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. 2017 Aug;134(1):41-53.
doi: 10.1007/s11060-017-2515-8. Epub 2017 Jun 15.

SOX2 immunity and tissue resident memory in children and young adults with glioma

Juan C Vasquez  1 , Anita Huttner  2 , Lin Zhang  3 , Asher Marks  1 , Amy Chan  3 , Joachim M Baehring  3 , Kristopher T Kahle  4 , Kavita M Dhodapkar  5
Affiliations

SOX2 immunity and tissue resident memory in children and young adults with glioma

Juan C Vasquez et al. J Neurooncol. 2017 Aug.

Abstract

Therapies targeting immune checkpoints are effective in tumors with a high mutation burden that express multiple neo-antigens. However, glial tumors including those seen in children carry fewer mutations and there is an unmet need to identify new antigenic targets of anti-tumor immunity. SOX2 is an embryonal stem cell antigen implicated in the biology of glioma initiating cells. Expression of SOX2 by pediatric glial tumors and the capacity of the immune system in these patients to recognize SOX2 has not been previously studied. We examined the expression of SOX2 on archived paraffin-embedded tissue from pediatric glial tumors. The presence of T-cell immunity to SOX2 was examined in both blood and tumor-infiltrating T-cells in children and young adults with glioma. The nature of tumor-infiltrating immune cells was analyzed with a 37-marker panel using single-cell mass cytometry. SOX2 is expressed by tumor cells but not surrounding normal tissue in pediatric gliomas of all grades. T-cells against this antigen can be detected in blood and tumor tissue in glioma patients. Glial tumors are enriched for CD8/CD4 T-cells with tissue resident memory (TRM; CD45RO+, CD69+, CCR7-) phenotype, which co-express multiple inhibitory checkpoints including PD-1, PD-L1 and TIGIT. Tumors also contain natural killer cells with reduced expression of lytic granzyme. Our data demonstrate immunogenicity of SOX2, which is specifically overexpressed on pediatric glial tumor cells. Harnessing tumor immunity in glioma will likely require the combined targeting of multiple inhibitory checkpoints.

Keywords: Immune checkpoints; Immunotherapy; Pediatric glioma; SOX2.

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Conflict of interest statement

