Allogeneic gastric cancer cell-dendritic cell hybrids induce tumor antigen (carcinoembryonic antigen) specific CD8+ T cells

The development of protocols for the ex vivo generation of dendritic cells (DCs) has led to intensive research of their potential use in immunotherapy. Accumulating results show the efficacy of this treatment on melanomas which are highly immunogenic. However, its efficacy remains unclear in other tumors. In this study, allogeneic gastric cancer cell–DC hybrids were used to determine the efficacy of this type of immunotherapy in gastric cancer. Fusion cells of DC and allogeneic gastric cancer cells were generated by polyethylene glycol (PEG) and electrofusion. These hybrids were used to induce tumor associated antigen (TAA) specific cytotoxic T lymphocytes (CTLs). The DCs were successfully fused with the allogeneic gastric cancer cells resulting in hybrid cells. These hybrid cells were functional as antigen-presenting cell because they induced allogeneic CD4+ T cells proliferation. CD8+ T cells stimulated by the MKN-45-DC hybrid cells were able to kill MKN-45 when used for immunization. The CTLs killed another gastric cancer cell line, MKN-1, as well as a melanoma cell line, 888mel, suggesting the recognition of a shared tumor antigen. MKN-45 specific CTLs can recognize carcinoembryonic antigen (CEA), indicating that the killing is due to tumor antigens as well as alloantigens. This approach suggests the possible use of allogeneic gastric cancer cell–DC hybrids in DC based immunotherapy for gastric cancer treatment.     

Recognition of a shared human prostate cancer-associated antigen by nonclassical MHC-restricted CD8+ T cells

To identify prostate cancer-associated Ags, tumor-reactive T lymphocytes were generated using iterative stimulations of PBMC from a prostate cancer patient with an autologous IFN-gamma-treated carcinoma cell line in the presence of IL-2. A CD8+ T cell line and TCR alphabeta+ T cell clone were isolated that secreted IFN-gamma and TNF-alpha in response to autologous prostate cancer cells but not to autologous fibroblasts or lymphoblastoid cells. However, these T cells recognized several normal and malignant prostate epithelial cell lines without evidence of shared classical HLA molecules. The T cell line and clone also recognized colon cancers, but not melanomas, sarcomas, or lymphomas, suggesting recognition of a shared epithelium-associated Ag presented by nonclassical MHC or MHC-like molecules. Although Ag recognition by T cells was inhibited by mAb against CD8 and the TCR complex (anti-TCR alphabeta, CD3, Vbeta12), it was not inhibited by mAb directed against MHC class Ia or MHC class II molecules. Neither target expression of CD1 molecules nor HLA-G correlated with T cell recognition, but beta2-microglobulin expression was essential. Ag expression was diminished by brefeldin A, lactacystin, and cycloheximide, but not by chloroquine, consistent with an endogenous/cytosolic Ag processed through the classical class I pathway. These results suggest that prostate cancer and colon cancer cells can process and present a shared peptidic Ag to TCR alphabeta+ T cells via a nonclassical MHC I-like molecule yet to be defined.     

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.     

A MAGE-1 antigenic peptide recognized by human cytolytic T lymphocytes on HLA-A2 tumor cells

Cancer-germline genes such as those of the MAGE family are expressed in many tumors and in male germline cells, but are silent in normal tissues. They encode shared tumor-specific antigens that have been used in therapeutic vaccination trials of cancer patients. It was previously demonstrated that MAGE-1 peptide KVLEYVIKV was presented by HLA-A 0201 molecules on the surface of a human breast carcinoma cell line, but no human specific CTL had been isolated so far. Here, we have used HLA-A2/MAGE-1 fluorescent multimers to isolate from blood cells three human CTL clones that recognized the MAGE-1 peptide. These clones killed efficiently HLA-A2 tumor cells expressing MAGE-1, whether or not they were treated with IFN-, suggesting that the MAGE-1 antigen is processed efficiently by both the standard proteasome and the immunoproteasome. These results indicate that the MAGE-1.A2 peptide can be used for antitumoral vaccination.     

