Jacobus J. Bosch

Lung cancer patients’ CD4+ T cells are activated in vitro by MHC II cell-based vaccines despite the presence of myeloid-derived suppressor cells

BackgroundAdvanced non-small cell lung cancer (NSCLC) remains an incurable disease. Immunotherapies that activate patients’ T cells against resident tumor cells are being developed; however, these approaches may not be effective in NSCLC patients due to tumor-induced immune suppression. A major cause of immune suppression is myeloid-derived suppressor cells (MDSC). Because of the strategic role of CD4+ T lymphocytes in the activation of cytotoxic CD8+ T cells and immune memory, we are developing cell-based vaccines that activate tumor-specific CD4+ T cells in the presence of MDSC. The vaccines are NSCLC cell lines transfected with costimulatory (CD80) plus major histocompatibility complex class II (MHC II) genes that are syngeneic to the recipient. The absence of invariant chain promotes the presentation of endogenously synthesized tumor antigens, and the activation of MHC II-restricted, tumor-antigen-specific CD4+ T cells.MethodsPotential vaccine efficacy was tested in vitro by priming and boosting peripheral blood mononuclear cells from ten NSCLC patients who had varying levels of MDSC. CD4+ T cell activation was quantified by measuring Type 1 and Type 2 cytokine release.ResultsThe vaccines activated CD4+ T cells from all ten patients, despite the presence of CD33+CD11b+ MDSC. Activated CD4+ T cells were specific for NSCLC and did not cross-react with tumor cells derived from non-lung tissue or normal lung fibroblasts.ConclusionsThe NSCLC vaccines activate tumor-specific CD4+ T cells in the presence of potent immune suppression, and may be useful for the treatment of patients with NSCLC.

Activation of Tumor-specific CD4+ T Lymphocytes by Major Histocompatibility Complex Class II Tumor Cell Vaccines A Novel Cell-based Immunotherapy

Mouse tumor cells transfected with syngeneic MHC class II and costimulatory molecule genes are therapeutic vaccines in mice, provided they do not coexpress the class II-associated invariant chain (Ii). We demonstrated previously that the vaccine cells present tumor peptides via the endogenous antigen presentation pathway to activate CD4+ and CD8+ T cells. Because of their efficacy in mice, we are translating this vaccine strategy for clinical use. To obtain MHC class II+CD80+Ii− human tumor cells, we developed retroviruses encoding HLA-DR and CD80. The HLA-DR virus encodes the DRα and DRβ0101 chains using an internal ribosomal entry site to coordinate expression. SUM159PT mammary carcinoma and Mel 202 ocular melanoma cells transduced with the retroviruses DRB1/CD80 express high levels of DRB0101 and CD80 on the cell surface in the absence of Ii. Irradiated SUM159PT/DR1/CD80 vaccines stimulate proliferation of non-HLA-DRB0101 peripheral blood mononuclear cells and present an exogenous DR1-restricted tetanus toxoid (TT) peptide, indicating that the transduced DRB0101 is functional. SUM159PT/DR1/CD80 vaccines were further transduced with a retrovirus encoding the TT fragment C gene, as a model tumor antigen. These cells stimulate IFN-γ release from TT-primed human DRB0101 peripheral blood mononuclear cells, demonstrating their ability to present “endogenous” tumor antigen. Depletion and antibody blocking experiments confirm that MHC class II-restricted, endogenously synthesized epitopes are presented to CD4+ T cells. Therefore, the MHC class II vaccines are efficient antigen-presenting cells that activate tumor-specific MHC class II-restricted, CD4+ T lymphocytes, and they are a novel and potential immunotherapeutic for metastatic cancers.

Prognostic Significance of Tumor-Associated Lymphangiogenesis in Malignant Melanomas of the Conjunctiva

