NKT cells provide help for dendritic cell-dependent priming of MHC class I-restricted CD8+ T cells in vivo

Dendritic cells (DC) are potent APCs for naive T cells in vivo. This is evident by inducing T cell responses through adoptive DC transfer. Priming specific CTL responses in vivo often requires "help". We study alternative sources of help in DC-dependent priming of MHC class I-restricted CTL. Priming an anti-viral CTL response in naive B6 mice by adoptive transfer of antigenic peptide-pulsed DC required CD4(+) T cell help. CTL priming was facilitated by providing MHC class II-dependent specific help. Furthermore, transfers of MHC class II-deficient pulsed DC into naive, normal hosts, or DC transfers into naive, CD4(+) T cell-depleted hosts primed CTL inefficiently. Pretreatment of DC with immune-stimulating oligodeoxynucleotides rendered them more efficient for CD4(+) T cell-independent priming of CTL. DC copresenting a K(b)-binding antigenic peptide and the CD1d-binding glycolipid alpha-galactosyl-ceramide efficiently primed CTL in a class II-independent way. To obtain NKT cell-dependent help in CTL priming, the same DC had to present both the peptide and the glycolipid. CTL priming by adoptive DC transfer was largely NK cell-dependent. The requirement for NK cells was only partially overcome by recruiting NKT cell help into DC-dependent CTL priming. NKT cells thus are potent helper cells for DC-dependent CTL priming.     

Acidosis improves uptake of antigens and MHC class I-restricted presentation by dendritic cells

It is widely appreciated that inflammatory responses in peripheral tissues are usually associated to the development of acidic microenvironments. Despite this, there are few studies aimed to analyze the effect of extracellular pH on immune cell functions. We analyzed the impact of acidosis on the behavior of dendritic cells (DCs) derived from murine bone marrow. We found that extracellular acidosis (pH 6.5) markedly stimulated the uptake of FITC-OVA, FITC-dextran, and HRP by DCs. In fact, to reach similar levels of endocytosis, DCs cultured at pH 7.3 required concentrations of Ag in the extracellular medium almost 10-fold higher compared with DCs cultured at pH 6.5. Not only the endocytic capacity of DCs was up-regulated by extracellular acidosis, but also the expression of CD11c, MHC class II, CD40, and CD86 as well as the acquisition of extracellular Ags by DCs for MHC class I-restricted presentation. Importantly, DCs pulsed with Ag under acidosis showed an improved efficacy to induce both specific CD8(+) CTLs and specific Ab responses in vivo. Our results suggest that extracellular acidosis improves the Ag-presenting capacity of DCs.     

Mice can recover from pulmonary influenza virus infection in the absence of class I-restricted cytotoxic T cells.

Intranasal exposure of athymic (nu/nu) BALB/c mice to influenza virus leads to a persistent infection of the respiratory tract from which the mice die, usually within 3 to 4 wk with symptoms of general cachexia. However, if these nude mice were injected 1 day after infection, with approximately 10(6) cells from individual virus-specific MHC class II-restricted Th cell clones, they showed greatly reduced mortality and the titers of infectious virus in their lungs were reduced, often to undetectable levels. By coinfecting mice with pairs of antigenically distinct viruses and subsequently determining the extent of clearance of each type of virus, it could be shown first that the clearance mechanism was immunologically specific but did not display the typical crossreaction of class I-restricted cytotoxic T (Tc) cells. In addition, neither primary nor memory Tc responses could be detected in these mice. Second, Th cell clones promoted clearance solely of those viruses that contained the specific Th cell determinant, i.e., Th cell-nonreactive bystander viruses were not cleared. These findings were compatible with virus clearance being effected either directly after recognition of infected class II-positive cells by the transferred Th cells or indirectly via promotion of a glycoprotein-specific antibody response. The latter seems to be the case because transfer of Th cells into infected T and B cell-deficient SCID mice did not result in virus clearance, although transfer of an anti-hemagglutinin antibody cocktail did. Thus, a virus-specific Tc cell response is not a requirement for recovery from a pulmonary influenza virus infection.     