CD103 is a marker for alloantigen-induced regulatory CD8+ T cells

The alphaEbeta7 integrin CD103 may direct lymphocytes to its ligand E-cadherin. CD103 is expressed on T cells in lung and gut and on allograft-infiltrating T cells. Moreover, recent studies have documented expression of CD103 on CD4+ regulatory T cells. Approximately 4% of circulating CD8+ T cells bear the CD103 molecule. In this study, we show that the absence or presence of CD103 was a stable trait when purified CD103- and CD103+ CD8+ T cell subsets were stimulated with a combination of CD3 and CD28 mAbs. In contrast, allostimulation induced CD103 expression on approximately 25% of purified CD103- CD8+ T cells. Expression of CD103 on alloreactive cells was found to be augmented by IL-4, IL-10, or TGF-beta and decreased by addition of IL-12 to MLCs. The alloantigen-induced CD103+ CD8+ T cell population appeared to be polyclonal and retained CD103 expression after restimulation. Markedly, in vitro-expanded CD103+ CD8+ T cells had low proliferative and cytotoxic capacity, yet produced considerable amounts of IL-10. Strikingly, they potently suppressed T cell proliferation in MLC via a cell-cell contact-dependent mechanism. Thus, human alloantigen-induced CD103+ CD8+ T cells possess functional features of regulatory T cells.     

Defective adhesion in tumor infiltrating CD8+ T cells

CD8(+) tumor-infiltrating lymphocytes (TIL) are defective in cytolysis due to tumor-induced inhibition of proximal TCR-mediated signaling, a defect that is relieved upon purification and brief culture. We show in this study that frequency of conjugation in vitro of nonlytic TIL with tumor cells is low in comparison with their lytic counterparts, and the strength of interaction and duration of conjugation are also reduced. Previous reports show that p56(lck) activation is required for TCR-initiated LFA-1 avidity up-regulation, raising the question: is low LFA-1 avidity the basis of reduced TIL conjugation frequency? When stimulated with phorbol ester, nonlytic TIL bind purified ICAM-1 equivalently as lytic TIL, suggesting that LFA-1 can be activated if proximal TCR signaling is bypassed. However, when treated with phorbol ester, the conjugation frequency of nonlytic TIL does not increase. CD2 and CD8 also mediate T cell adhesion to cognate target cells and are both expressed at lower levels in nonlytic TIL in addition to being excluded from the immune synapse formed upon conjugation. Collectively, these results imply that adhesion defects in nonlytic TIL result from a combination of decreased cell surface levels of adhesion molecules, deficient LFA-1 activation, and the failure to recruit essential adhesion receptors to the membrane contact site formed with cognate target cells.     

MHC-unrestricted recognition of bacteria-infected target cells by human CD8+ cytotoxic T lymphocytes.

A CD8+ alpha beta TCR+ T cell clone (A35) was isolated from the synovial fluid of a patient with post-enteric reactive arthritis caused by Yersinia enterocolitica. This clone efficiently killed autologous and allogeneic target cells that had been preincubated with live but not with heat-killed bacteria. There was no restriction by polymorphic parts of HLA-A, -B, or -C molecules and a HLA class II-deficient mutant cell line was lysed as efficiently as its normal counterpart, whereas infected HLA class I-deficient cells (Daudi cells) were not. The clone showed crossreaction between Yersinia enterocolitica, Escherichia coli, Pseudomonas aeruginosa, and Streptococcus pyogenes, but did not lyse target cells preincubated with Staphylococcus epidermidis. MAb to CD2, CD3, and CD8 efficiently blocked A35, whereas the addition of mAb to HLA class II or to HLA class I did not. This clone apparently represents a novel effector mechanism against bacteria-infected or -modified cells that could be involved in the immunopathology of reactive arthritis.     