PURPOSE To evaluate whether tumor-associated lymphangiogenesis contributes to prognosis of conjunctival malignant melanomas and to study its association with other tumor characteristics. DESIGN Nonrandomized, retrospective case series. PARTICIPANTS A total of 109 consecutive patients with primary conjunctival malignant melanoma. METHODS Proliferating lymphatic vessels were identified immunohistochemically using lymphatic vascular endothelial hyaluronan receptor-1 and podoplanin as specific lymphatic endothelial markers and Ki-67 as proliferation marker. Baseline tumor characteristics included tumor location, tumor thickness, tumor diameter, tumor origin, and tumor growth pattern. Kaplan-Meier and Cox regression analyses of the risk of local recurrence, lymphatic spread, distant metastasis, and melanoma-related death were performed. MAIN OUTCOME MEASURES Intratumoral lymphatic vascular density and its association with tumor characteristics and recurrence-free, lymphatic spread-free, distant metastasis-free, and melanoma-specific survival. RESULTS Intratumoral and peritumoral proliferating lymphatic vessels could be detected in all of the 109 conjunctival melanoma samples. High intratumoral lymphatic density was significantly associated with palpebral tumor location (P<0.001), greater tumor thickness (P<0.001), larger tumor diameter (P = 0.001), tumor origin de novo (P = 0.002), and nodular tumor growth pattern (P = 0.037). Patients with high intratumoral lymphatic density revealed significantly lower recurrence-free, lymphatic spread-free, distant metastasis-free, and melanoma-specific survival rates (P<0.001 for all). By multivariate Cox regression, factors predictive of local recurrence included palpebral tumor location (hazard ratio [HR] 2.66, P = 0.014), large tumor diameter (HR 5.48, P<0.001), and high intratumoral lymphatic density (HR 2.48, P = 0.043); factors predictive of lymphatic spread included palpebral tumor location (HR 4.13, P = 0.009), high tumor thickness (HR 12.17, P<0.001), and high intratumoral lymphatic density (HR 6.79, P = 0.019); factors predictive of distant metastasis included palpebral tumor location (HR 7.63, P<0.001), high tumor thickness (HR 8.60, P<0.001), large tumor diameter (HR 0.30, P = 0.029), and high intratumoral lymphatic density (HR 8.90, P = 0.047); and factors predictive of melanoma-related death included palpebral tumor location (HR 7.74, P<0.001), high tumor thickness (HR 10.88, P<0.001), large tumor diameter (HR 0.28, P = 0.018), and, with borderline significance, high intratumoral lymphatic density (HR 8.46, P = 0.052). CONCLUSIONS Tumor-associated lymphangiogenesis seems to be associated with an increased risk of local recurrence, lymphatic spread, distant metastasis, and melanoma-related death in patients with conjunctival malignant melanomas. FINANCIAL DISCLOSURE(S) The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Tumor cell programmed death ligand 1-mediated T cell suppression is overcome by coexpression of CD80.

Programmed death ligand 1 (PDL1, or B7-H1) is expressed constitutively or is induced by IFN-γ on the cell surface of most human cancer cells and acts as a “molecular shield” by protecting tumor cells from T cell-mediated destruction. Using seven cell lines representing four histologically distinct solid tumors (lung adenocarcinoma, mammary carcinoma, cutaneous melanoma, and uveal melanoma), we demonstrate that transfection of human tumor cells with the gene encoding the costimulatory molecule CD80 prevents PDL1-mediated immune suppression by tumor cells and restores T cell activation. Mechanistically, CD80 mediates its effects through its extracellular domain, which blocks the cell surface expression of PDL1 but does not prevent intracellular expression of PDL1 protein. These studies demonstrate a new role for CD80 in facilitating antitumor immunity and suggest new therapeutic avenues for preventing tumor cell PDL1-induced immune suppression.

The absence of invariant chain in MHC II cancer vaccines enhances the activation of tumor-reactive type 1 CD4+ T lymphocytes

Activation of tumor-reactive T lymphocytes is a promising approach for the prevention and treatment of patients with metastatic cancers. Strategies that activate CD8+ T cells are particularly promising because of the cytotoxicity and specificity of CD8+ T cells for tumor cells. Optimal CD8+ T cell activity requires the co-activation of CD4+ T cells, which are critical for immune memory and protection against latent metastatic disease. Therefore, we are developing “MHC II” vaccines that activate tumor-reactive CD4+ T cells. MHC II vaccines are MHC class I+ tumor cells that are transduced with costimulatory molecules and MHC II alleles syngeneic to the prospective recipient. Because the vaccine cells do not express the MHC II-associated invariant chain (Ii), we hypothesized that they will present endogenously synthesized tumor peptides that are not presented by professional Ii+ antigen presenting cells (APC) and will therefore overcome tolerance to activate CD4+ T cells. We now report that MHC II vaccines prepared from human MCF10 mammary carcinoma cells are more efficient than Ii+ APC for priming and boosting Type 1 CD4+ T cells. MHC II vaccines consistently induce greater expansion of CD4+ T cells which secrete more IFNγ and they activate an overlapping, but distinct repertoire of CD4+ T cells as measured by T cell receptor Vβ usage, compared to Ii+ APC. Therefore, the absence of Ii facilitates a robust CD4+ T cell response that includes the presentation of peptides that are presented by traditional APC, as well as peptides that are uniquely presented by the Ii− vaccine cells.

