Immunogenicity of apoptotic cells in vivo: role of antigen load, antigen-presenting cells, and cytokines.

Apoptosis allows the clearance of unwanted cells from living tissues without causing inflammation. Processing of phagocytosed apoptotic cells yields Ags that access the cytosol and the MHC class I pathway of engulfing cells and are recognized by Ag-specific CTL. We show here that injection of apoptotic RMA cells, a syngeneic T cell lymphoma, into C57BL/6 mice results in priming of a functional and long-lasting tumor-specific immune response. Cross-priming of CTLs by apoptotic cells requires CD4+ T cell help. Apoptotic cells, however, are at least 20-fold less immunogenic than nonreplicating live cells. Immunogenicity of apoptotic cells is proportional to the number of cells injected, correlates with the serum concentration of IL-10 and IL-1beta cytokines, and is enhanced in IL-10 knockout mice. Moreover, immunization with dendritic cells (DCs), but not macrophages (Mphi), pulsed with apoptotic cells primes tumor-specific CTLs and confers protection against a tumor challenge. Our findings demonstrate that tumor cells undergoing apoptosis are, though scarcely, immunogenic in vivo, outline the different roles of Mphi and DCs in the physiologic clearance of unwanted cells, and have implications in designing immunomodulating vaccines.     

Dendritic cell editing by activated natural killer cells results in a more protective cancer-specific immune response

Over the last decade, several studies have extensively reported that activated natural killer (NK) cells can kill autologous immature dendritic cells (DCs) in vitro, whereas they spare fully activated DCs. This led to the proposal that activated NK cells might select a more immunogenic subset of DCs during a protective immune response. However, there is no demonstration that autologous DC killing by NK cells is an event occurring in vivo and, consequently, the functional relevance of this killing remains elusive. Here we report that a significant decrease of CD11c(+) DCs was observed in draining lymph nodes of mice inoculated with MHC-devoid cells as NK cell targets able to induce NK cell activation. This in vivo DC editing by NK cells was perforin-dependent and it was functionally relevant, since residual lymph node DCs displayed an improved capability to induce T cell proliferation. In addition, in a model of anti-cancer vaccination, the administration of MHC-devoid cells together with tumor cells increased the number of tumor-specific CTLs and resulted in a significant increase in survival of mice upon challenge with a lethal dose of tumor cells. Depletion of NK cells or the use of perforin knockout mice strongly decreased the tumor-specific CTL expansion and its protective role against tumor cell challenge. As a whole, our data support the hypothesis that NK cell-mediated DC killing takes place in vivo and is able to promote expansion of cancer-specific CTLs. Our results also indicate that cancer vaccines could be improved by strategies aimed at activating NK cells.     

Rejection of mouse sarcoma cells after transfection of MHC class II genes

Th cells are stimulated by peptide Ag presented in the context of MHC class II molecules. We have reasoned that immune responses against tumors may be more efficient if tumor cells were class II Ag positive, and thereby able to directly function as APC to stimulate tumor-specific Th cell proliferation. We have tested this hypothesis by using DNA-mediated gene transfer to generate syngeneic MHC class II Ag-expressing mouse Sal sarcoma cells (Sal/Ak transfectants). Autologous A/J mice challenged i.p. or s.c. with Sal/Ak transfectants do not develop tumors, whereas A/J mice challenged with the class II negative parental Sal tumor have a high tumor incidence. Furthermore, immunization of the autologous host with Sal/Ak transfectants completely protects against subsequent challenge with wild-type Sal cells. MHC class II-expressing tumor cells, therefore, stimulate an improved tumor-specific immune response, and the immunity is cross-reactive with the class II negative tumor. Inasmuch as the transfected MHC class II gene product is not functioning as a target molecule for autologous tumor rejection, the improved immunogenicity of the Sal/Ak cells is probably due to stimulation of a tumor-specific Th cell population. The increased immunogenicity of Sal/Ak cells is, therefore, probably the result of direct presentation of Sal tumor-associated Ag in the context of tumor cell MHC class II molecules to Th lymphocytes. These studies demonstrate that induction of tumor cell MHC class II Ag expression is a potential strategy for tumor-specific immunotherapy, and suggest that tumor immunity may be enhanced by improved Th cell generation.     