Induction of central deletional T cell tolerance by gene therapy

Transgenic mice expressing an alloreactive TCR specific for the MHC class I Ag K(b) were used to examine the mechanism by which genetic engineering of bone marrow induces T cell tolerance. Reconstitution of lethally irradiated mice with bone marrow infected with retroviruses carrying the MHC class I gene H-2K(b) resulted in lifelong expression of K(b) on bone marrow-derived cells. While CD8 T cells expressing the transgenic TCR developed in control mice reconstituted with mock-transduced bone marrow, CD8 T cells expressing the transgenic TCR failed to develop in mice reconstituted with H-2K(b) transduced bone marrow. Analysis of transgene-expressing CD8 T cells in the thymus and periphery of reconstituted mice revealed that CD8 T cells expressing the transgenic TCR underwent negative selection in the thymus of mice reconstituted with K(b) transduced bone marrow. Negative selection induced by gene therapy resulted in tolerance to K(b). Thus, genetic engineering of bone marrow can be used to alter T cell education in the thymus by inducing negative selection.     

Peptide-loaded dendritic cells prime and activate MHC-class I-restricted T cells more efficiently than protein-loaded cross-presenting DC

Undifferentiated and differentiated dendritic cells (uDC and dDC, respectively), derived from the bone marrow, were studied in vitro and in vivo. Ovalbumin (OVA) and two OVA-derived peptides binding to H-2K(b) and I-A(b), respectively, were used. Two IL-2 secreting T cell hybridomas specific for the OVA-derived epitopes were used in the in vitro read-out. The ability to cross-present the H-2K(b) binding OVA(257-264)-peptide (SIINFEKL) was restricted to dDC, which express CD11c(+), CD86(+), and MHC-II(+). In vitro, the antigenicity of SIINFEKL-loaded DC declined at a slower rate than that of OVA-pulsed DC. Moreover, SIINFEKL-loaded DC were up to 50 times more efficient than DC-pulsed with OVA-protein for generation of an H-2K(b)-restricted response. Immunization of mice with SIINFEKL-loaded DC resulted in a much stronger H-2K(b)-restricted response than immunization with OVA-pulsed DC. These data might have important implications for the choice of antigen source in the design of DC-based vaccines.     

APC stimulated by CpG oligodeoxynucleotide enhance activation of MHC class I-restricted T cells

Oligonucleotides containing unmethylated CpG motifs (cytosine-phosphorothioate-guanine oligodeoxynucleotide (CpG ODN)) are potent immunostimulatory agents capable of enhancing the Ag-specific Th1 response when used as immune adjuvants. We evaluated the cellular mechanisms responsible for this effect. Development of a CTL response was enhanced when mice were immunized with peptide-pulsed dendritic cells (DCs) treated with CpG ODN. However, in vitro, CpG ODN had no direct effect on highly purified T cells. In vitro, CpG ODN treatment of peptide- or protein-pulsed DCs enhanced the ability of the DCs to activate class I-restricted T cells. The presence of helper T cells enhanced this effect, indicating that treatment with CpG ODN does not obviate the role of T cell help. The enhanced ability of CpG ODN-treated DCs to activate T cells was present but blunted when DCs derived from IL-12 knockout mice were used. Fixation of Ag-pulsed, CpG ODN-treated DCs limited their ability to activate T cells. In contrast, fixation had little effect on DC activation of T cells when DCs were not exposed to CpG ODN. This indicates that production of soluble factors by DCs stimulated with CpG ODN plays a particularly important role in their ability to activate class I-restricted T cells. We conclude that CpG ODN enhances the development of a cellular immune response by stimulating APCs such as DCs, to produce IL-12 and other soluble factors.     

Alloreactive CD8 T cell tolerance requires recipient B cells, dendritic cells, and MHC class II

Allogeneic bone marrow chimerism induces robust systemic tolerance to donor alloantigens. Achievement of chimerism requires avoidance of marrow rejection by pre-existing CD4 and CD8 T cells, either of which can reject fully MHC-mismatched marrow. Both barriers are overcome with a minimal regimen involving anti-CD154 and low dose (3 Gy) total body irradiation, allowing achievement of mixed chimerism and tolerance in mice. CD4 cells are required to prevent marrow rejection by CD8 cells via a novel pathway, wherein recipient CD4 cells interacting with recipient class II MHC tolerize directly alloreactive CD8 cells. We demonstrate a critical role for recipient MHC class II, B cells, and dendritic cells in a pathway culminating in deletional tolerance of peripheral alloreactive CD8 cells.     

Inhibition of na?ve class I-restricted T cells by altered peptide ligands.

