVA or MVA-B and following a period of 48 h or 72 h of maturation were able to migrate toward CCL19 or CCL21 chemokine gradients. MVA-B infection induced apoptosis of the infected cells and the resulting apoptotic bodies were engulfed by the uninfected MDDC, which cross-presented HIV-1 antigens to autologous CD8+ T lymphocytes. MVA-B-infected MDDC co-cultured with autologous T lymphocytes induced a highly functional HIV-specific CD8+ T cell response including proliferation, secretion of IFN-γ, IL-2, TNF-α, MIP-1β, MIP-1α, RANTES and IL-6, and strong cytotoxic activity against autologous HIV-1-infected CD4+ T lymphocytes. These results evidence the adjuvant role of the vector itself (MVA) and support the clinical development of prophylactic and therapeutic anti-HIV vaccines based on MVA-B.

Viral superantigen-induced negative selection of TCR transgenic CD4+ CD8+ thymocytes depends on activation, but not proliferation.

T-cell negative selection, a process by which intrathymic immunological tolerance is induced, involves the apoptosis-mediated clonal deletion of potentially autoreactive T cells. Although different experimental approaches suggest that this process is triggered as the result of activation-mediated cell death, the signal transduction pathways underlying this process is not fully understood. In the present report we have used an in vitro system to analyze the cell activation and proliferation requirements for the deletion of viral superantigen (SAg)-reactive Vbeta8.1 T-cell receptor (TCR) transgenic (TG) thymocytes. Our results indicate that in vitro negative selection of viral SAg-reactive CD4+ CD8+ thymocytes is dependent on thymocyte activation but does not require the proliferation of the negatively signaled thymocytes.

CpG are efficient adjuvants for specific CTL induction against tumor antigen-derived peptide.

The identification of CTL-defined tumor-associated Ags has allowed the development of new strategies for cancer immunotherapy. To potentiate the CTL responses, peptide-based vaccines require the coadministration of adjuvants. Because oligodeoxynucleotides (ODN) containing CpG motifs are strong immunostimulators, we analyzed the ability of CpG ODN to act as adjuvant of the CTL response against tumor-derived synthetic peptide in the absence or presence of IFA. Mice transgenic for a chimeric MHC class I molecule were immunized with a peptide analog of MART-1/Melan-A(26-35) in the presence of CpG ODN alone or CpG ODN emulsified in IFA. The CTL response was monitored ex vivo by tetramer staining of lymphocytes. In blood, spleen, and lymph nodes, peptide mixed with CpG ODN alone was able to elicit a stronger systemic CTL response as compared with peptide emulsified in IFA. Moreover, CpG ODN in combination with IFA further enhanced the CTL response in terms of the frequency of tetramer+CD8+ T cells ex vivo. The CTL induced in vivo against peptide analog in the presence of CpG ODN are functional, as they were able to recognize and kill melanoma cells in vitro. Overall, these results indicate that CpG ODN by itself is a good candidate adjuvant of CTL response and can also enhance the effect of classical adjuvant.

Magnitude of CD8 T-cell responses against hepatitis C virus and severity of hepatitis do not necessarily determine outcomes in acute hepatitis C virus infection.

Aim: We investigated the relationship between the magnitude of comprehensive hepatitis C virus (HCV)-specific CD8(+) T-cell responses and the clinical course of acute HCV infection. Methods: Six consecutive patients with acute HCV infection were studied. Analysis of HCV-specific CD8(+) T-cell responses was performed using an interferon-gamma-based enzyme-linked immunospot assay using peripheral CD8(+) T-cells, monocytes and 297 20-mer synthetic peptides overlapping by 10 residues and spanning the entire HCV sequence of genotype 1b. Results: Five patients presented detectable HCV-specific CD8(+) T-cell responses against a single and different peptide, whereas 1 patient showed responses against three different peptides. Neither the magnitude of HCV-specific CD8(+) T-cell responses nor the severity of hepatitis predicts the outcome of acute hepatitis. The maximum number of HCV-specific CD8(+) T-cells correlated with maximum serum alanine aminotransferase level during the course (r = 0.841, P = 0.036). Conclusions: HCV-specific CD8(+) T-cell responses were detectable in all 6 patients with acute HCV infection, and 6 novel HCV-specific CTL epitopes were identified. Acute HCV infection can resolve with detectable HCV-specific CD8(+) T-cell responses, but without development of antibody against HCV.

Clinical Implications of CD8+ T-cell infiltration in frequent and rare cancers.

Recent studies of cancer patients revealed high diversity in oncogenic mechanisms, leading to increased treatment individualization for subgroups of patients with frequent cancers. A similar development may not be possible for patients with rare cancers, such as Merkel cell carcinoma (MCC). Finding shared disease mechanisms may open new options to understanding and treating such tumors. Tumor-infiltrating CD8+ T cells are frequently associated with favorable clinical outcome in a remarkably large spectrum of cancers. In this issue, Afanasiev et al. suggest a mechanism that may hinder the tumor homing of CD8+ T cells in MCC patients. It is possible that therapeutic mobilization of anti-cancer T cells may be useful in patients who share this specific immune biological feature.

An optimized method for establishing high purity murine CD8(+) T cell cultures.

Establishing CD8(+) T cell cultures has been empirical and the published methods have been largely individual laboratory based. In this study, we optimized culturing conditions and show that IL-2 concentration is the most critical factor for the success of establishing CD8(+) T cell cultures. High IL-2 concentration encouraged T cells to non-specifically proliferate, express a B cell marker, B220, and undergo apoptosis. These cells also lose typical irregular T cell morphology and are incapable of sustaining long-term cultures. Using tetramer and intracellular cytokine assessments, we further demonstrated that many antigen-specific T cells have been rendered nonfunctional when expanded under high IL-2 concentration. When IL-2 is used in the correct range, B220-mediated cell depletion greatly enhanced the success rate of such T cell cultures.

DNA/NYVAC vaccine regimen induces HIV-specific CD4 and CD8 T-cell responses in intestinal mucosa.

In the present study, we have investigated the anatomic distribution in blood and gut mucosal tissues of memory poxvirus-specific CD4 and CD8 T cells in subjects vaccinated with smallpox and compared it with vector (NYVAC)-specific and HIV insert-specific T-cell responses induced by an experimental DNA-C/ NYVAC-C vaccine regimen. Smallpox-specific CD4 T-cell responses were present in the blood of 52% of the subjects studied, while smallpox-specific CD8 T cells were rarely detected (12%). With one exception, smallpox-specific T cells were not measurable in gut tissues. Interestingly, NYVAC vector-specific and HIV-specific CD4 and CD8 T-cell responses were detected in almost 100% of the subjects immunized with DNA-C/NYVAC-C in blood and gut tissues. The large majority (83%) of NYVAC-specific CD4 T cells expressed α4β7 integrins and the HIV coreceptor CCR5. These results demonstrate that the experimental DNA-C/NYVAC-C HIV vaccine regimen induces the homing of potentially protective HIV-specific CD4 and CD8 T cells in the gut, the port of entry of HIV and one of the major sites for HIV spreading and the depletion of CD4 T cells.