Induction of tumor immunity by removing CD25+CD4+ T cells: a common basis between tumor immunity and autoimmunity.

This study shows that removal of a T cell subpopulation can evoke effective tumor immunity in otherwise nonresponding animals. Elimination of CD25-expressing T cells, which constitute 5-10% of peripheral CD4+ T cells in normal naive mice, elicited potent immune responses to syngeneic tumors in vivo and eradicated them. The responses were mediated by tumor-specific CD8+ CTLs and tumor-nonspecific CD4-8- cytotoxic cells akin to NK cells. Furthermore, in vitro culture of CD25+4+ T cell-depleted splenic cell suspensions prepared from tumor-unsensitized normal mice led to spontaneous generation of similar CD4-8- cytotoxic cells capable of killing a broad spectrum of tumors; reconstitution of CD25+4+ T cells inhibited the generation. In this culture, self-reactive CD25-4+ T cells responding to self peptides/class II MHC complexes on APCs spontaneously proliferated upon removal of CD25+4+ T cells, secreting large amounts of IL-2. The IL-2 thus produced appeared to be responsible for the generation of CD4-8- NK cells as lymphokine-activated killer cells, because direct addition of an equivalent amount of IL-2 to the culture of CD4-8- cells generated similar lymphokine-activated killer/NK cells, whereas coculture of normal CD4-8- cells with CD25-4+ T cells from IL-2-deficient mice did not. Thus, removal of immunoregulatory CD25+4+ T cells can abrogate immunological unresponsiveness to syngeneic tumors in vivo and in vitro, leading to spontaneous development of tumor-specific effector cells as well as tumor-nonspecific ones. This novel way of evoking tumor immunity would help to devise effective immunotherapy for cancer in humans.     

A human NK cell activation/inhibition threshold allows small changes in the target cell surface phenotype to dramatically alter susceptibility to NK cells

NK cell activation is negatively regulated by the expression of target cell MHC class I molecules. We show that this relationship is nonlinear due to an NK cell activation/inhibition threshold. Ewing's sarcoma family tumor cell monolayers, which were highly susceptible to NK cells in vitro, developed a highly resistant phenotype when cultured as three-dimensional multicellular tumor spheroid structures. This suggested that tumor architecture is likely to influence the susceptibility to NK cells in vivo. Resistance of the multicellular tumor spheroid was associated with the increased expression of MHC class I molecules and greatly reduced NK cell activation, implying that a threshold of NK cell activation/inhibition had been crossed. Reducing MHC class I expression on Ewing's sarcoma family tumor monolayers did not alter their susceptibility to NK cells, whereas increased expression of MHC class I rendered them resistant and allowed the threshold point to be identified. This threshold, as defined by MHC class I expression, was predictive of the number of NK-resistant target cells within a population. A threshold permits modest changes in the target cell surface phenotype to profoundly alter the susceptibility to NK cells. Whereas this allows for the efficient detection of target cells, it also provides a route for pathogens and tumors to evade NK cell attack.     

HSV-2 Specifically Down Regulates HLA-C Expression to Render HSV-2-Infected DCs Susceptible to NK Cell Killing