Amino acid variants of an antigenic peptide or altered peptide ligands have previously been investigated with CD4+ and CD8+ T cells. However, for CD8+ T cells, only clones (which are continually restimulated in vitro) have been assessed. Using TCR transgenic mice specific for a class I Kb-restricted OVA peptide (OVAp; OT-I mice) as a source of na?ve CD8+ T cells, single amino acid variants of the OVAp were analysed in vitro for their ability to antagonize the proliferative and cytotoxic function of na?ve OT-I cells. Peptides with substitutions at TCR contact residues were found to be the most potent antagonists of OT-I cell function. Those peptides that inhibited activation of cells to proliferate also inhibited activation of cells to become killers. Inhibition was inversely correlated with interferon (IFN)-gamma production. It was found that levels of antagonist peptide required for inhibition were higher than that described for T cell clones, presumably due to affinity differences.     

Down-regulation of T cell receptors on self-reactive T cells as a novel mechanism for extrathymic tolerance induction.

By generating two types of transgenic mice we have investigated how extrathymic events can contribute to self tolerance. The major histocompatibility complex class I gene Kb was expressed under the control of the glial fibrillary acidic protein promoter in cells of neuroectodermal origin outside the thymus. These mice were tolerant to Kb. When crossed to transgenic mice expressing a Kb-specific T cell receptor (TCR), clonotype+, CD8+CD4- mature T cells could be detected in normal numbers in the thymus of the double-transgenic mice but were strongly reduced in spleen and lymph nodes in comparison with TCR single-transgenic mice. After isolation of clonotype negative splenic T cells and activation in vitro, reappearance of the clonotype+, CD8+CD4- cells was observed. These results indicate that down-regulation of TCR and CD8 molecules on the antigen-specific T cells is a novel mechanism, by which peripheral tolerance to this antigen can occur.     

Rapid deletional peripheral CD8 T cell tolerance induced by allogeneic bone marrow: role of donor class II MHC and B cells

Mixed chimerism and donor-specific tolerance are achieved in mice receiving 3 Gy of total body irradiation and anti-CD154 mAb followed by allogeneic bone marrow (BM) transplantation. In this model, recipient CD4 cells are critically important for CD8 tolerance. To evaluate the role of CD4 cells recognizing donor MHC class II directly, we used class II-deficient donor marrow and were not able to achieve chimerism unless recipient CD8 cells were depleted, indicating that directly alloreactive CD4 cells were necessary for CD8 tolerance. To identify the MHC class II(+) donor cells promoting this tolerance, we used donor BM lacking certain cell populations or used positively selected cell populations. Neither donor CD11c(+) dendritic cells, B cells, T cells, nor donor-derived IL-10 were critical for chimerism induction. Purified donor B cells induced early chimerism and donor-specific cell-mediated lympholysis tolerance in both strain combinations tested. In contrast, positively selected CD11b(+) monocytes/myeloid cells did not induce early chimerism in either strain combination. Donor cell preparations containing B cells were able to induce early deletion of donor-reactive TCR-transgenic 2C CD8 T cells, whereas those devoid of B cells had reduced activity. Thus, induction of stable mixed chimerism depends on the expression of MHC class II on the donor marrow, but no requisite donor cell lineage was identified. Donor BM-derived B cells induced early chimerism, donor-specific cell-mediated lympholysis tolerance, and deletion of donor-reactive CD8 T cells, whereas CD11b(+) cells did not. Thus, BM-derived B cells are potent tolerogenic APCs for alloreactive CD8 cells.     

Activation and control of self-reactive gammadelta T cells

Mammalian and avian CD3+ T cells can be separated into two lymphocyte subsets bearing heterodimeric T-cell receptors (TCR) composed of either alphabeta or gammadelta chains. Although it is now widely accepted that gammadelta and alphabeta T cells fulfill mandatory and nonredundant roles, the generality of this assumption and the exact functions played by gammadelta T cells remain uncertain. While an early protective role of gammadelta T cells has long been suspected, recent observations drawn in particular from transgenic models suggest their implication in the homeostatic control of immune and nonimmune processes. This hypothesis is also supported by the existence of several self-reactive gammadelta T-cell subsets in rodents and humans, whose specificity and effector properties will be detailed and discussed here. The present review will also describe several mechanisms that could allow efficient control of these self-reactive subsets while permitting expression of their regulatory and/or protective properties.     

