Monoclonal antibodies targeted against melanoma and ovarian tumors enhance dendritic cell-mediated cross-presentation of tumor-associated antigens and efficiently cross-prime CD8+ T cells

Dendritic cells (DCs) constitute very attractive vectors for cancer immunotherapy due to their ability to efficiently capture and present tumor antigens, which initiates tumor-directed T-cell responses. Because the initiation of cytotoxic anti-tumor immune responses requires the cross-presentation mechanism, antigen targeting to DCs represents a very important step in the chain of events that constitutes the cross-priming immune process. In the current study, we explored the ability of DCs loaded with antibody-coated melanoma and ovarian carcinoma tumor cells to cross-present tumor antigens to CD8+ T cells and elicit in vitro anti-tumor immune responses. Coating melanoma and ovarian cancer cells with monoclonal antibodies against different surface antigens (CD44, ME491, LFA-3, and CD24) expressed by the tumor cells promoted the cross-presentation of the tumor-associated antigens as MART-1, gp100, tyrosinase, and NY-ESO-1 by DCs to CD8+ T. These tumor antigen-specific CD8+ T-cell populations resulting from the DC-mediated cross-priming process were identified using specific immune tetramers and were a few fold larger than the ones generated using peptide-pulsed or apoptotic tumor cell-loaded DCs. The CD8+ T cells generated by DCs loaded with monoclonal antibody-coated tumor cells were cytotoxic against the primary melanoma and ovarian carcinoma cells. Thus, targeting monoclonal antibody-coated tumor cells to DCs is a novel method that opens new perspectives for immunotherapy strategies.     

Dendritic cells cultured in anti-CD40 antibody-immobilized plates elicit a highly efficient peptide-specific T-cell response

The function of dendritic cells (DCs), antigen-presenting cells that can initiate and regulate cellular and humoral responses, is highly influenced by their level of maturation. Immature DCs may be harmful in anti-tumor immunotherapy, because they can induce immunotolerance rather than immunostimulation. In this study, the authors sought to determine the optimal culture conditions for obtaining fully mature DCs. When DCs were cultured in agonistic anti-CD40 monoclonal antibody-immobilized plates, they showed a higher expression of the maturation marker CD83 than DCs cultured without CD40 ligation or those cultured in medium supplemented with anti-CD40 monoclonal antibody. In addition, when interferon-gamma (IFN-gamma) was added to the medium, additive up-regulation of CD83 expression was observed. These DCs treated with both maturation signals showed a higher secretion of interleukin-12. To evaluate the capacity of antigen presentation, specific cytotoxic T lymphocytes were generated using autologous DC pulsed with a human lymphocyte antigen-A24-restricted peptide epitope derived from carcinoembryonic antigen. Interferon-gamma-secreting CD8+ T cells were analyzed by flow cytometry using the cellular affinity matrix technology. Dendritic cells, matured with CD40 ligation and IFN-gamma, were more efficient at eliciting an antigen-specific T-cell response in vitro than DCs stimulated with anti-CD40 monoclonal antibody or IFN-gamma alone. A cytotoxicity assay using carcinoembryonic antigen-expressing tumor cell lines also showed that DCs matured with both signals were more efficient at inducing cytotoxic T lymphocytes. These results demonstrate that DC culture in an anti-CD40 monoclonal antibody-immobilized plate in medium supplemented with IFN-gamma has a positive impact on DC maturation and may be optimal for eliciting an antigen-specific T-cell response without the need for CD4+ T-helper epitopes.     

Antitumor monoclonal antibodies enhance cross-presentation ofcCellular antigens and the generation of myeloma-specific killer T cells by dendritic cells.

