STEAP, a prostate tumor antigen, is a target of human CD8+ T cells

STEAP is a recently identified protein shown to be particularly overexpressed in prostate cancer and also present in numerous human cancer cell lines from prostate, pancreas, colon, breast, testicular, cervical, bladder and ovarian carcinoma, acute lymphocytic leukemia and Ewing sarcoma. This expression profile renders STEAP an appealing candidate for broad cancer immunotherapy. In order to investigate if STEAP is a tumor antigen that can be targeted by specific CD8⁺ T cells, we identified two high affinity HLA-A*0201 restricted peptides (STEAP_86–94 and STEAP_262–270). These peptides were immunogenic in vivo in HLA-A*0201 transgenic HHD mice. Peptide specific murine CD8 T cells recognized COS-7 cells co-transfected with HHD (HLA-A*0201) and STEAP cDNA constructs and also HLA-A*0201⁺ STEAP⁺ human tumor cells. Furthermore, STEAP_86–94 and STEAP_262–270 stimulated specific CD8⁺ T cells from HLA-A*0201⁺ healthy donors, and these peptide specific CD8⁺ T cells recognized STEAP positive human tumor cells in an HLA-A*0201-restricted manner. Importantly, STEAP_86–94-specific T cells were detected and reactive in the peripheral blood mononuclear cells in NSCLC and prostate cancer patients ex vivo. These results show that STEAP can be a target of anti-tumor CD8⁺ T cells and that STEAP peptides can be used for a broad-spectrum-tumor immunotherapy.     

The predominance of CD8+ T cells after infection with measles virus suggests a role for CD8+ class I MHC-restricted cytotoxic T lymphocytes (CTL) in recovery from measles. Clonal analyses of human CD8+ class I MHC-restricted CTL

Stimulation of PBMC, in children recovering from acute measles, with autologous EBV-transformed and measles virus (MV)-infected lymphoblastoid cell lines (B-LCL) expanded primarily MV-specific CD8+ T cells. A large number of CD8+ T cell clones were obtained either by passaging of bulk cultures at limiting dilutions or by direct cloning of PBMC without previous stimulation in bulk culture. The MV-specific CD8+ T cell clones responding in a proliferative and a CTL assay were found to be class I MHC restricted. In contrast, CD4+ MV-specific T cell clones, which were generated by the same protocol, recognized MV in association with class II MHC molecules. Analysis of processing requirements for Ag presentation to CD8+ and CD4+ T cell clones, measured by the effect of chloroquine in a proliferative T cell response, revealed that both types of T cells recognized MV Ag processed via the endogenous/cytoplasmic pathway. Thus, these studies indicate that, as in most other viral infections and in contrast to previous suggestions, the class I MHC-restricted CTL response by CD8+ T cells may be an important factor in the control and elimination of MV infection. Therefore, the role proposed for CD4+ class II-restricted T cells in recovery from measles needs to be reevaluated.     

Polyclonal MHC Ib-restricted CD8+ T cells undergo homeostatic expansion in the absence of conventional MHC-restricted T cells

Naive T cells have the capacity to expand in a lymphopenic environment in a process called homeostatic expansion, where they gain a memory-like phenotype. Homeostatic expansion is dependent on competition for a number of factors, including growth factors and interactions with their selecting self-MHC molecules. In contrast to conventional T cells, it is unclear whether class Ib-restricted CD8+ T cells have a capacity to undergo homeostatic expansion. In this study, we demonstrate that polyclonal MHC Ib-restricted CD8+ T cells can undergo homeostatic expansion and that their peripheral expansion is suppressed by conventional MHC-restricted T cells. The acute depletion of CD4+ T cells in MHC class Ia-deficient Kb-/-Db-/- mice led to the substantial expansion of class Ib-restricted CD8+ T cells. Adoptive transfer of class Ib-restricted CD8+ T cells to congenic lymphopenic recipients revealed their ability to undergo homeostatic expansion in a MHC Ib-dependent manner. To further study the homeostatic expansion of MHC Ib-restricted T cells in the absence of all conventional MHC-restricted T cells, we generated mice that express only MHC Ib molecules by crossing H-2Kb-/-Db-/- with CIITA-/- mice. CD8+ T cells in these mice exhibit all of the hallmarks of naive T cells actively undergoing homeostatic expansion with constitutive memory-like surface and functional phenotype. These findings provide direct evidence that MHC Ib-restricted CD8+ T cells have the capacity to undergo homeostatic expansion. Their peripheral expansion is suppressed under normal conditions by a numerical excess of conventional MHC class Ia- and class II-restricted T cells.     