Effects of lipofectin-antigen complexes on major histocompatibility complex class I-restricted antigen presentation pathway in murine dendritic cells and on dendritic cell maturation

We previously reported that exogenous antigens complexed with the cationic liposome lipofectin (LF) were efficiently presented via major histocompatibility complex (MHC) class I molecules on pulsed dendritic cells (DCs) in vitro. In the present study, we demonstrated that MHC class I-restricted antigen presentation on DC2.4 cells, a murine immature DC line, treated with LF-antigen complexes was remarkably suppressed through the inhibition of endocytosis, proteasome catalysis, and Golgi transport. We also found that LF did not influence expression of interleukin-12 p40 mRNA, MHC molecules, or co-stimulatory molecules in DC2.4 cells. These findings suggest that an antigen-loading procedure using LF could enhance delivery of exogenous antigens to the classical MHC class I pathway in DCs, but it does not initiate DC maturation.     

Retrovirally transduced human dendritic cells can generate T cells recognizing multiple MHC class I and class II epitopes from the melanoma antigen glycoprotein 100

Involvement of tumor-Ag specific CD4(+) and CD8(+) T cells could be critical in the generation of an effective immunotherapy for cancer. In an attempt to optimize the T cell response against defined tumor Ags, we previously developed a method allowing transgene expression in human dendritic cells (DCs) using retroviral vectors. One advantage of using gene-modified DCs is the potential ability to generate CD8(+) T cells against multiple class I-restricted epitopes within the Ag, thereby eliciting a broad antitumor immune response. To test this, we generated tumor-reactive CD8(+) T cells with DCs transduced with the melanoma Ag gp100, for which a number of HLA-A2-restricted epitopes have been described. Using gp100-transduced DCs, we were indeed able to raise T cells recognizing three distinct HLA-A2 epitopes within the Ag, gp100(154-162), gp100(209-217), and gp100(280-288). We next tested the ability of transduced DCs to raise class II-restricted CD4(+) T cells. Interestingly, stimulation with gp100-transduced DCs resulted in the generation of CD4(+) T cells specific for a novel HLA-DRbeta1*0701-restricted epitope of gp100. The minimal determinant of this epitope was defined as gp100(174-190) (TGRAMLGTHTMEVTVYH). These observations suggest that retrovirally transduced DCs have the capacity to present multiple MHC class I- and class II-restricted peptides derived from a tumor Ag, thereby eliciting a robust immune response against that Ag.     

Tumor-infiltrating dendritic cells are potent antigen-presenting cells able to activate T cells and mediate tumor rejection

Dendritic cells (DC) are potent inducers of immune responses. DC have been shown to infiltrate tumors, but very little is known about the functional status of these naturally occurring tumor-infiltrating DC (TIDC). In this study, the status and function of TIDC from several types of mouse melanoma were investigated in detail. CD11c+/MHC II+ cells, consistent with a DC phenotype, were found in all of transplantable or spontaneous melanomas studied. These TIDC were predominantly myeloid (CD11c+/CD8alpha-/B220-) in nature with small numbers of plasmacytoid (CD11c+/B220+). TIDC had an intermediate maturation phenotype with some expression of costimulatory molecules and the capacity to take up particles. Upon culture overnight ex vivo, the TIDC markedly up-regulated the expression of costimulatory molecules and also increased IL-12 production. Importantly, such ex vivo-matured TIDC pulsed with OVA were able to migrate to lymph nodes, to activate naive OVA-specific CD4+ and CD8+ T cells, and to confer protection against a challenge with OVA-expressing tumor cells. In conclusion, melanomas are infiltrated by functional DC that can act as fully competent APC. These APC have the potential to be manipulated and may therefore represent a promising target for cancer immunotherapy.     

Melanoma-reactive class I-restricted cytotoxic T cell clones are stimulated by dendritic cells loaded with synthetic peptides, but fail to respond to dendritic cells pulsed with melanoma-derived heat shock proteins in vitro

Immunization with heat shock proteins (hsp) isolated from cancer cells has been shown to induce a protective antitumor response. The mechanism of hsp-dependent cellular immunity has been attributed to a variety of immunological activities mediated by hsp. Hsp have been shown to bind antigenic peptides, trim the bound peptides by intrinsic enzymatic activity, improve endocytosis of the chaperoned peptides by APCs, and enhance the ability of APCs to stimulate peptide-specific T cells. We have investigated the potential capacity of hsp70 and gp96 to function as a mediator for Ag-specific CTL stimulation in an in vitro model for human melanoma. Repetitive stimulation of PBLs by autologous DCs loaded with melanoma-derived hsp did not increase the frequency of T cells directed against immunodominant peptides of melanoma-associated Ags Melan-A and tyrosinase. In contrast, repeated T cell stimulation with peptide-pulsed DCs enhanced the number of peptide-specific T cells, allowing HLA/peptide multimer-guided T cell cloning. We succeeded in demonstrating that the established HLA-A2-restricted CTL clones recognized HLA-A2(+) APCs exogenously loaded with the respective melanoma peptide as well as melanoma cells processing and presenting these peptides in the context of HLA-A2. We were not able to show that these melanoma-reactive CTL clones were stimulated by autologous dendritic cells pulsed with melanoma-derived hsp. These results are discussed with respect to various models for proving the role of hsp in T cell stimulation and to recent findings that part of the immunological antitumor activities reported for hsp are independent of the chaperoned peptides.     

