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

Abstract

Background: Advanced 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.

Methods: Potential 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.

Results: The 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.

Conclusions: The 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.

Fig. 1

Lung cancer patients have immunosuppressive CD11b⁺CD33⁺ cells in their peripheral blood. a PBMC from healthy donor BC123104 and from NSCLC patients 3, 11 and 6 were stained with mAbs for CD11b, CD33, CD15, CD3, CD68, CD11c, DEC205, CD56, and CD80, or isotype control mAbs. Viable cells were gated (largest gate in the right-hand panels) and analyzed by flow cytometry. b H&E stained, MACS purified CD11b⁺CD33⁺ cells. c PBMC from lung cancer patients 6, 7, and 11 were either depleted or not depleted for CD11b⁺CD33⁺ MDSC cells and activated with anti-CD3 and anti-CD28 mAbs. T cell proliferation was assessed by ³H-thymidine uptake. The ratio of T cells (CD3⁺CD4⁺ plus CD3⁺CD8⁺) to CD11b⁺CD33⁺ MDSC was 1:5, 1:0.5, and 1:0.8 for patients 6, 7, and 11, respectively. The data of a and b are one of two independent experiments using PBMC from two healthy donors and ten patients. The data of c are one of two independent experiments using PBMC from three patients

Fig. 2

Bronchioloalveolar adenocarcinoma (H358) and large cell carcinoma (H177) cells transfected with costimulatory molecule CD80 and HLA-DR7 or HLA-DR1 genes express cell surface HLA-DR and CD80 and do not express Ii and are not inducible by IFNγ for Ii or HLA-DR. a Western blots of NSCLC and control cells stained for Ii. b IFNγ- treated or untreated tumor cells were stained for cell surface MHC I (mAb W6/32), MHC II (mAb L243), or CD80 (anti-human CD80 mAb), and analyzed by flow cytometry. c H358 and H177 NSCLC cells were transfected with pLNCX2/DR7, pLNCX2/DR1, pLNCX2/DR4, and/or pLHCX2/CD80 plasmids using nucleofection technology (Amaxa), stained for HLA-DR and CD80, and analyzed by flow cytometry. Profiles are for transfectants that have been in cultures for >9–12 months

Fig. 3

Major histocompatibility complex class II (MHC II) lung cancer vaccines prime and boost type 1 CD4⁺ T cells from healthy donors that react with lung cancer cells and do not react with non-lung tumor cells. a Time line for in vitro priming and boosting of PBMC. b HLA-DR7⁺ healthy donor PBMC BC123104 were primed with irradiated H358/DR7/CD80 (MHC II vaccine) or H358 cells and boosted with the indicated cells. IFNγ production was measured by ELISA. c HLA-DR7⁺ PBMC from healthy donor BC123104 were either not depleted or depleted for CD4⁺ or CD8⁺ T cells before priming. IFNγ release by non-depleted PBMC was 450–500 pg/ml. Non-depleted PBMC contained 16–20% CD8⁺ T cells and 40–50% CD4⁺ T cells. PBMC depleted for CD4⁺ and CD8⁺ T cells contained <1 and <2% CD4⁺ and CD8⁺ T cells, respectively. d Supernatants from primed and boosted PBMC from panel B were assayed for cytokines by multiplex luminex analysis. e, f PBMC from healthy donor BC123104 were primed with the H358/DR7/CD80 MHC II lung cancer vaccine and boosted with the indicated cells. IFNγ production by activated CD4⁺ T cells was measured by ELISA. Data for each panel are representative of two to three independent experiments with PBMC from healthy donors BC123104 and BC011405

Fig. 4

Major histocompatibility complex class II lung cancer vaccines prime and boost tumor-specific T cells from lung cancer patients’ PBMC despite the presence of CD11b⁺CD33⁺ MDSC. HLA-DR7⁺ PBMC from healthy donor BC123104 or BC011405 and from patient 6 (a), patient 3 (b), or patient 1 (c) were primed with the H358/DR7/CD80 or H177/DR7/CD80 MHC II lung cancer vaccine and boosted with the indicated cells. IFNγ production by activated CD4⁺ T cells was measured by ELISA. d HLA-DR7⁺ PBMC from healthy donor BC123104 or from patient 11 (depleted or not depleted for CD11b⁺CD33⁺ cells) were primed with H358/DR7/CD80 vaccine cells and T cell proliferation following priming was measured by ³H-thymidine uptake. e HLA-DR4⁺ PBMC from patient 9 were primed with H358/DR4/CD80 vaccine cells and boosted with the priming vaccine, parental H358 cells, or lung fibroblasts in the absence or presence of purified autologous CD11b⁺CD33⁺ cells. f HLA-DR7⁺ PBMC from healthy donor BC123104 and from patient 10 were primed with H358/DR7/CD80 vaccine cells and boosted with the priming vaccine cells, parental H358 cells, or non-malignant lung fibroblasts. IFNγ production by activated CD4⁺ T cells was measured by ELISA. Data for panels d, and e are from one of two independent experiments. Data for panels a, b, c, and f are representative of one to two independent experiments with PBMC from ten NSCLC patients