Both NK cells and CTLs kill virus-infected and tumor cells. However, the ways by which these killer cells recognize the infected or the tumorigenic cells are different, in fact almost opposite. CTLs are activated through the interaction of the TCR with MHC class I proteins. In contrast, NK cells are inhibited by MHC class I molecules. The inhibitory NK receptors recognize mainly MHC class I proteins and in this regard practically all of the HLA-C proteins are recognized by inhibitory NK cell receptors, while only certain HLA-A and HLA-B proteins interact with these receptors. Sophisticated viruses developed mechanisms to avoid the attack of both NK cells and CTLs through, for example, down regulation of HLA-A and HLA-B molecules to avoid CTL recognition, leaving HLA-C proteins on the cell surface to inhibit NK cell response. Here we provide the first example of a virus that through specific down regulation of HLA-C, harness the NK cells for its own benefit. We initially demonstrated that none of the tested HSV-2 derived microRNAs affect NK cell activity. Then we show that surprisingly upon HSV-2 infection, HLA-C proteins are specifically down regulated, rendering the infected cells susceptible to NK cell attack. We identified a motif in the tail of HLA-C that is responsible for the HSV-2-meduiated HLA-C down regulation and we show that the HLA-C down regulation is mediated by the viral protein ICP47. Finally we show that HLA-C proteins are down regulated from the surface of HSV-2 infected dendritic cells (DCs) and that this leads to the killing of DC by NK cells. Thus, we propose that HSV-2 had developed this unique and surprising NK cell-mediated killing strategy of infected DC to prevent the activation of the adaptive immunity.     

The brain parenchyma is permissive for full antitumor CTL effector function, even in the absence of CD4 T cells

Effective antitumor immune responses against cerebral malignancies have been demonstrated in several models, but precise cellular function of specific effector cells is poorly understood. We have explored this topic by analyzing the MHC class I-restricted T cell response elicited after implantation of HLA-CW3-transfected P815 mastocytoma cells (P815-CW3) in syngeneic mice. In this model, tumor-specific CTLs use a distinctive repertoire of TCRs that allows ex vivo assessment of the response by immunophenotyping and TCR spectratyping. Thus, for the first time in a brain tumor model, we are able to directly visualize ex vivo CTLs specific for a tumor-expressed Ag. Tumor-specific CTLs are detected in the CNS after intracerebral implantation of P815-CW3, together with other inflammatory cells. Moreover, despite observations in other models suggesting that CTLs infiltrating the brain may be functionally compromised and highly dependent upon CD4 T cells, in this syngeneic P815-CW3 model, intracerebral tumors were efficiently rejected, whether or not CD4 T cells were present. This observation correlated with potent ex vivo cytotoxicity of brain-infiltrating CTLs, specific for the immunodominant epitope CW3170-179 expressed on P815-CW3 tumor cells.     

MHC class I antigens,immune surveillance,and tumor immune escape

Oncogenic transformation in human and experimental animals is not necessarily followed by the appearance of a tumor mass. The immune system of the host can recognize tumor antigens by the presentation of small antigenic peptides to the receptor of cytotoxic T-lymphocytes (CTLs) and reject the nascent tumor. However, cancer cells can sometimes escape these specific T-cell immune responses in the course of somatic (genetic and phenotypic) clonal evolution. Among the tumor immune escape mechanisms described to date, the alterations in the expression of major histocompatibility complex (MHC) molecules play a crucial step in tumor development due to the role of MHC antigens in antigen presentation to T-lymphocytes and the regulation of natural killer cell (NK) cell function. In this work, we have (1) updated information on the mechanisms that allow CTLs to recognize tumor antigens after antigen processing by transformed cells, (2) described the altered MHC class I phenotypes that are commonly found in human tumors, (3) summarized the molecular mechanisms responsible for MHC class I alteration in human tumors, (4) provided evidence that these altered human leukocyte antigens (HLA) class I phenotypes are detectable as result of a T-cell immunoselection of HLA class I-deficient variants by an immunecompetent host, and (5) presented data indicating the MHC class I phenotype and the immunogenicity of experimental metastatic tumors change drastically when tumors develop in immunodeficient mice.     