Development of memory-like autoregulatory CD8+ T cells is CD4+ T cell dependent

Progression of spontaneous autoimmune diabetes is associated with development of a disease-countering negative-feedback regulatory loop that involves differentiation of low-avidity autoreactive CD8(+) cells into memory-like autoregulatory T cells. Such T cells blunt diabetes progression by suppressing the presentation of both cognate and noncognate Ags to pathogenic high-avidity autoreactive CD8(+) T cells in the pancreas-draining lymph nodes. In this study, we show that development of autoregulatory CD8(+) T cell memory is CD4(+) T cell dependent. Transgenic (TG) NOD mice expressing a low-affinity autoreactive TCR were completely resistant to autoimmune diabetes, even after systemic treatment of the mice with agonistic anti-CD40 or anti-4-1BB mAbs or autoantigen-pulsed dendritic cells, strategies that dramatically accelerate diabetes development in TG NOD mice expressing a higher affinity TCR for the same autoantigenic specificity. Furthermore, whereas abrogation of RAG-2 expression, hence endogenous CD4(+) T cell and B cell development, decelerated disease progression in high-affinity TCR-TG NOD mice, it converted the low-affinity TCR into a pathogenic one. In agreement with these data, polyclonal CD4(+) T cells from prediabetic NOD mice promoted disease in high-affinity TCR-TG NOD.Rag2(-/-) mice, but inhibited it in low-affinity TCR-TG NOD.Rag2(-/-) mice. Thus, in chronic autoimmune responses, CD4(+) Th cells contribute to both promoting and suppressing pathogenic autoimmunity.     

NKG2D is a costimulatory receptor for human naive CD8+ T cells

In humans, all alpha beta CD8+ T cells express NKG2D, but in mouse, it is only expressed by activated and memory CD8+ T cells. We purified human naive CD8+ T cells to show that NKG2D serves as a costimulatory receptor for TCR induced Ca2+ mobilization and proliferation. The resulting effector cells are skewed toward a type 1 phenotype and produce high levels of IFN-gamma and TNF-alpha. NKG2D ligands, MHC class I chain-related (MIC)A, MICB, and UL16-binding proteins are expressed on the proliferating cells and NKG2D is down-regulated. The addition of the homeostatic cytokines IL-7 and IL-15 to the culture medium not only enhances proliferation but also counteracts the down-regulation of NKG2D, more so than the addition of IL-2. These results indicate that NKG2D can regulate the priming of human naive CD8+ T cells, which may provide an alternative mechanism for potentiating and channeling the immune response.     

CD8+ T cell immunity against a tumor/self-antigen is augmented by CD4+ T helper cells and hindered by naturally occurring T regulatory cells

CD4(+) T cells control the effector function, memory, and maintenance of CD8(+) T cells. Paradoxically, we found that absence of CD4(+) T cells enhanced adoptive immunotherapy of cancer when using CD8(+) T cells directed against a persisting tumor/self-Ag. However, adoptive transfer of CD4(+)CD25(-) Th cells (Th cells) with tumor/self-reactive CD8(+) T cells and vaccination into CD4(+) T cell-deficient hosts induced autoimmunity and regression of established melanoma. Transfer of CD4(+) T cells that contained a mixture of Th and CD4(+)CD25(+) T regulatory cells (T(reg) cells) or T(reg) cells alone prevented effective adoptive immunotherapy. Maintenance of CD8(+) T cell numbers and function was dependent on Th cells that were capable of IL-2 production because therapy failed when Th cells were derived from IL-2(-/-) mice. These findings reveal that Th cells can help break tolerance to a persisting self-Ag and treat established tumors through an IL-2-dependent mechanism, but requires simultaneous absence of naturally occurring T(reg) cells to be effective.     

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.     