Functional tolerance of CD8+ T cells induced by muscle-specific antigen expression

Skeletal muscles account for more than 30% of the human body, yet mechanisms of immunological tolerance to this tissue remain mainly unexplored. To investigate the mechanisms of tolerance to muscle-specific proteins, we generated transgenic mice expressing the neo-autoantigen OVA exclusively in skeletal muscle (SM-OVA mice). SM-OVA mice were bred with OT-I or OT-II mice that possess a transgenic TCR specific for OVA peptides presented by MHC class I or class II, respectively. Tolerance to OVA did not involve clonal deletion, anergy or an increased regulatory T cell compartment. Rather, CD4+ T cell tolerance resulted from a mechanism of ignorance revealed by their response following OVA immunization. In marked contrast, CD8+ T cells exhibited a loss of OVA-specific cytotoxic activity associated with up-regulation of the immunoregulatory programmed death-1 molecule. Adoptive transfer experiments further showed that OVA expression in skeletal muscle was required to maintain this functional tolerance. These results establish a novel asymmetric model of immunological tolerance to muscle autoantigens involving Ag ignorance for CD4+ T cells, whereas muscle autoantigens recognized by CD8+ T cells results in blockade of their cytotoxic function. These observations may be helpful for understanding the breakage of tolerance in autoimmune muscle diseases.     

Cutting edge: intravenous soluble antigen is presented to CD4 T cells by CD8- dendritic cells, but cross-presented to CD8 T cells by CD8+ dendritic cells

Mouse spleen contains three distinct mature dendritic cell (DC) populations (CD4(+)8(-), CD4(-)8(-), and CD4(-)8(+)) which retain a capacity to take up particulate and soluble AGS: Although the three splenic DC subtypes showed similar uptake of injected soluble OVA, they differed markedly in their capacity to present this Ag and activate proliferation in OVA-specific CD4 or CD8 T cells. For class II MHC-restricted presentation to CD4 T cells, the CD8(-) DC subtypes were more efficient, but for class I MHC-restricted presentation to CD8 T cells, the CD8(+) DC subtype was far more effective. This differential persisted when the DC were activated with LPS. The CD8(+) DC are therefore specialized for in vivo cross-presentation of exogenous soluble Ags into the class I MHC presentation pathway.     

Activation of CD8+ regulatory T cells by human placental trophoblasts

The immunological basis by which a mother tolerates her semi-allogeneic fetus remains poorly understood. Several mechanisms are likely to contribute to this phenomenon including active immune regulation by regulatory T cells. In this article, we report that human placental trophoblasts activate a clonal population of CD8(+) T cells with regulatory function. These cells are not MHC class I restricted, but require costimulation through a member of the carcinoembryonic Ag family present on early gestation trophoblasts. These regulatory T cells express the mucosal markers CD101 and CD103 and display selective usage of the TCR gene Vbeta9. CD8(+) T cells isolated from the peripheral blood of pregnant mothers (16-28 wk) also demonstrate expansions in the same Vbeta family (Vbeta9), signaling a possible role for these cells in preventing fetal rejection in vivo. We have previously characterized a subset of CD8(+) regulatory T cells activated by the combination of the nonclassical class I molecule CD1d and a costimulatory molecule of the carcinoembryonic Ag family present on the intestinal epithelium. These data support the concept that distinct regulatory T cell populations exist at different sites and may be regulated locally by unique restriction elements, costimulatory signals, and Ags.     