Evidence for an involvement of T4+ cytotoxic T cells in tumor immunity

Cell-mediated immunity against cancer cells primarily involves class I major histocompatibility complex (MHC)-restricted cytotoxic T cells and natural killer (NK) cells. To investigate whether T4+ cytotoxic T cells also have a role in tumor-specific immunity, mice were immunized with a B cell lymphoma. T cell hybridomas were constructed from the immune spleen cells and analyzed for their cytotoxic ability against the immunizing lymphoma. A T4+, Lyt-1+ hybridoma cell line was developed (103L2) which specifically killed the immunizing tumor cells but not normal B cells or a range of other tumor cells of B or non-B origin. This cytotoxic hybridoma cell line differed from Lyt-2+ cytotoxic T lymphocyte cells and NK cells, commonly identified with cytotoxicity, in a number of important ways. First, the cells were class II MHC restricted; second, interleukin-2 was released from activated effector cells; and finally but most importantly, innocent nonparticipating bystander cells were also killed. The significance of this observation was that normal cells were protected, although a broad range of tumor cell types, including tumor antigen-negative mutants, were killed. It is therefore conceivable that T4+ cytotoxic T cells might play an important role in tumor immunity through the direct recognition and lysis of tumor cells while any tumor variants, arising due to antigen loss, would remain susceptible through the bystander killing effect and normal cells would remain unaffected. These results strongly suggest that tumor-reactive T4+ cytotoxic T cells belong to a new category of effector cells with an important role in tumor-specific immunity.     

Depletion of IL-10- and TGF-beta-producing regulatory gamma delta T cells by administering a daunomycin-conjugated specific monoclonal antibody in early tumor lesions augments the activity of CTLs and NK cells.

It has been demonstrated that gamma delta T cells accumulating in early tumor lesions and those purified from spleen cells of tumor-bearing mice attenuate the activity of CTLs and NK cells. We, therefore, investigated whether depletion of gamma delta T cells from early lesions of tumors results in restoration of CTL and NK cell activities and subsequent regression of tumors. A daunomycin-conjugated anti-gamma delta TCR mAb UC7-13D5 (Dau-UC7) was prepared to efficiently deplete gamma delta T cells. An in vitro study revealed that Dau-UC7 specifically lysed gamma delta TCR+ cells and effectively inhibited splenic gamma delta T cells from tumor-bearing mice to produce cytotoxic cell-suppressive factors. Furthermore, intralesional injections of Dau-UC7 at an early stage of tumor development led to augmentation of tumor-specific CTL as well as NK cell activities and to the resultant regression or growth inhibition of the tumors. On analysis of cytokine profile, gamma delta T cells transcribed mRNAs for IL-10 and TGF-beta, but not IL-4 or IFN-gamma, suggesting the T regulatory 1-like phenotype. Finally, a blocking study with mAbs showed that the inhibitory action of gamma delta T cells on CTLs and NK cells was at least partly mediated by IL-10 and TGF-beta. These results clearly demonstrated the novel mechanism by which T regulatory 1-like gamma delta T cells suppress anti-tumor CTL and NK activities by their regulatory cytokines in early tumor formation.     

CD1d-independent NKT cells in beta 2-microglobulin-deficient mice have hybrid phenotype and function of NK and T cells

Unlike CD1d-restricted NK1.1(+)TCRalphabeta(+) (NKT) cells, which have been extensively studied, little is known about CD1d-independent NKT cells. To characterize their functions, we analyzed NKT cells in beta(2)-microglobulin (beta(2)m)-deficient B6 mice. They are similar to NK cells and expressed NK cell receptors, including Ly49, CD94/NKG2, NKG2D, and 2B4. NKT cells were found in normal numbers in mice that are deficient in beta(2)m, MHC class II, or both. They were also found in the male HY Ag-specific TCR-transgenic mice independent of positive or negative selection in the thymus. For functional analysis of CD1d-independent NKT cells, we developed a culture system in which CD1d-independent NKT cells, but not NK, T, or most CD1d-restricted NKT cells, grew in the presence of an intermediate dose of IL-2. IL-2-activated CD1d-independent NKT cells were similar to IL-2-activated NK cells and efficiently killed the TAP-mutant murine T lymphoma line RMA-S, but not the parental RMA cells. They also killed beta(2)m-deficient Con A blasts, but not normal B6 Con A blasts, indicating that the cytotoxicity is inhibited by MHC class I on target cells. IL-2-activated NKT cells expressing transgenic TCR specific for the HY peptide presented by D(b) killed RMA-S, but not RMA, cells. They also killed RMA (H-2(b)) cells that were preincubated with the HY peptide. NKT cells from beta(2)m-deficient mice, upon CD3 cross-linking, secreted IFN-gamma and IL-2, but very little IL-4. Thus, CD1d-independent NKT cells are significantly different from CD1d-restricted NKT cells. They have hybrid phenotypes and functions of NK cells and T cells.     