Tumor-specific CD4+ T cells render the tumor environment permissive for infiltration by low-avidity CD8+ T cells

CD4+ T cells enhance tumor destruction by CD8+ T cells. One benefit that underlies CD4+ T cell help is enhanced clonal expansion of newly activated CD8+ cells. In addition, tumor-specific CD4+ help is also associated with the accumulation of greater numbers of CD8+ T cells within the tumor. Whether this too is attributable to the effects of help delivered to the CD8+ cells during priming within secondary lymphoid tissues, or alternatively is due to the action of CD4+ cells within the tumor environment has not been examined. In this study, we have evaluated separately the benefits of CD4+ T cell help accrued during priming of tumor-specific CD8+ T cells with a vaccine, as opposed to the benefits delivered by the presence of cognate CD4+ cells within the tumor. The presence of CD4+ T cell help during priming increased clonal expansion of tumor-specific CD8+ T cells in secondary lymphoid tissue; however, CD8+ T cells that have low avidity for tumor Ag were inefficient in tumor invasion. CD4+ T cells that recognized tumor Ag were required to facilitate accumulation of CD8+ T cells within the tumor and enhance tumor lysis during the acute phase of the response. These experiments highlight the ability of tumor-specific CD4+ T cells to render the tumor microenvironment receptive for CD8+ T cell immunotherapy, by facilitating the accumulation of all activated CD8+ T cells, including low-avidity tumor-specific and noncognate cells.     

Targeting an anti-viral CD8+ T cell response to a growing tumor facilitates its rejection

 We demonstrate in a murine model that targeting an anti-viral T cell response to a growing tumor facilitates priming of a tumor-associated antigen (TAA)-specific, rejecting T cell response. Murine P815 mastocytoma cells grow aggressively in a syngeneic host. Transfected P815/S cells (expressing the hepatitis B surface antigen, HBsAg) also grow as subcutaneous tumors, but occasional ‘spontaneous’ rejections after transient growth are observed. Growth of P815/S tumors (but not of P815 tumors) is efficiently suppressed by a CD8⁺ cytotoxic T lymphocyte (CTL)-dependent immune mechanism in mice primed to HBsAg by DNA–immunization. In hosts immunized against HBsAg by DNA vaccination, HBsAg-specific CTL are generated. This specific CTL reactivity was targeted to s.c.-growing P815 tumors by intra tumor injections of either HBsAg-encoding plasmid DNA or viable P815/S cells; this treatment led to tumor rejection in 70–80% of the tumor-bearing animals. All rejecting animals showed a CD8⁺ CTL-dependent resistance to subsequent challenges by native, non-transfected P815 tumors. Targeting an established anti-viral (‘strong’) CTL response to a growing tumor hence is an efficient strategy to facilitate priming of a rejecting CTL response against (‘weak’) TAA in this system. Received: 18 December 1996 / Accepted: 6 February 1997     

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.     

Suppressor effector function of CD4+CD25+ immunoregulatory T cells is antigen nonspecific

CD4+CD25+ T cells represent a unique population of "professional" suppressor T cells that prevent induction of organ-specific autoimmune disease. In vitro, CD4+CD25+ cells were anergic to simulation via the TCR and when cultured with CD4+CD25- cells, markedly suppressed polyclonal T cell proliferation by specifically inhibiting the production of IL-2. Suppression was cytokine independent, cell contact dependent, and required activation of the suppressors via their TCR. Further characterization of the CD4+CD25+ population demonstrated that they do not contain memory or activated T cells and that they act through an APC-independent mechanism. CD4+CD25+ T cells isolated from TCR transgenic (Tg) mice inhibited responses of CD4+CD25- Tg T cells to the same Ag, but also inhibited the Ag-specific responses of Tg cells specific for a distinct Ag. Suppression required that both peptide/MHC complexes be present in the same culture, but the Ags could be presented by two distinct populations of APC. When CD4+CD25+ T cells were cultured with anti-CD3 and IL-2, they expanded, remained anergic, and in the absence of restimulation via their TCR, suppressed Ag-specific responses of CD4+CD25- T cells from multiple TCR transgenics. Collectively, these data demonstrate that CD4+CD25+ T cells require activation via their TCR to become suppressive, but once activated, their suppressor effector function is completely nonspecific. The cell surface molecules involved in this T-T interaction remain to be characterized.     

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.     