A shared epitope of the interphotoreceptor retinoid-binding protein recognized by the CD4+ and CD8+ autoreactive T cells

We previously demonstrated that cultures of rat uveitogenic T cells rapidly become dominated by CD4+ cells, but activation of CD8+ autoreactive T cells also occurred during the in vitro culture of in vivo-primed T cells. In the present study, we show that the commonly used uveitogenic peptide, interphotoreceptor retinoid-binding protein (IRBP) 1-20, generated both CD4+ and CD8+ autoreactive T cells in the C57BL/6 (B6) mouse and that this 20-mer contains at least two distinct antigenic epitopes. To determine whether the CD8 response was Ag-specific and whether CD4+ and CD8+ IRBP1-20-specific T cells recognize distinct antigenic epitopes, we prepared highly purified CD4+ and CD8+ T cells from IRBP1-20-primed mice and tested their proliferative response to a large panel of truncated peptides derived from IRBP1-20. The results showed that both CD4+ and CD8+ T cells recognized the same spectrum of peptides. In addition, peptides P10-18 were found to bind effectively to CD8+ IRBP1-20-specific T cells when complexed with recombinant H-2K(b) and also stimulate the proliferation and cytokine production of CD4+ IRBP1-20-specific T cells. Our results document for the first time that CD8+ and CD4+ autoreactive T cells display characteristic epitope recognition and they both recognize the same core epitope.     

Delayed clearance of Sendai virus in mice lacking class I MHC-restricted CD8+ T cells.

The role and interdependence of CD8+ and CD4+ alpha beta-T cells in the acute response after respiratory infection with the murine parainfluenza type 1 virus, Sendai virus, has been analyzed for H-2b mice. Enrichment of CD8+ virus-specific CTL effectors in the lungs of immunologically intact C57BL/6 animals coincided with the clearance of the virus from this site by day 10 after infection. Removal of the CD4+ T cells by in vivo mAb treatment did not affect appreciably either the recruitment of CD8+ T cells to the infected lung, or their development into virus-specific cytotoxic effectors. In contrast, depletion of the CD8+ subset delayed virus clearance, although most mice survived the infection. Transgenic H-2b F3 mice homozygous (-/-) for a beta 2 microglobulin (beta 2-m) gene disruption, which lack both class I MHC glycoproteins and mature CD8+ alpha beta-T cells, showed a comparable, delayed clearance of Sendai virus from the lung. Virus-specific, class II MHC-restricted CTL were demonstrated in both freshly isolated bronchoalveolar lavage populations and cultured lymph node and spleen tissue from the beta 2-m (-/-) transgenics. Treatment of the beta 2-m (-/-) mice with the mAb to CD4 led to delayed virus clearance and death, which was also the case for normal mice that were depleted simultaneously of the CD4+ and CD8+ subsets. These results indicate that, although classical class I MHC-restricted CD8+ cytotoxic T cells normally play a dominant role in the recovery of mice acutely infected with Sendai virus, alternative mechanisms involving CD4+ T cells exist and can compensate, in time, for the loss of CD8+ T cell function.     

Unrestricted recognition of a nonpeptide antigen by CD8+ cytolytic T lymphocytes

CD8+ murine CTL that are specific for an unusual nonpeptide Ag, the heme moiety of hemoglobin, have been derived by in vitro stimulation of spleen cells with hemin. Such CTL demonstrate a requirement for the expression of class I Ag on target cells, yet appear to be unrestricted to the extent that both syngeneic and allogeneic targets precoated with hemin are sensitive to lysis. A series of CTL clones with specificity for hemin was derived from C57BL/6 mice. They exhibited the same type of promiscuous recognition that was observed in CTL populations from a number of different strains. The possibility that hemin acts as a nonspecific mediator of lysis by CTL was ruled out by the fact that a variety of CTL populations and clones specific for different Ag did not exhibit hemin-specific lysis. Some explanations offered to explain these results include 1) the possibility that hemin is recognized by binding to a site on the MHC other than the Ag-binding groove, and 2) the possibility that TCR recognition of a rigid molecule, such as hemin, may be less sensitive to polymorphic variation in the MHC than is recognition of a conventional peptide Ag whose conformation may differ significantly when bound to MHC molecules whose sequences differ within the Ag-binding groove.     