Protective immunity against disparate tumors is mediated by a nonpolymorphic MHC class I molecule

Current peptide-based immunotherapies for treatment of model cancers target tumor Ags bound by the classical MHC class I (class Ia) molecules. The extensive polymorphism of class Ia loci greatly limits the effectiveness of these approaches. We demonstrate in this study that the murine nonpolymorphic, nonclassical MHC class I (class Ib) molecule Q9 (Qa-2) promotes potent immune responses against multiple syngeneic tumors. We have previously shown that ectopic expression of Q9 on the surface of class Ia-negative B78H1 melanoma led to efficient CTL-mediated rejection of this tumor. In this study, we report that surface-expressed Q9 on 3LLA9F1 Lewis lung carcinoma and RMA T cell lymphoma also induces potent antitumor CTL responses. Importantly, CTL harvested from animals surviving the initial challenge with Q9-positive 3LLA9F1, RMA, or B78H1 tumors recognized and killed their cognate tumors as well as the other cancer lines. Furthermore, immunization with Q9-expressing 3LLA9F1 or RMA tumor cells established immunological memory that enhanced protection against subsequent challenge with a weakly immunogenic, Q9-bearing melanoma variant. Collectively, the generation of cross-reactive CTL capable of eliminating multiple disparate Q9-expressing tumors suggests that this nonpolymorphic MHC class I molecule serves as a restriction element for a shared tumor Ag(s) common to lung carcinoma, T cell lymphoma, and melanoma.     

In situ detection of antigen-specific T cells in cryo-sections using MHC class I tetramers after dendritic cell vaccination of melanoma patients

Application of tetrameric MHC class I-peptide complexes has significantly improved the monitoring of antigen-specific T cell immune responses in mouse models as well as in clinical studies. Especially MHC class I tetramer analysis of tumor-specific T cells in suspension or on thick vibratome sections from viable tissue has been proven extremely useful. Using the well-characterized mouse tyrosinase-related-protein-2 specific cytotoxic T cell (CTL) clone LP9, we now developed a method that allows for specific identification of T cells with MHC class I tetramers in 8 mum thick, chemically fixed cryosections. The protocol was validated in a murine influenza virus-infection model. Moreover, analysis of delayed type hypersensitivity (DTH) skin biopsies from melanoma patients vaccinated with peptide-loaded mature dendritic cells, revealed the presence and location of anti-tumor CTLs. The specificity of the CTLs detected in situ correlated with both the DTH challenge specificity and reactivity of cell suspensions derived from the same biopsies. Collectively, our data demonstrate that in situ MHC class I tetramer staining provides a valuable tool to reveal the presence and anatomical location of specific CTLs in frozen tissue following immune-based treatment strategies in cancer patients.     

Immunization with a mimotope of GD2 ganglioside induces CD8+ T cells that recognize cell adhesion molecules on tumor cells

The GD2 ganglioside expressed on neuroectodermal tumor cells has been used as a target for passive and active immunotherapy in patients with malignant melanoma and neuroblastoma. We have reported that immunization of mice with a 47-LDA mimotope of GD2, isolated from a phage display peptide library with anti-GD2 mAb 14G2a, induces MHC class I-restricted CD8(+) T cell responses to syngeneic neuroblastoma tumor cells. The cytotoxic activity of the vaccine-induced CTLs was independent of GD2 expression, suggesting recognition of a novel tumor-associated Ag cross-reacting with 47-LDA. Glycan microarray and immunoblotting studies using 14G2a mAb demonstrated that this Ab is highly specific for the entire carbohydrate motif of GD2 but also cross-reacts with a 105 kDa glycoprotein expressed by GD2(+) and GD2(-) neuroblastoma and melanoma cells. Functional studies of tumor cells grown in three-dimensional collagen cultures with 14G2a mAb showed decreases in matrix metalloproteinase-2 activation, a process regulated by the 105 kDa-activated leukocyte cell adhesion molecule (ALCAM/CD166). A recombinant CD166 glycoprotein was shown to be recognized by 14G2a Ab and inhibition of CD166 expression by RNA interference ablated the cell sensitivity to lysis by 47-LDA-induced CD8(+) T cells in vitro and in vivo. The binding of 14G2a to CD166 was not disruptable by a variety of exo- and endo-glycosidases, implying recognition of a non-glycan epitope on CD166. These results suggest that the vaccine-induced CTLs recognize a 47-LDA cross-reactive epitope expressed by CD166, and reveal a novel mechanism of induction of potent tumor-specific cellular responses by mimotopes of tumor-associated carbohydrate Ags.     