The mechanism of antitumor effect of monoclonal antibodies (mAbs) is not fully understood. Here we show that coating myeloma cells with anti-syndecan-1 antibody promotes cross-presentation of cellular antigens by dendritic cells (DCs) to autologous T cells from healthy donors. The tumor cells treated with anti-syndecan-1 or isotype-matched control antibody were fed to HLA-mismatched monocyte-derived immature DCs. Tumor cell-loaded mature DCs induced a strong CD8(+) T cell response that was specific for the cancer-testis (C-T) antigens expressed in the tumor. The CD8(+) T cells killed peptide-pulsed targets, as well as myeloma tumor cells. Importantly, mAbs-coated tumor-loaded DCs were consistently superior to DCs loaded with peptides or dying cells for eliciting tumor-specific killer T cells. This enhanced cross-presentation was not due to enhanced tumor cell uptake or to DC maturation. When mixtures of NY-Eso-1-positive and -negative myeloma cells were captured by DCs, the anti-syndecan-1 antibody had to be on the NY-Eso-1-positive cells to elicit NY-Eso-1-specific response. Cross-presentation was inhibited by pretreatment of DCs with Fc gamma receptor blocking antibodies. Targeting of mAb-coated tumors to DCs may contribute to the efficacy of tumor-reactive mAb and offers a new strategy for immunotherapy.     

LF 15-0195 generates synergistic tolerance by promoting formation of CD4+CD25+CTLA4+ T cells

The purpose of the present study was to investigate the influence of 15-deoxyspergualin (LF) on the phenotypes and functions of dendritic cells (DCs) and T cells and to further illustrate the mechanism of LF-inducing immunologic tolerance. DCs from mice were cultured and treated with varying doses of LF at specific time-points. Fluorescence-activated cell sorting (FACS) was used to verify the changes of phenotypes in the cultured DCs labeled with fluorescent antibody. DCs were also used as stimulators in mixed leukocyte reaction to detect their ability to stimulate T-cell proliferation. DCs and T cells, treated with or without LF, were cultured together; phenotypes and cytokine profile of the T cells were identified and assayed by FACS and enzyme-linked immunosorbent assay. LF induced a dose- and time-dependent suppression of maturation of DCs and a dose-dependent suppression of T-cell proliferation in mixed leukocyte reaction when LF-treated DCs were used as stimulators. LF-treated DCs, cultured with naive T cells, could promote the formation of CD4+CD25+CTLA4+ T-cell subtypes and the production of higher levels of interleukin-10. It was suggested that the mechanism of LF-induced tolerance was inhibiting maturation and function of DC and inducing the formation of regulatory T-cell subtype by "suppressor DCs" to achieve a new immune balance.     

Role of CD44 and hyaluronic acid (HA) in activation of alloreactive and antigen-specific T cells by bone marrow-derived dendritic cells

In the current study, the role played by hyaluronic acid (HA) and its receptor CD44 on the activation and functions of dendritic cells (DCs) was investigated. Activation of DCs with HA enhanced their ability to stimulate allogeneic and antigen (Ag)-specific T cells markedly. HA treatment upregulated the expression of costimulatory molecules such as CD40, CD80, and CD86 on DCs. Cell mixing experiments using DC or T cells from CD44 wild-type or CD44 knockout mice as well as blocking studies with anti-CD44 monoclonal antibodies revealed that CD44 expression on T cells but not DC played a critical role in Ag-specific T-cell responsiveness. Also, the HA-induced activation of DC was independent of CD44. When conjugate formation between Ag-pulsed DCs and Ag-specific T cells was studied, the deficiency of CD44 on T cells rather than on DCs was found to play a key role in T-cell-DC interaction. Together, these data demonstrated that HA can activate DC independently of CD44; however, CD44 expressed on Ag-specific T cells plays a critical role in its interaction with DC and resultant expansion of T cells.     

