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The prognosis for patients with glioblastoma is grim. Ex vivo expanded tumor-associated antigen (TAA)-reactive T-cells from patients with glioma may represent a viable source for anticancer-directed cellular therapies. Immunohistochemistry was used to test the survivin (n = 40 samples) and NY-ESO-1 (n = 38 samples) protein expression in tumor specimens. T-cells from peripheral blood were stimulated with TAAs (synthetic peptides) in IL-2 and IL-7, or using a combination of IL-2, IL-15 and IL-21. CD4+ and CD8+ T-cells were tested for antigen-specific proliferation by flow cytometry, and IFN-γ production was tested by ELISA. Twenty-eight out of 38 cancer specimens exhibited NY-ESO-1 protein expression, 2/38 showed a strong universal (4+) NY-ESO-1 staining, and 9/40 cancer lesions exhibited a strong (4+) staining for survivin. We could detect antigen-specific IFN-γ responses in 25% blood samples for NY-ESO-1 and 30% for survivin. NY-ESO-1-expanded T-cells recognized naturally processed and presented epitopes. NY-ESO-1 or survivin expression in glioma represents viable targets for anticancer-directed T-cells for the biological therapy of patients with glioma. [ABSTRACT FROM AUTHOR]

Cytomegalovirus (CMV) infection remains a life-threatening condition in individuals with a suppressed immune system. CMV may also represent a clinically relevant target for immune responses in CMV-positive malignancies. We established a protocol to expand CMV-specific T cells (CMV-T) using peripheral blood mononuclear cells (PBMCs). PBMCs from 16 HLA-A*0201 donors were cultured with a cytokine cocktail comprising IL-2/IL-15/IL-21 along with overlapping peptides from CMV-pp65. Ten days later, T cells were stimulated with anti-CD3 (OKT3) and irradiated autologous PBMCs. CMV-T were detected by HLA-A*0201 CMV-pp65 NLVPMVATV wild type and q226a mutant tetramers (for high-affinity T cells), intracellular cytokine staining, a CD107a mobilization assays as well as IFN-γ and TNF-α production in cell culture supernatants. We reliably obtained 50.25 ± 27.27% of CD8+ and 22.08 ± 21.83% of CD4+ T cells post-CMV-pp65 stimulation of PBMCs with a Th1-polarized phenotype and decreased Th2/Th17 responses. Most CD3 + CD8 + tetramer+ T cells were effector-memory cells, particularly among high-affinity CMV-T (q226a CMV-tetramer+). High-affinity CMV-T cells, compared to WT-tetramer+ cells, expressed higher IL-21R and lower FasL post-stimulation with CMV-pp65. The IL-2/IL-15/IL-21 cocktail also promoted CCR6 and CXCR3 expression necessary for T-cell migration into tissues. We have optimized methods for generating high-affinity CMV-specific T cells that can be used for adoptive cellular therapy in clinical practice. [ABSTRACT FROM AUTHOR]

Tumor-infiltrating lymphocytes (TILs) may represent a viable source of T cells for the biological treatment of patients with gliomas. Glioma tissue was obtained from 16 patients, tumor cell lines were established, and TILs were expanded in 16/16 cases using a combination of IL-2/IL-15/IL-21. Intracellular cytokine staining (ICS, IL-2, IL-17, TNFα and IFNγ production) as well as a cytotoxicity assay was used to detect TIL reactivity against autologous tumor cells or shared tumor-associated antigens (TAAs; i.e., NY-ESO-1, Survivin or EGFRvIII). TILs were analyzed by flow cytometry, including T-cell receptor (TCR) Vβ family composition, exhaustion/activation and T-cell differentiation markers (CD45RA/CCR7). IL-2/IL-15/IL-21 expanded TILs exhibited a mixture of CD4+, CD8+, as well as CD3+CD4−CD8−T cells with a predominant central memory CD45RA−CCR7+phenotype. TIL showed low frequencies of T cells testing positive for PD-1, TIM-3 and CTLA-4. LAG3 tested positive in up to 30% of CD8+TIL, with low (1.25%) frequencies in CD4+T cells. TIL cultures exhibited preferential usage of Vβ families and recognition of autologous tumor cells defined by cytokine production and cytotoxicity. IL-2/IL-15/IL-21 expanded TILs represent a viable source for the cellular therapy of patients with gliomas. [ABSTRACT FROM AUTHOR]