Cytotoxic T lymphocytes induce caspase-dependent and -independent cell death in neuroblastomas in a MHC-nonrestricted fashion

The MHC class I- restricted processing and presentation pathway is frequently nonfunctional in tumor cells; therefore, the direct targeting of tumor cells by CTLs may be difficult, if at all possible, to achieve. We used neuroblastoma (NB), which represents a striking example of a tumor with an impaired MHC class I pathway, as a model to study bystander effects of activated T lymphocytes on tumor cells. We found that NB cell lines are susceptible to killing by differentiated CD8(+) CTL clones in a MHC class I-nonrestricted manner that involves two programs of cell death distinguished on the basis of different kinetics, sensitivities to caspase inhibitors, and cytokine-blocking reagents. The "early" death exhibited characteristic features of apoptosis, whereas the "delayed" caspase-independent death exhibited features associated with necrosis and was partially inhibited by TNF-alpha-blocking and prevented by overexpression of Bcl-2 or Bcl-x(L). Our data reveal a previously unappreciated complexity of death pathways induced in tumor cells by immune activation and suggest that redirecting nonspecific effector CTLs to even a small proportion of NB cells or activating CTLs in a tumor's proximity may have therapeutic effects in patients with NB.     

NK cell tolerance in mixed allogeneic chimeras

Alterations in inhibitory receptor expression on NK cells have been detected in mixed allogeneic chimeras and in mosaic MHC class I-expressing transgenic mice. However, it is not known whether or not NK cells are tolerant to host and donor Ags in mixed chimeras. In vitro studies have shown a lack of mutual tolerance of separated donor and host NK cells obtained from mixed chimeras. Using BALB/c-->B6 fully MHC-mismatched mixed chimeras, we have now investigated this question in vivo. Neither donor nor host NK cells in mixed chimeras showed evidence for activation, as indicated by expression of B220 and Thy-1.2 on NK cells in chimeric mice at levels similar to those in nonchimeric control mice. Lethally irradiated, established mixed BALB/c--> B6 chimeras rejected a low dose of beta(2)-microglobulin-deficient bone marrow cells (BMC) efficiently but did not reject BALB/c or B6 BMCs. In contrast, similarly conditioned B6 mice rejected both BALB/c and beta(2)-microglobulin-deficient BMCs. Thus, NK cells were specifically tolerant to the donor and the host in mixed allogeneic chimeras. The similar growth of RMA lymphoma cells in both chimeric and control B6 mice further supports the conclusion that donor BALB/c NK cells are tolerant to B6 Ags in chimeras. Administration of a high dose of exogenous IL-2 could not break NK cell tolerance in chimeric mice, suggesting that NK cell tolerance in chimeras is not due to a lack of activating cytokine. No reduction in the level of expression of the activating receptor Ly-49D, recognizing a donor MHC molecule, was detected among recipient NK cells in mixed chimeras. Thus, the present studies demonstrate that NK cells in mixed chimeras are stably tolerant to both donor and host Ags, by mechanisms that are as yet unexplained.     