Chemosensitization of pancreatic carcinoma cells to enhance T cell-mediated cytotoxicity induced by tumor lysate-pulsed dendritic cells

Dendritic cells (DCs) can induce cytotoxic T-cell (CTL) responses against tumor antigens in vitro and in vivo, yet few cancer patients experience tumor regression after DC-based vaccination. Combination with other treatment modalities, such as radiation or pharmacologic anticancer agents, may reduce tumor cell resistance against immune responses. The authors tested whether treatment with gemcitabine or cyclooxygenase-2 (COX-2) inhibitors increases the sensitivity of pancreatic carcinoma cells to CTL-mediated killing. Monocyte-derived DCs of HLA-A2+ donors were loaded with lysate from the HLA-A2+ pancreatic carcinoma cell line Panc-1 and co-cultured with autologous CD3+ T cells. ELISPOT and cytotoxicity assays performed after two rounds of in vitro stimulation confirmed induction of a tumor-specific CTL response. Changes in the magnitude and the effector mechanism of the CTL response were analyzed after treatment of Panc-1 cells with gemcitabine and COX-2 inhibitors. Compared with gemcitabine, COX-2 inhibitors more effectively sensitized Panc-1 cells to CTL-mediated killing and showed less inhibition of T-cell activation by DCs in vitro. Using anti-CD95 blocking antibody, the authors showed that the increase in CTL-mediated tumor cell killing observed after treatment with COX-2 inhibitors is dependent on CD95/CD95 ligand interaction. Increased apoptosis of Panc-1 cells treated with COX-2 inhibitor was also observed after incubation with agonistic anti-CD95 antibody. Sensitization of cancer cells to CD95-dependent killing by CTLs represents a novel mechanism of action for COX-2 inhibitors and provides a rationale for their concomitant use with immunotherapeutic strategies such as DC-based vaccination.     

Consequences of cell death: exposure to necrotic tumor cells, but not primary tissue cells or apoptotic cells, induces the maturation of immunostimulatory dendritic cells

Cell death by necrosis is typically associated with inflammation, in contrast to apoptosis. We have identified additional distinctions between the two types of death that occur at the level of dendritic cells (DCs) and which influence the induction of immunity. DCs must undergo changes termed maturation to act as potent antigen-presenting cells. Here, we investigated whether exposure to apoptotic or necrotic cells affected DC maturation. We found that immature DCs efficiently phagocytose a variety of apoptotic and necrotic tumor cells. However, only exposure to the latter induces maturation. The mature DCs express high levels of the DC-restricted markers CD83 and lysosome-associated membrane glycoprotein (DC-LAMP) and the costimulatory molecules CD40 and CD86. Furthermore, they develop into powerful stimulators of both CD4(+) and CD8(+) T cells. Cross-presentation of antigens to CD8(+) T cells occurs after uptake of apoptotic cells. We demonstrate here that optimal cross-presentation of antigens from tumor cells requires two steps: phagocytosis of apoptotic cells by immature DCs, which provides antigenic peptides for major histocompatibility complex class I and class II presentation, and a maturation signal that is delivered by exposure to necrotic tumor cells, their supernatants, or standard maturation stimuli, e.g., monocyte-conditioned medium. Thus, DCs are able to distinguish two types of tumor cell death, with necrosis providing a control that is critical for the initiation of immunity.     

Constitutive versus activation-dependent cross-presentation of immune complexes by CD8(+) and CD8(-) dendritic cells in vivo

Murine splenic dendritic cells (DCs) can be divided into two subsets based on CD8alpha expression, but the specific role of each subset in stimulation of T cells is largely unknown. An important function of DCs is the ability to take up exogenous antigens and cross-present them in the context of major histocompatibility complex (MHC) class I molecules to CD8(+) T cells. We previously demonstrated that, when cell-associated ovalbumin (OVA) is injected into mice, only the CD8(+) DC subset cross-presents OVA in the context of MHC class I. In contrast to this selectivity with cell-associated antigen, we show here that both DC subsets isolated from mice injected with OVA/anti-OVA immune complexes (OVA-IC) cross-present OVA to CD8(+) T cells. The use of immunoglobulin G Fc receptor (Fc(gamma)R) common gamma-chain-deficient mice revealed that the cross-presentation by CD8(-) DCs depended on the expression of gamma-chain-containing activating FcgammaRs, whereas cross-presentation by CD8(+) DCs was not reduced in gamma-chain-deficient mice. These results suggest that although CD8(+) DCs constitutively cross-present exogenous antigens in the context of MHC class I molecules, CD8(-) DCs only do so after activation, such as via ligation of Fc(gamma)Rs. Cross-presentation of immune complexes may play an important role in autoimmune diseases and the therapeutic effect of antitumor antibodies.     