The role of dendritic cells in selection of classical and nonclassical CD8+ T cells in vivo

T cell development is determined by positive and negative selection events. An intriguing question is how signals through the TCR can induce thymocyte survival and maturation in some and programmed cell death in other thymocytes. This paradox can be explained by the hypothesis that different thymic cell types expressing self-MHC/peptide ligands mediate either positive or negative selection events. Using transgenic mice that express MHC class I (MHC-I) selectively on DC, we demonstrate a compartmentalization of thymic functions and reveal that DC induce CTL tolerance to MHC-I-positive hemopoietic targets in vivo. However, in normal and bone marrow chimeric mice, MHC-I+ DC are sufficient to positively select neither MHC-Ib (H2-M3)- nor MHC-Ia (H2-K)-restricted CD8+ T cells. Thus, thymic DC are specialized in tolerance induction, but cannot positively select the vast majority of MHC-I-restricted CD8+ T cells.     

Cutting edge: cross-presented intracranial antigen primes CD8+ T cells

The CNS is considered immune privileged due to the blood-brain barrier and the absence of conventional lymphatics. Nonetheless, T cell immune responses specific for CNS Ag have been documented. Where these events are initiated and what cellular mechanisms are involved remain unknown. In this study, we established an experimental mouse model to evaluate the requirements for priming CD8+ T cells following the cross-presentation of intracranial Ag. Surprisingly, we find that even with a damaged blood-brain barrier, Ag presentation occurs in regional lymph nodes and not within the CNS itself. Only once the responding cells have expanded can they traffic to the site of CNS injury. Cross-presentation of intracranial Ag is efficient and the subsequent priming of CD8+ T cells is dependent on CD4+ T cell help and CD40 signaling in host APCs. Our findings have important implications for the initiation of T cell immune responses toward CNS Ags.     

Staphylococcal superantigens induce lymphotactin production by human CD4+ and CD8+ T cells.

Lymphotactin is a potent chemotactic cytokine (chemokine) that is produced by and also attracts T and natural killer (NK) cells. We are studying whether chemokines that affect mainly T cells might also regulate immune responses by preferentially recruiting individual subsets or by affecting cytokine or other chemokine responses. In order to pursue these questions, we need to learn more about the mechanisms regulating lymphotactin production and the cell types capable of releasing this factor. We used new monoclonal antibodies against human lymphotactin to develop a sensitive antigen-capture enzyme linked immunoabsorbent assay (ELISA) that measures chemokine levels in culture fluids. Using this capture ELISA, we showed that lymphotactin could be produced by CD4+ and CD8+ T cells, but only after T cell-receptor-dependent stimulation using bacterial superantigens and not after treatment by inflammatory cytokines or lipopolysaccharide (LPS). Our data show that lymphotactin production responds mainly to T cell-receptor signals in CD4+ and CD8+ T cells, and suggests a mechanism whereby this chemokine could help to regulate T cell immune responses.     

Targeting antigen in mature dendritic cells for simultaneous stimulation of CD4+ and CD8+ T cells

Due to their potent immunostimulatory capacity, dendritic cells (DC) have become the centerpiece of many vaccine regimens. Immature DC (DCimm) capture, process, and present Ags to CD4(+) lymphocytes, which reciprocally activate DCimm through CD40, and the resulting mature DC (DCmat) loose phagocytic capacity, but acquire the ability to efficiently stimulate CD8(+) lymphocytes. Recombinant vaccinia viruses (rVV) provide a rapid, easy, and efficient method to introduce Ags into DC, but we observed that rVV infection of DCimm results in blockade of DC maturation in response to all activation signals, including CD40L, monocyte-conditioned medium, LPS, TNF-alpha, and poly(I:C), and failure to induce a CD8(+) response. By contrast, DCmat can be infected with rVV and induce a CD8(+) response, but, having lost phagocytic activity, fail to process the Ag via the exogenous class II pathway. To overcome these limitations, we used the CMV protein pp65 as a model Ag and designed a gene containing the lysosomal-associated membrane protein 1 targeting sequence (Sig-pp65-LAMP1) to target pp65 to the class II compartment. DCmat infected with rVV-Sig-pp65-LAMP1 induced proliferation of pp65-specific CD4(+) clones and efficiently induced a pp65-specific CD4(+) response, suggesting that after DC maturation the intracellular processing machinery for class II remains intact for at least 16 h. Moreover, infection of DCmat with rVV-Sig-pp65-LAMP1 resulted in at least equivalent presentation to CD8(+) cells as infection with rVV-pp65. These results demonstrate that despite rVV interference with DCimm maturation, a single targeting vector can deliver Ags to DCmat for the effective simultaneous stimulation of both CD4(+) and CD8(+) cells.     