Mouse Hepa-1 tumor is rejected by H-2Db-restricted CTL despite decreased MHC class I antigen expression

It has recently been hypothesized that tumor cells with reduced levels of MHC class I antigens are more susceptible to NK-mediated lysis and are rejected by NK cells, whereas tumor cells with normal levels of class I are rejected by tumor-specific CTL. We have tested this hypothesis using a mouse hepatoma system. The Hepa-1 tumor is a spontaneous H-2Kb loss variant that arose from the BW7756 tumor, when BW7756 was adapted to growth in culture. Our studies have shown that despite the loss of H-2Kb antigen, Hepa-1 is not more susceptible to NK lysis than its H-2Kb-transfected variants. These studies also suggested that NK cells were not responsible for rejection of the Hepa-1 tumor. The Hepa-1 tumor, therefore, appears to contradict the hypothesized linkage of MHC levels and NK susceptibility. Because NK cells are not involved in immunity to this tumor, we have sought to identify the effector cell responsible for Hepa-1 rejection. Cytotoxic T lymphocyte assays demonstrate that in vitro, Hepa-1 cells are lysed by Hepa-1-specific H-2Db-restricted CD4-CD8+ T lymphocytes. Footpad assays demonstrate that in vivo, Hepa-1 rejection requires CD4+CD8- and CD4-CD8+ Hepa-1-primed splenocytes. These results indicate that immunity to Hepa-1 is T cell mediated. Hepa-1 is therefore an example of an unusual tumor in that down-regulation of MHC class I antigen expression is associated with increased CTL susceptibility.     

Intraocular tumor antigen drains specifically to submandibular lymph nodes, resulting in an abortive cytotoxic T cell reaction

Ocular immune privilege is considered essential in the protection against sight-threatening immune responses, as illustrated by the ability of the ocular environment to permit the growth of tumors that are rejected when implanted at other sites. Although several studies indicate that soluble Ag can drain directly into the spleen when injected into the anterior chamber, the primary site of intraocular tumor Ag presentation to tumor-specific CTLs has not been studied. To gain a better understanding of the mechanism involved in ocular immune privilege, we examined to which lymphoid organs anterior chamber tumor Ags primarily drain. Our data show that intraocular tumor Ag drains exclusively to the submandibular lymph nodes, resulting in activation of tumor-specific CTLs, whereas no Ag drainage was found in spleen. However, these tumor-specific CTLs do not distribute systemically and, as a consequence, intraocular tumor growth is unhampered. A similar lack of CTL efficacy has been observed in mice bearing s.c. tumors, which is converted to a systemic tumoricidal CTL response by administration of agonistic anti-CD40 mAb. In contrast, systemic anti-CD40 treatment of eye tumor-bearing mice did not result in mobilizing tumor-specific CTLs or tumor eradication. Together, these results show that intraocular tumor Ag drains to regional lymph nodes for activation of tumor-specific CTLs. However, the induced tumor-specific immunity is insufficient for tumor clearance, even combined with otherwise highly effective immune intervention protocols.     

Identification and characterization of the immunodominant rat HER-2/neu MHC class I epitope presented by spontaneous mammary tumors from HER-2/neu-transgenic mice

The HER-2/neu (neu-N)-transgenic mice are a clinically relevant model of breast cancer. They are derived from the parental FVB/N mouse strain and are transgenic for the rat form of the proto-oncogene HER-2/neu (neu). In this study, we report the identification of a MHC class I peptide in the neu protein that is recognized by CD8(+) T cells derived from vaccinated FVB/N mice. This 10-mer was recognized by all tumor-specific FVB/N T cells generated regardless of the TCR Vbeta region expressed by the T cell or the method of vaccination used, establishing it as the immunodominant MHC class I epitope in neu. T cells specific for this epitope were able to cure FVB/N mice of transplanted neu-expressing tumor cells, demonstrating that this is a naturally processed peptide. Altered peptide analogs of the epitope were analyzed for immunogenicity. Vaccination with dendritic cells pulsed with a heteroclitic peptide provided FVB/N and neu-N mice with increased protection against tumor challenge as compared with mice immunized with dendritic cells loaded with either wild-type or irrelevant peptide. Discovery of this epitope allows for better characterization of the CD8(+) T cell responses in the neu-N mouse model in which neu-specific tolerance must be overcome to produce effective antitumor immunity.