CTLA4 blockade and GM-CSF combination immunotherapy alters the intratumor balance of effector and regulatory T cells

CTL-associated antigen 4 (CTLA4) blockade releases inhibitory controls on T cell activation and proliferation, inducing antitumor immunity in both preclinical and early clinical trials. We examined the mechanisms of action of anti-CTLA4 and a GM-CSF-transduced tumor cell vaccine (Gvax) and their impact on the balance of effector T cells (Teffs) and Tregs in an in vivo model of B16/BL6 melanoma. Tumor challenge increased the frequency of Tregs in lymph nodes, and untreated tumors became infiltrated by CD4+Foxp3- and CD4+Foxp3+ T cells but few CD8+ T cells. Anti-CTLA4 did not deplete Tregs or permanently impair their function but acted in a cell-intrinsic manner on both Tregs and Teffs, allowing them to expand, most likely in response to self antigen. While Gvax primed the tumor-reactive Teff compartment, inducing activation, tumor infiltration, and a delay in tumor growth, the combination with CTLA4 blockade induced greater infiltration and a striking change in the intratumor balance of Tregs and Teffs that directly correlated with tumor rejection. The data suggest that Tregs control both CD4+ and CD8+ T cell activity within the tumor, highlight the importance of the intratumor ratio of effectors to regulators, and demonstrate inversion of the ratio and correlation with tumor rejection during Gvax/anti-CTLA4 immunotherapy.     

An ITAM-signaling pathway controls cross-presentation of particulate but not soluble antigens in dendritic cells

Dendritic cells (DC) possess a unique capacity for presenting exogenous antigen on major histocompatibility class I, a process that is referred to as cross-presentation, which serves a critical role in microbial and tumor immunity. During cross-presentation, antigens derived from pathogen-infected or tumor cells are internalized and processed by DCs for presentation to cytotoxic T lymphocytes (CTLs). We demonstrate that a signaling pathway initiated by the immunoreceptor tyrosine-based activation motif (ITAM)-containing adaptors DAP12 and FcRgamma utilizes the Vav family of Rho guanine nucleotide exchange factors (GEFs) for processing and cross-presentation of particulate, but not soluble, antigens by DCs. Notably, this novel pathway is crucial for processing and presentation of particulate antigens, such as those associated with Listeria monocytogenes bacteria, yet it is not required for antigen uptake. Mechanistically, we provide evidence that in DCs, Vav GEFs are essential to link ITAM-dependent receptors with the activation of the NOX2 complex and production of reactive oxygen species (ROS), which regulate phagosomal pH and processing of particulate antigens for cross-presentation. Importantly, we show that genetic disruption of the DAP12/FcRgamma-Vav pathway leads to antigen presentation defects that are more profound than in DCs lacking NOX2, suggesting that ITAM signaling also controls cross-presentation in a ROS-independent manner.     

Therapeutic effects of tumor reactive CD4+ cells generated from tumor-primed lymph nodes using anti-CD3/anti-CD28 monoclonal antibodies

T-cell activation involves multiple signaling pathways. In this report, we conducted in vitro and in vivo immune function analysis of tumor-draining lymph node (TDLN) cells after anti-CD3/anti-CD28 activation versus anti-CD3 activation alone in a murine tumor model. In cytokine release assays, the doubly activated TDLN cells secreted significantly greater amounts of IFN-gamma and GM-CSF in response to specific tumor antigen compared with anti-CD3 activated cells. In adoptive immunotherapy, the doubly activated TDLN cells were more effective in mediating regression of 3-day pulmonary metastases compared with anti-CD3 activated cells. Although there was predominant proliferation of CD8+ cells after either activation procedure, the mean-fold expansion of CD4+ cells was significantly greater after anti-CD3/anti-CD28 activation than anti-CD3 activation alone. Using magnetic bead-enriched T-cell subsets, we found that either CD4+ or CD8+ doubly activated TDLN cells could independently mediate tumor regression. Furthermore, the doubly activated CD4+ cells were more effective than CD8+ cells in adoptive immunotherapy on a per-cell basis. The antitumor activity mediated by CD4+ or CD8+ cells could be significantly enhanced with the exogenous administration of IL-2. CD28 co-stimulation of tumor-primed lymphoid cells promotes the generation of potent tumor reactive effector cells, particularly CD4+ T cells, with antitumor activity in adoptive immunotherapy.     