Tumor-associated lymphangiogenesis in the development of conjunctival melanoma.

PURPOSE To analyze whether tumor-associated lymphangiogenesis is concurrent with the progression of premalignant conjunctival melanocytic intraepithelial neoplasia (C-MIN) into invasive conjunctival melanoma (CM) and to study its association with prognosis. METHODS Twenty patients with CM were closely matched with 20 patients with C-MIN with atypia and 20 with C-MIN without atypia regarding tumor size, tumor location, tumor extension, and patients age. All conjunctival specimens were analyzed for the immunohistochemical presence of proliferating lymphatic vessels, with LYVE-1 and podoplanin used as specific lymphatic endothelial markers and Ki-67 as a proliferation marker. Lymphatic vascular density was measured within the mass (intratumoral) and within an area ≤ 500 μm from the tumor border (peritumoral) and was correlated with recurrence, metastasis, and survival rates. RESULTS Intratumoral and peritumoral proliferating lymphatic vessels were detected in none of the C-MINs without atypia, in 10 of the 20 C-MINs with atypia, and in all 20 CMs. Invasive CM showed a significantly higher intra- and peritumoral density of proliferating lymphatics than did C-MIN with atypia (P ≤ 0.001). Patients with high intratumoral lymphatic density revealed significantly lower recurrence-free survival rates (P = 0.041) in C-MIN with atypia and significantly lower recurrence-free (P = 0.006), lymphatic-spread-free (P = 0.041), distant-metastasis-free (P = 0.029), and melanoma-specific survival rates (P = 0.029) in CM. CONCLUSIONS Development of CM from premalignant precursors is concurrent with the outgrowth of lymphatic vessels. This active lymphangiogenesis seems to be associated with an increased risk of local recurrence in patients with C-MIN with atypia and with an increased risk of local recurrence, lymphatic spread, distant metastasis, and tumor-related death in patients with invasive CM.

MHC Class II–Transduced Tumor Cells Originating in the Immune-Privileged Eye Prime and Boost CD4+ T Lymphocytes that Cross-react with Primary and Metastatic Uveal Melanoma Cells

Uveal melanoma, the most common malignancy of the eye, has a 50% rate of liver metastases among patients with large primary tumors. Several therapies prolong survival of metastatic patients; however, none are curative and no patients survive. Therefore, we are exploring immunotherapy as an alternative or adjunctive treatment. Uveal melanoma may be particularly appropriate for immunotherapy because primary tumors arise in an immune-privileged site and may express antigens to which the host is not tolerized. We are developing MHC class II (MHC II)-matched allogeneic, cell-based uveal melanoma vaccines that activate CD4(+) T lymphocytes, which are key cells for optimizing CD8(+) T-cell immunity, facilitating immune memory, and preventing tolerance. Our previous studies showed that tumor cells genetically modified to express costimulatory and MHC II molecules syngeneic to the recipient are potent inducers of antitumor immunity. Because the MHC II-matched allogeneic vaccines do not express the accessory molecule, Invariant chain, they present MHC II-restricted peptides derived from endogenously encoded tumor antigens. We now report that MHC II-matched allogeneic vaccines, prepared from primary uveal melanomas that arise in the immune-privileged eye, prime and boost IFNgamma-secreting CD4(+) T cells from the peripheral blood of either healthy donors or uveal melanoma patients that cross-react with primary uveal melanomas from other patients and metastatic tumors. In contrast, vaccines prepared from metastatic cells in the liver are less effective at activating CD4(+) T cells, suggesting that tumor cells originating in immune-privileged sites may have enhanced capacity for inducing antitumor immunity and for serving as immunotherapeutic agents.

MHC II lung cancer vaccines prime and boost tumor-specific CD4+ T cells that cross-react with multiple histologic subtypes of nonsmall cell lung cancer cells.