Antigen-specific T cells can mediate demyelination in organotypic central nervous system cultures

To investigate a role for T lymphocytes in primary demyelination of central nervous system (CNS) tissue, antigen-specific T cell lines sensitized to myelin-associated and myelin-unrelated antigens were developed from SJL mice and tested on myelinated organotypic cultures of syngeneic spinal cord. Demyelination was assessed morphologically by electron microscopy. Antigen responsiveness and specificity, and the phenotypes of the cell lines, were determined by thymidine uptake (3H-TdR) assays and flow cytometry (FC), respectively. Although all T cell lines caused pathologic changes in myelin, the CNS-antigen-specific line induced the most pronounced effects. 3H-TdR uptake assays and FC showed that after three cycles of incubation in the presence of interleukin-2 (IL-2) or antigen, the T cell lines had increased specificity and responsiveness to the priming antigen and were enriched for the L3T4 (helper/inducer) phenotype. This represents the first direct demonstration of T-cell-mediated demyelination, supports a role for the helper/inducer subset in CNS lesion development, and may prove relevant to the human demyelinating disease multiple sclerosis.     

Generation of T cell help through a MHC class I-restricted TCR

CD4+ T cells that are activated by a MHC class II/peptide encounter can induce maturation of APCs and promote cytotoxic CD8+ T cell responses. Unfortunately, the number of well-defined tumor-specific CD4+ T cell epitopes that can be exploited for adoptive immunotherapy is limited. To determine whether Th cell responses can be generated by redirecting CD4+ T cells to MHC class I ligands, we have introduced MHC class I-restricted TCRs into postthymic murine CD4+ T cells and examined CD4+ T cell activation and helper function in vitro and in vivo. These experiments indicate that Ag-specific CD4+ T cell help can be induced by the engagement of MHC class I-restricted TCRs in peripheral CD4+ T cells but that it is highly dependent on the coreceptor function of the CD8beta-chain. The ability to generate Th cell immunity by infusion of MHC class I-restricted Th cells may prove useful for the induction of tumor-specific T cell immunity in cases where MHC class II-associated epitopes are lacking.     

Immunologic self tolerance maintained by T-cell-mediated control of self-reactive T cells: implications for autoimmunity and tumor immunity

T-cell-mediated dominant control of self-reactive T cells is one mechanism for maintaining immunologic self tolerance. It also hampers the generation of immunity to autologous tumor cells. Abrogation of the control can evoke potent tumor immunity as well as autoimmunity in normal animals. This common regulatory mechanism for autoimmunity and tumor immunity can be exploited to devise a novel immunotherapy against cancer.     

Generation of antiviral major histocompatibility complex class I-restricted T cells in the absence of CD8 coreceptors

The CD8 coreceptor is important for positive selection of major histocompatibility complex I (MHC-I)-restricted thymocytes and in the generation of pathogen-specific T cells. However, the requirement for CD8 in these processes may not be essential. We previously showed that mice lacking beta(2)-microglobulin are highly susceptible to tumors induced by mouse polyoma virus (PyV), but CD8-deficient mice are resistant to these tumors. In this study, we show that CD8-deficient mice also control persistent PyV infection as efficiently as wild-type mice and generate a substantial virus-specific, MHC-I-restricted, T-cell response. Infection with vesicular stomatitis virus (VSV), which is acutely cleared, also recruited antigen-specific, MHC-I-restricted T cells in CD8-deficient mice. Yet, unlike in VSV infection, the antiviral MHC-I-restricted T-cell response to PyV has a prolonged expansion phase, indicating a requirement for persistent infection in driving T-cell inflation in CD8-deficient mice. Finally, we show that the PyV-specific, MHC-I-restricted T cells in CD8-deficient mice, while maintained long term at near-wild-type levels, are short lived in vivo and have extremely narrow T-cell receptor repertoires. These findings provide a possible explanation for the resistance of CD8-deficient mice to PyV-induced tumors and have implications for the maintenance of virus-specific MHC-I-restricted T cells during persistent infection.