IL-2 is required for the activation of memory CD8+ T cells via antigen cross-presentation

Dendritic cells (DCs) are capable of capturing exogenous Ag for the generation of MHC class I/peptide complexes. For efficient activation of memory CD8(+) T cells to occur via a cross-presentation pathway, DCs must receive helper signals from CD4(+) T cells. Using an in vitro system that reflects physiologic recall memory responses, we have evaluated signals that influence helper-dependent cross-priming, while focusing on the source and cellular target of such effector molecules. Concerning the interaction between CD4(+) T cells and DCs, we tested the hypothesis that CD40 engagement on DCs is critical for IL-12p70 (IL-12) production and subsequent stimulation of IFN-gamma release by CD8(+) T cells. Although CD40 engagement on DCs, or addition of exogenous IL-12 are both sufficient to overcome the lack of help, neither is essential. We next evaluated cytokines and chemokines produced during CD4(+) T cell/DC cross talk and observed high levels of IL-2 produced within the first 18-24 h of Ag-specific T cell engagement. Functional studies using blocking Abs to CD25 completely abrogated IFN-gamma production by the CD8(+) T cells. Although required, addition of exogenous IL-2 did not itself confer signals sufficient to overcome the lack of CD4(+) T cell help. Thus, these data support a combined role for Ag-specific, cognate interactions at the CD4(+) T cell/DC as well as the DC/CD8(+) T cell interface, with the helper effect mediated by soluble noncognate signals.     

Inefficient cross-presentation limits the CD8+ T cell response to a subdominant tumor antigen epitope

CD8(+) T lymphocytes (T(CD8)) responding to subdominant epitopes provide alternate targets for the immunotherapy of cancer, particularly when self-tolerance limits the response to immunodominant epitopes. However, the mechanisms that promote T(CD8) subdominance to tumor Ags remain obscure. We investigated the basis for the lack of priming against a subdominant tumor epitope following immunization of C57BL/6 (B6) mice with SV40 large tumor Ag (T Ag)-transformed cells. Immunization of B6 mice with wild-type T Ag-transformed cells primes T(CD8) specific for three immunodominant T Ag epitopes (epitopes I, II/III, and IV) but fails to induce T(CD8) specific for the subdominant T Ag epitope V. Using adoptively transferred T(CD8) from epitope V-specific TCR transgenic mice and immunization with T Ag-transformed cells, we demonstrate that the subdominant epitope V is weakly cross-presented relative to immunodominant epitopes derived from the same protein Ag. Priming of naive epitope V-specific TCR transgenic T(CD8) in B6 mice required cross-presentation by host APC. However, robust expansion of these T(CD8) required additional direct presentation of the subdominant epitope by T Ag-transformed cells and was only significant following immunization with T Ag-expressing cells lacking the immunodominant epitopes. These results indicate that limited cross-presentation coupled with competition by immunodominant epitope-specific T(CD8) contributes to the subdominant nature of a tumor-specific epitope. This finding has implications for vaccination strategies targeting T(CD8) responses to cancer.     

Human embryonal carcinoma tumor antigen,Gp200/GCTM-2, is podocalyxin

We previously characterized a peanut agglutinin-binding tumor antigen, gp200, a surface membrane glycoprotein expressed on human embryonal carcinoma, a malignant stem cell of testicular tumors. Gp200 is remarkably similar to another embryonal carcinoma antigen, GCTM-2, a cell differentiation marker that is also detected in blood of testis cancer patients, yet neither molecular identity is known. We now report the identity of gp200 as podocalyxin. Protein sequence results of gp200 peptides match with podocalyxin sequence. Furthermore, two distinct monoclonal antibodies, specific for podocalyxin, react positively with gp200. Therefore, gp200 is a testicular tumor form of podocalyxin, a surface membrane glycoprotein that was originally discovered as a scaffolding extracellular matrix protein of kidney podocyte cells. Podocalyxin is also expressed on subsets of hematopoietic cells where it has a putative function as a cell adhesion protein. This is the first report of podocalyxin expression on malignant cells.