Plasmacytoid dendritic cells from mouse tumor-draining lymph nodes directly activate mature Tregs via indoleamine 2,3-dioxygenase

A small population of plasmacytoid DCs (pDCs) in mouse tumor-draining LNs can express the immunoregulatory enzyme indoleamine 2,3-dioxygenase (IDO). We show that these IDO+ pDCs directly activate resting CD4+CD25+Foxp3+ Tregs for potent suppressor activity. In vivo, Tregs isolated from tumor-draining LNs were constitutively activated and suppressed antigen-specific T cells immediately ex vivo. In vitro, IDO+ pDCs from tumor-draining LNs rapidly activated resting Tregs from non-tumor-bearing hosts without the need for mitogen or exogenous anti-CD3 crosslinking. Treg activation by IDO+ pDCs was MHC restricted, required an intact amino acid-responsive GCN2 pathway in the Tregs, and was prevented by CTLA4 blockade. Tregs activated by IDO markedly upregulated programmed cell death 1 ligand 1 (PD-L1) and PD-L2 expression on target DCs, and the ability of Tregs to suppress target T cell proliferation was abrogated by antibodies against the programmed cell death 1/PD-L (PD-1/PD-L) pathway. In contrast, Tregs activated by anti-CD3 crosslinking did not cause upregulation of PD-Ls, and suppression by these cells was unaffected by blocking the PD-1/PD-L pathway. Tregs isolated from tumor-draining LNs in vivo showed potent PD-1/PD-L-mediated suppression, which was selectively lost when tumors were grown in IDO-deficient hosts. We hypothesize that IDO+ pDCs create a profoundly suppressive microenvironment within tumor-draining LNs via constitutive activation of Tregs.     

Dendritic cells cross-present latency gene products from Epstein-Barr virus-transformed B cells and expand tumor-reactive CD8(+) killer T cells

Dendritic cells (DCs) are not targets for infection by the transforming Epstein-Barr virus (EBV). To test if the adjuvant role of DCs could be harnessed against EBV latency genes by cross-presentation, DCs were allowed to process either autologous or human histocompatibility leukocyte antigen (HLA)-mismatched, transformed, B lymphocyte cell lines (LCLs) that had been subject to apoptotic or necrotic cell death. After phagocytosis of small numbers of either type of dead LCL, which lacked direct immune-stimulatory capacity, DCs could expand CD8(+) T cells capable of killing LCLs that were HLA matched to the DCs. Necrotic EBV-transformed, major histocompatibility complex (MHC) class I-negative LCLs, when presented by DCs, also could elicit responses to MHC class II-negative, EBV-transformed targets that were MHC class I matched to the DCs, confirming efficient cross-presentation of LCL antigens via MHC class I on the DC. Part of this EBV-specific CD8(+) T cell response, in both lytic and interferon gamma secretion assays, was specific for the EBV nuclear antigen (EBNA)3A and latent membrane protein (LMP)2 latency antigens that are known to be expressed at low levels in transformed cells. The induced CD8(+) T cells recognized targets at low doses, 1-10 nM, of peptide. Therefore, the capacity of DCs to cross-present antigens from dead cells extends to the expansion of high affinity T cells specific for viral latency antigens involved in cell transformation.     

Modulation of immune response with cytotoxic T-lymphocyte-associated antigen 4 immunoglobulin-induced anergic T cells in chronic idiopathic thrombocytopenic purpura.