CD8 blockade promotes antigen responsiveness to nontolerizing antigen in tolerant mice by inhibiting apoptosis of CD4+ T cells

Using the DO11.10 CD4+ TCR-transgenic mouse system, we have recently shown that CD8 blockade promotes the expansion of Ag-specific regulatory CD4+ T cells in mice made tolerant to OVA with anti-CD4 mAb. We now show that CD8 blockade is also critical to promoting responses to nontolerizing Ag in anti-CD4 mAb-treated tolerant mice. Previously published work shows that treatment with anti-CD4 mAb without CD8 blockade induces Ag-specific tolerance. We now show that, in addition to inducing tolerance, anti-CD4 mAb treatment also significantly reduces responsiveness to irrelevant, nontolerizing Ag, and this unresponsiveness is associated with significant apoptosis of the CD4+ T cells. Anti-CD4 mAb-induced apoptosis is inhibited by cotreatment with anti-CD8 mAb and responsiveness to irrelevant Ag is restored, while Ag-specific tolerance is maintained. These data suggest that CD8 blockade promotes responsiveness to nontolerizing Ags in tolerant mice by inhibiting CD4+ T cell apoptosis.     

HIV-specific IL-10-positive CD8+ T cells suppress cytolysis and IL-2 production by CD8+ T cells

IL-10 producing T cells inhibit Ag-specific CD8+ T cell responses and may play a role in the immune dysregulation observed in HIV infection. We have previously observed the presence of HIV-specific IL-10-positive CD8+ T cells in advanced HIV disease. In this study, we examined the suppressive function of the Gag-specific IL-10-positive CD8+ T cells. Removal of these IL-10-positive CD8+ T cells resulted in increased cytolysis and IL-2, but not IFN-gamma, production by both HIV- and human CMV-specific CD8+ T cells. In addition, these IL-10-positive CD8+ T cells mediated suppression through direct cell-cell contact, and had a distinct immunophenotypic profile compared with other regulatory T cells. We describe a new suppressor CD8+ T cell population in advanced HIV infection that may contribute to the immune dysfunction observed in HIV infection.     

Differential suppression of tumor-specific CD8+ T cells by regulatory T cells

In the CT26 BALB/c murine model of colorectal carcinoma, depletion of regulatory T cells (Tregs) prior to tumor inoculation results in protective immunity to both CT26 and other BALB/c-derived tumors of diverse histological origin. In this paper, we show that cross-protection can be conferred by adoptively transferred CD8(+) CTLs. Other schedules for inducing immunity to CT26 have been described, but they do not lead to cross-protection. We show that Treg ablation facilitates the development of new CTL specificities that are normally cryptic, and have mapped the root epitope of one of these responses. This work has allowed us to demonstrate how the specificity of CTL responses to tumor Ags can be controlled via differential suppression of CTL specificities by Tregs, and how this can result in very different physiological outcomes.     

Ex vivo expansion of human CD8+ T cells using autologous CD4+ T cell help

Background

Using in vivo mouse models, the mechanisms of CD4⁺ T cell help have been intensively investigated. However, a mechanistic analysis of human CD4⁺ T cell help is largely lacking. Our goal was to elucidate the mechanisms of human CD4⁺ T cell help of CD8⁺ T cell proliferation using a novel in vitro model.

Methods/Principal Findings

We developed a genetically engineered novel human cell-based artificial APC, aAPC/mOKT3, which expresses a membranous form of the anti-CD3 monoclonal antibody OKT3 as well as other immune accessory molecules. Without requiring the addition of allogeneic feeder cells, aAPC/mOKT3 enabled the expansion of both peripheral and tumor-infiltrating T cells, regardless of HLA-restriction. Stimulation with aAPC/mOKT3 did not expand Foxp3⁺ regulatory T cells, and expanded tumor infiltrating lymphocytes predominantly secreted Th1-type cytokines, interferon-γ and IL-2. In this aAPC-based system, the presence of autologous CD4⁺ T cells was associated with significantly improved CD8⁺ T cell expansion in vitro. The CD4⁺ T cell derived cytokines IL-2 and IL-21 were necessary but not sufficient for this effect. However, CD4⁺ T cell help of CD8⁺ T cell proliferation was partially recapitulated by both adding IL-2/IL-21 and by upregulation of IL-21 receptor on CD8⁺ T cells.

