Expression of immune checkpoint proteins on T cells in bone marrow of myeloma bearing mice over time. KaLwRij mice were inoculated with 2×106 5T33-GFP cells iv. Myeloma bearing mice were euthanized between days 7 and 28 and when moribund at days 29-40 after myeloma injection. Femoral bone marrow cells were harvested and (A) GFP+ tumor cell accumulation was monitored by flow cytometry. CD4+ and CD8+ T cells in the bone marrow were analyzed by flow cytometry for expression of (B) PD-1, TIM-3, LAG-3, BTLA and 2B4 at each of the time points indicated, or for surface expression of (C) CTLA4 when animals were moribund. T cells harvested from naïve non-myeloma bearing mice were analyzed as controls. Immune checkpoint protein percentages were calculated based on isotype controls. (D) Expression of TIM-3 and PD-1, LAG-3 and PD-1 or 2B4 and PD-1 on gated CD8+ or CD4+ T cells harvested from the BM of moribund mice. Data shown are representative of more than four independent analyses. *p < 0.05, **p < 0.01, ***p < 0.001 as compared to T cells from naïve non-myeloma bearing mice.