BACKGROUND: Platelet glycoprotein (GP)-reactive CD4+ T cells are essential for the stimulation and maintenance of antiplatelet autoantibody production in chronic idiopathic thrombocytopenic purpura (ITP). Blocking costimulatory signals could result in platelet-specific T-cell anergy. METHODS: GP-specific CD4+ T cells from patients with ITP were made anergic using cytotoxic T-lymphocyte-associated antigen 4 immunoglobulin (CTLA4-Ig). The CTLA4-Ig-induced GP-specific anergic T cells were investigated for their inhibitory function on GP-reactive T-cell proliferation and antibody production with in vitro culture systems. To further analyze their tolerizing mechanisms, we cocultured GP-anergic T cells with dendritic cells (DCs) from patients with ITP. RESULTS: Our studies demonstrated that the anergized GP-specific T cells have profound effects on both GP-specific T-cell proliferation and antibody production. These anergic T cells exerted their suppressive effects mainly in a cell contact-dependent manner, and they were not constitutively suppressive but required specific antigen stimulation to make DCs tolerogenic. The anergic T-cell-modulated DCs could induce the autoreactive T cells to be tolerant, and this effect was not restricted to T cells of the same specificity. CONCLUSION: Our studies demonstrate the efficacy of CTLA4-Ig in suppressing the pathologic autoimmune responses in ITP. These findings provide new insights into the underlying mechanisms of anergy induction in chronic ITP.     

Immunolipoplexes: an efficient, nonviral alternative for transfection of human dendritic cells with potential for clinical vaccination.

Genetic manipulation of dendritic cells (DCs) is important in the context of using either mature DCs to immunize patients or immature DCs to induce tolerance. Here, we describe a novel method of transfecting monocyte-derived human DCs using immunolipoplexes containing anti-CD71 or anti-CD205 monoclonal Abs. This results in up to 20% transfection, which can be increased to 20-30% if the immunolipoplexes are used to transfect CD14+ monocytes prior to differentiation into DCs. Transfected DCs can be substantially enriched using a drug-selection protocol during differentiation. Unlike adenoviral transduction, this nonviral transfection does not alter the expression of costimulatory molecules or the production of proinflammatory cytokines by DCs. In addition, DC function is unaltered, as assessed by mixed lymphocyte reactions. To test the feasibility of the immunolipoplexes and selection protocol for therapeutic intervention, we transfected DCs with the immunomodulatory enzyme indoleamine 2,3-dioxygenase (IDO). Allogeneic T cells exposed to IDO-expressing DCs did not proliferate, secreted more IL-10 and less Th1 and Th2 cytokines, and had a higher amount of apoptosis than T cells incubated with control DCs. Furthermore the remaining T cells were rendered anergic to further stimulation by allogeneic DC. These immunolipoplexes, which can be easily and rapidly assembled, have potential for clinical immunization, in particular for tolerance induction protocols.     

The CD8+ dendritic cell subset selectively endocytoses dying cells in culture and in vivo

Dendritic cells (DCs) are able in tissue culture to phagocytose and present antigens derived from infected, malignant, and allogeneic cells. Here we show directly that DCs in situ take up these types of cells after fluorescent labeling with carboxyfluorescein succinimidyl ester (CFSE) and injection into mice. The injected cells include syngeneic splenocytes and tumor cell lines, induced to undergo apoptosis ex vivo by exposure to osmotic shock, and allogeneic B cells killed by NK cells in situ. The CFSE-labeled cells in each case are actively endocytosed by DCs in vivo, but only the CD8+ subset. After uptake, all of the phagocytic CD8+ DCs can form major histocompatibility complex class II-peptide complexes, as detected with a monoclonal antibody specific for these complexes. The CD8+ DCs also selectively present cell-associated antigens to both CD4+ and CD8+ T cells. Similar events take place with cultured DCs; CD8+ DCs again selectively take up and present dying cells. In contrast, both CD8+ and CD8- DCs phagocytose latex particles in culture, and both DC subsets present soluble ovalbumin captured in vivo. Therefore CD8+ DCs are specialized to capture dying cells, and this helps to explain their selective ability to cross present cellular antigens to both CD4+ and CD8+ T cells.     

