Using the Spleen as an In Vivo Systemic Immune Barometer Alongside Osteosarcoma Disease Progression and Immunotherapy with α-PD-L1

Figure 3

Treatment with α-PD-L1 significantly reverses osteosarcoma-induced immunomodulation in LLC and MLC populations in the spleens of tumor-bearing mice. Spleens were harvested at time of metastatic disease (approximately 11 weeks) after inoculation and analyzed via flow cytometry for LLC and MLC immunophenotyping from n = 5 (−) α-PD-L1 and n = 3 (+) α-PD-L1 TF tumor-bearing mice using Antibody Panels 3 (mod) and 4, respectively. For treated mice, 20 μg of α-PD-L1 was given in phosphate buffered saline via intraperitoneal injection twice a week (Mondays and Thursdays) starting when tumors were first palpable. The data spreads of (a) TCES and (b) CTLA-4⁺ CTLs from Antibody Panel 3 (mod) are shown with significance being assigned to those populations presenting value < 0.01 from a two-tailed Student’s T-test with Bonferroni correction and 95% confidence as denoted by an asterisk. The data spreads of (c) monocytes/macrophages, (d) Tgm2⁺ macrophages, (e) Cxcl9⁺ macrophages, and (f) Nos2⁺ macrophages from Antibody Panel 4 are shown with significance being assigned to those populations presenting value < 0.00714 from a two-tailed Student’s T-test with Bonferroni correction and 95% confidence as denoted by an asterisk. PD-L1: programmed death-ligand; LLC: lymphoid lineage cell; MLC: myeloid lineage cell; TF: transfected; TCES: T-cell exhaustion status; CTLA-4: cytotoxic T-lymphocyte-associated protein 4; CTL: cytotoxic T lymphocyte; Tgm2: transglutaminase 2; Cxcl9: chemokine (C-X-C motif) ligand 9; Nos2: nitric oxide synthase 2.

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