Conflicts of interest: The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
SOX2 expression and function in pediatric brain tumors. a. Immunohistochemistry was performed on archived paraffin embedded tumor tissue from 27 pediatric glial tumors. Figure shows SOX2 expression in a representative patient with i) juvenile pilocytic astrocytoma, ii) glioblastoma iii) oligodendroglioma. Arrows in red shows nuclear SOX2 staining in tumor cells, blue arrows show absence of SOX2 staining in vascular cells and black arrows show normal glial cells that do not stain for SOX2. b. Primary patient derived short-term culture of JPA cells (JPA) and pediatric anaplastic astrocytoma cell line CHLA-01 (AA) were electroporated with either non-targeting siRNA smart pool (NT) or SOX2 siRNA smart pool (SOX2). Figure shows decrease in cell growth following SOX2 knock down compared to cells treated with NT
Figure 2
Figure 2
Detection of SOX2 specific T-cell response in peripheral blood and tumor tissue. Peripheral blood mononuclear cells (PBMCs, n=14) obtained from patients were cultured alone (NEG) or with an overlapping peptide library from SOX2 (5 μg/ml, Mix 1, 2, 3, 4), phytohemaglutanin (PHA) and either viral peptide mix or Candida as positive control (CEF/Candida). M1 peptides cover SOX2 residues 1–89, M2 residues 79–171, M3 residues 161–246 and M4 residues 236–321. After 48 hrs., the culture supernatant was examined for the presence of CXCL10. In 2 patients we were able to examine reactivity to SOX2 peptide mixes in the blood as well as the tumor. a. Representative SOX2 T-cell reactivity to mix 2 in a patient using CXCL10 Luminex assay. *Positive T-cell reactivity to SOX2 b. PBMCs were labelled with CFSE and co-cultured with SOX2 peptide mixes. Figure shows T-cell proliferation in response to SOX2 mix 2 in a representative patient (black arrow). c. SOX2 T-cell reactivity in LGG (n=8) and HGG (n=6). d. Reactivity to the different regions of the SOX2 protein in SOX2-immune patients (n = 5). Some patients were reactive to more than one region of the protein. e. Reactivity to PHA as well as positive control with either CEF or Candida in patients who did and did not have T-cell immune response to SOX2 antigen. f. Figure shows T-cell reactivity in paired blood and tumor tissue in two patients. In patient 1, SOX2 reactivity was detected in the tumor but not in the blood. In patient 2, SOX2 reactivity was not detected in the blood or the tumor. *Positive T-cell reactivity to SOX2
Figure 3
Figure 3
Characteristics of the glial tumor immune microenvironment. Paired blood and fresh tumor tissue was obtained from children with glioma (n=4). PBMCs from healthy pediatric donors (HD PBMCs; n=3) were used as an additional control. PBMCs were isolated and tumor tissue was processed to obtain a single cell suspension. Immune cells were examined with single cell mass cytometry using a panel of 37 different antibodies. All plots show mean and SEM. *p<0.05 a. Composition of the CD45+ cells in HD PBMCs as well as paired peripheral blood and tumor tissue from glioma patients. b. Percentage of CD4+ and CD8+ T-cells in HD PBMCs and paired blood and tumor tissue from glioma patients. c. Naïve (CCR7+RO−) and memory (CCR7−RO+) phenotype of T-cells in HD PBMCs and paired blood and tumor tissue from glioma patients. d. viSNE plot showing phenotype of T-cells in paired blood and tumor tissue from a representative patient. Figure shows expression of CD4, CD8, CCR7, CD45RO, CD69 and CD103. e. Expression of immune checkpoints in HD PBMCS as well as paired blood and tumor CD4+ and CD8+ T-cells. f. Representative heat plot showing median fluorescence intensity of immune checkpoint expression in paired blood and tumor T-cells. g. Expression of immune checkpoints on tumor CD4+ and CD8+ TRM and non-TRM cells. h. Representative heat plot showing median fluorescence intensity of immune checkpoint expression in CD4 and CD8+ tumor TRM and non-TRM cells. i. We analyzed the expression of PD-1, PD-L1 and TIGIT on CD4+ and CD8+ TRM cells in the tumor. Figure shows mean percent of cells expressing none, one, two or three checkpoints. j. Figure shows expression of granzyme B and CD 16 by CD56+NK cells in HD PBMCs as well as paired blood and tumor tissue from glioma patients. Panel on the left is a representative healthy donor and patient. Panel on the right shows data for all healthy donors and patients studied (n=4). k. Granzyme B expression in CD8+ T-cells in the HD blood as well as paired blood and tumor from a glioma patient. Panel on the left is a representative healthy donor and patient. Panel on the right shows data for all healthy donors and patients studied(n=4)
Figure 3
Figure 3
Characteristics of the glial tumor immune microenvironment. Paired blood and fresh tumor tissue was obtained from children with glioma (n=4). PBMCs from healthy pediatric donors (HD PBMCs; n=3) were used as an additional control. PBMCs were isolated and tumor tissue was processed to obtain a single cell suspension. Immune cells were examined with single cell mass cytometry using a panel of 37 different antibodies. All plots show mean and SEM. *p<0.05 a. Composition of the CD45+ cells in HD PBMCs as well as paired peripheral blood and tumor tissue from glioma patients. b. Percentage of CD4+ and CD8+ T-cells in HD PBMCs and paired blood and tumor tissue from glioma patients. c. Naïve (CCR7+RO−) and memory (CCR7−RO+) phenotype of T-cells in HD PBMCs and paired blood and tumor tissue from glioma patients. d. viSNE plot showing phenotype of T-cells in paired blood and tumor tissue from a representative patient. Figure shows expression of CD4, CD8, CCR7, CD45RO, CD69 and CD103. e. Expression of immune checkpoints in HD PBMCS as well as paired blood and tumor CD4+ and CD8+ T-cells. f. Representative heat plot showing median fluorescence intensity of immune checkpoint expression in paired blood and tumor T-cells. g. Expression of immune checkpoints on tumor CD4+ and CD8+ TRM and non-TRM cells. h. Representative heat plot showing median fluorescence intensity of immune checkpoint expression in CD4 and CD8+ tumor TRM and non-TRM cells. i. We analyzed the expression of PD-1, PD-L1 and TIGIT on CD4+ and CD8+ TRM cells in the tumor. Figure shows mean percent of cells expressing none, one, two or three checkpoints. j. Figure shows expression of granzyme B and CD 16 by CD56+NK cells in HD PBMCs as well as paired blood and tumor tissue from glioma patients. Panel on the left is a representative healthy donor and patient. Panel on the right shows data for all healthy donors and patients studied (n=4). k. Granzyme B expression in CD8+ T-cells in the HD blood as well as paired blood and tumor from a glioma patient. Panel on the left is a representative healthy donor and patient. Panel on the right shows data for all healthy donors and patients studied(n=4)
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