Lymphocytes genetically modified to express tumor antigens target DCs in vivo and induce antitumor immunity

The exploitation of the physiologic processing and presenting machinery of DCs by in vivo loading of tumor-associated antigens may improve the immunogenic potential and clinical efficacy of DC-based cancer vaccines. Here we show that lymphocytes genetically modified to express self/tumor antigens, acting as antigen carriers, efficiently target DCs in vivo in tumor-bearing mice. The infusion of tyrosinase-related protein 2-transduced (TRP-2-transduced) lymphocytes induced the establishment of protective immunity and long-term memory in tumor-bearing mice. Analysis of the mechanism responsible for the induction of such an immune response allowed us to demonstrate that cross-presentation of the antigen mediated by the CD11c(+)CD8alpha(+) DC subset had occurred. Furthermore, we demonstrated in vivo and in vitro that DCs had undergone activation upon phagocytosis of genetically modified lymphocytes, a process mediated by a cell-to-cell contact mechanism independent of CD40 triggering. Targeting and activation of secondary lymphoid organ-resident DCs endowed antigen-specific T cells with full effector functions, which ultimately increased tumor growth control and animal survival in a therapeutic tumor setting. We conclude that the use of transduced lymphocytes represents an efficient method for the in vivo loading of tumor-associated antigens on DCs.     

Generating renal cancer-reactive T cells using dendritic cells (DCs) to present autologous tumor

Dendritic cells (DCs) have been used as professional antigen-presenting cells in vitro to prime T-cell responses. In this study, we generated both CD8+ and CD4+ renal cell carcinoma (RCC)-reactive T cells using a completely autologous system of DCs presenting engulfed whole-tumor cells. We compared DCs presenting RCC tumor cells in different preparations and found ultraviolet-irradiated apoptotic tumor cells to be more immunogenic than necrotic tumor cells or live untreated tumor cells in generating tumor-reactive T cells. In analyzing the T cells generated in this fashion, a CD8+ RCC-reactive T-cell clone generated from a patient recognized an epitope derived from fibroblast growth factor 5 in the context of human leukocyte antigen (HLA) B44*02. CD4+ T cells generated from another patient recognized multiple allogeneic RCC lines expressing HLA-DRbeta1*04, indicating a common shared tumor antigen presented by HLA-DRbeta1*04. The technique of using DCs to present whole-tumor cells can consistently generate both CD4+ and CD8+ RCC-reactive T cells for use in both antigen identification and therapeutic protocols.     

A therapeutic cancer vaccine delivers antigens and adjuvants to lymphoid tissues using genetically modified T cells

Therapeutic vaccines that augment T cell responses to tumor antigens have been limited by poor potency in clinical trials. In contrast, the transfer of T cells modified with foreign transgenes frequently induces potent endogenous T cell responses to epitopes in the transgene product, and these responses are undesirable, because they lead to rejection of the transferred T cells. We sought to harness gene-modified T cells as a vaccine platform and developed cancer vaccines composed of autologous T cells modified with tumor antigens and additional adjuvant signals (Tvax). T cells expressing model antigens and a broad range of tumor neoantigens induced robust and durable T cell responses through cross-presentation of antigens by host DCs. Providing Tvax with signals such as CD80, CD137L, IFN-β, IL-12, GM-CSF, and FLT3L enhanced T cell priming. Coexpression of IL-12 and GM-CSF induced the strongest CD4⁺ and CD8⁺ T cell responses through complimentary effects on the recruitment and activation of DCs, mediated by autocrine IL-12 receptor signaling in the Tvax. Therapeutic vaccination with Tvax and adjuvants showed antitumor activity in subcutaneous and metastatic preclinical mouse models. Human T cells modified with neoantigens readily activated specific T cells derived from patients, providing a path for clinical translation of this therapeutic platform in cancer.