Conclusions

We have developed an in vitro model that advances our understanding of the immunobiology of human CD4⁺ T cell help of CD8⁺ T cells. Our data suggests that human CD4⁺ T cell help can be leveraged to expand CD8⁺ T cells in vitro.     

Recognition of a sequestered self peptide by influenza virus-specific CD8+ cytolytic T lymphocytes

The Ag receptors on CD8+ CTL recognize foreign antigenic peptides associated with cell surface MHC class I molecules. Peptides derived from self proteins are also normally presented by MHC class I molecules. Here we report that an H-2Kd-restricted murine CD8+ CTL clone directed to an influenza hemagglutinin epitope can recognize a peptide derived from the murine mitochondrial aconitase enzyme in association with H-2Kd molecules. Surprisingly, this self peptide is not normally displayed on the cell surface associated with the restricting MHC class I molecule. Several lines of evidence suggest that this self peptide, although requiring association with the Kd molecule for CTL recognition, is not associated with this or other MHC class I allele under physiologic conditions in intact cells. Rather, it is sequestered in the cytoplasm associated with a carrier protein and is released only upon cell disruption. These results suggest a means of restricting the entry of self peptide into the class I pathway. In addition, this finding raises the possibility that self peptides sequestered within the cell can, after release from damaged cells, interact with MHC class I molecules on bystander cells and trigger autoimmune injury by virus-specific CTLs during viral infection.     

Recognition of homo- and heterosubtypic variants of influenza A viruses by human CD8+ T lymphocytes

In the present study, the recognition of epitope variants of influenza A viruses by human CTL was investigated. To this end, human CD8(+) CTL clones, specific for natural variants of the HLA-B*3501-restricted epitope in the nucleoprotein (NP(418-426)), were generated. As determined in (51)Cr release assays and by flow cytometry with HLA-B*3501-peptide tetrameric complexes, CTL clones were found to be specific for epitopes within one subtype or cross-reactive with heterosubtypic variants of the epitope. Using eight natural variants of the epitope, positions in the 9-mer important for T cell recognition and involved in escape from CTL immunity were identified and visualized using multidimensional scaling. It was shown that positions 4 and 5 in the 9-mer epitope were important determinants of T cell specificity. The in vivo existence of CD8(+) cells cross-reactive with homo- and heterosubtypic variants of the epitope was further confirmed using polyclonal T cell populations obtained after stimulation of PBMC with different influenza A viruses. Based on the observed recognition patterns of the clonal and polyclonal T cell populations and serology, it is hypothesized that consecutive infections with influenza viruses containing different variants of the epitope select for cross-reactive T cells in vivo.     

Proliferation requirements of cytomegalovirus-specific,effector-type human CD8+ T cells

Two prototypic types of virus-specific CD8(+) T cells can be found in latently infected individuals: CD45R0(+)CD27(+)CCR7(-) effector-memory, and CD45RA(+)CD27(-)CCR7(-) effector-type cells. It has recently been implied that CD45RA(+)CD27(-)CCR7(-) T cells are terminally differentiated effector cells and as such have lost all proliferative capacity. We show in this study, however, that stimulation of CMV-specific CD45RA(+)CD27(-)CCR7(-) T cells with their cognate peptide in concert with either CD4(+) help or IL-2, IL-15, or IL-21 in fact induces massive clonal expansion. Concurrently, these stimulated effector T cells change cell surface phenotype from CD45RA to CD45R0 and regain CCR7, while effector functions are maintained. Our data imply that CD45RA(+)CD27(-)CCR7(-) effector-type T cells contribute to immunity not only by direct execution of effector functions, but also by yielding progeny in situations of viral reinfection or reactivation.