Nonsmall cell lung cancer (NSCLC) is the major cause of lung cancer‐related deaths in the United States. We are developing cell‐based vaccines as a new approach for the treatment of NSCLC. NSCLC is broadly divided into 3 histologic subtypes: adenocarcinoma, squamous cell carcinoma and large cell carcinoma. Since these subtypes are derived from the same progenitor cells, we hypothesized that they share common tumor antigens, and vaccines that induce immune reactivity against 1 subtype may also induce immunity against other subtypes. Our vaccine strategy has focused on activating tumor‐specific CD4+ T cells, a population of lymphocytes that facilitates the optimal activation of effector and memory cytotoxic CD8+ T cells. We now report that our NSCLC MHC II vaccines prepared from adeno, squamous or large cell carcinomas each activate CD4+ T cells that cross‐react with the other NSCLC subtypes and do not react with HLA‐DR‐matched normal lung fibroblasts or other HLA‐DR‐matched nonlung tumor cells. Using MHC II NSCLC vaccines expressing the DR1, DR4, DR7 or DR15 alleles, we also demonstrate that antigens shared among the different subtypes are presented by multiple HLA‐DR alleles. Therefore, MHC II NSCLC vaccines expressing a single HLA‐DR allele activate NSCLC‐specific CD4+ T cells that react with the 3 major classes of NSCLC, and the antigens recognized by the activated T cells are presented by several common HLA‐DR alleles, suggesting that the MHC II NSCLC vaccines are potential immunotherapeutics for a range of NSCLC patients.

Antigen-Specific Transfer of Functional Programmed Death Ligand 1 from Human APCs onto CD8+ T Cells via Trogocytosis

Upon specific interaction with APCs, T cells capture membrane fragments and surface molecules in a process termed trogocytosis. In this study, we demonstrate that human Ag-specific CD8+ T cells acquire the coinhibitory molecule programmed death ligand 1 (PD-L1) from mature dendritic cells (mDC) and tumor cells in an Ag-specific manner. Immature dendritic cells were less effective in transferring surface molecules onto CD8+ T cells than mDCs. Interestingly, trogocytosis of PD-L1 requires cell–cell contact and cannot be induced by uptake of soluble proteins obtained from mDC lysates. The transfer process is impaired by inhibition of vacuolar ATPases in T cells as well as by fixation of dendritic cells. Of importance, CD8+ T cells that acquired PD-L1 complexes were able to induce apoptosis of neighboring programmed death 1–expressing CD8+ T cells. In summary, our data demonstrate that human CD8+ T cells take up functionally active PD-L1 from APCs in an Ag-specific fashion, leading to fratricide of programmed death 1–expressing, neighboring T cells. The transfer of functionally active coinhibitory molecules from APCs onto human CD8+ T cells could have a regulatory role in immune responses.

Uveal melanoma cell-based vaccines express MHC II molecules that traffic via the endocytic and secretory pathways and activate CD8 + cytotoxic, tumor-specific T cells

We are exploring cell-based vaccines as a treatment for the 50% of patients with large primary uveal melanomas who develop lethal metastatic disease. MHC II uveal melanoma vaccines are MHC class I+ uveal melanoma cells transduced with CD80 genes and MHC II genes syngeneic to the recipient. Previous studies demonstrated that the vaccines activate tumor-specific CD4+ T cells from patients with metastatic uveal melanoma. We have hypothesized that vaccine potency is due to the absence of the MHC II-associated invariant chain (Ii). In the absence of Ii, newly synthesized MHC II molecules traffic intracellularly via a non-traditional pathway where they encounter and bind novel tumor peptides. Using confocal microscopy, we now confirm this hypothesis and demonstrate that MHC II molecules are present in both the endosomal and secretory pathways in vaccine cells. We also demonstrate that uveal melanoma MHC II vaccines activate uveal melanoma-specific, cytolytic CD8+ T cells that do not lyse normal fibroblasts or other tumor cells. Surprisingly, the CD8+ T cells are cytolytic for HLA-A syngeneic and MHC I-mismatched uveal melanomas. Collectively, these studies demonstrate that MHC II uveal melanoma vaccines are potent activators of tumor-specific CD4+ and CD8+ T cells and suggest that the non-conventional intracellular trafficking pattern of MHC II may contribute to their enhanced immunogenicity. Since MHC I compatibility is unnecessary for the activation of cytolytic CD8+ T cells, the vaccines could be used in uveal melanoma patients without regard to MHC I genotype.