The Exhausted Intratumoral T Cell Population in B-Cell Non-Hodgkin Lymphoma Is Defined By LAG-3, PD-1 andtim-3 Expression

T-cell exhaustion plays an important role in attenuating the function of immune cells in B-cell non-Hodgkin's lymphoma (NHL) and PD-1 expression is typically used to identify exhausted T-cells. We have however previously shown that not all PD-1⁺ cells are exhausted and that PD-1 is differentially expressed on two distinct T-cell subpopulations, with high expression on T follicular helper cells and dim expression on exhausted T cells. Other markers are therefore needed to more clearly identify exhausted intratumoral T cells. To further define exhaustion of intratumoral T cells, we determined the co-expression, regulation and function of PD-1, TIM-3 and LAG-3 on CD4⁺ or CD8⁺ T cells by flow cytometry. Using biopsy specimens from follicular B-cell NHL, we found that the percentages of PD-1⁺ and TIM-3⁺ T cells were 53.1% (range: 17.2-81.2%, n=32) and 34.5% (range: 14.9-62.6%, n=34) in CD4⁺ T cells and 46.8% (range: 12.8-81.7%, n=32) and 40.4% (range: 15.0-78.4%, n=34) in CD8⁺ T cells, respectively. We observed that TIM-3 was predominantly expressed on PD-1^dim T cells and TIM-3⁺ cells accounted for 40% of CD4⁺ PD-1^dim or 45% of CD8⁺ PD-1^dim T cells. Similarly, LAG-3 was variably expressed on intratumoral T cells from B-cell NHL. A median of 9.54% (range: 3.01-15.46, n=6) of CD4⁺ or 20.48% (7.93-33.9, n=8) of CD8⁺ T cells express LAG-3. We found that LAG-3⁺ T cells almost exclusively came from PD-1⁺ TIM-3⁺ cells, forming a defined population of intratumoral PD-1⁺ TIM-3⁺ LAG-3⁺ CD4⁺ or CD8⁺ T cells. While the majority of LAG-3⁺ T cells were effector memory T cells (CD45RA^- CCR7^-), some LAG-3-expressing T cells displayed a phenotype of terminally-differentiated T cells (CD45RA⁺ CCR7^-). Functionally, the intratumoral TIM-3⁺ LAG-3⁺ T cells exhibited reduced capacity to produce cytokines (IL-2, IFN-γ) and granules (perforin, granzyme B). Similar to TIM-3, LAG-3 expression was strongly up-regulated on CD4⁺ or CD8⁺ T cells by IL-12, a cytokine that has been shown to induce T-cell exhaustion. Interestingly, we observed that while expression of TIM-3 on CD8⁺ T cells was upregulated by IL-12 at an early time point (day 1), LAG-3 was only induced after TIM-3 up-regulation (day 3) and almost exclusively on TIM-3⁺ T cells. Furthermore, we found that blockade of both TIM-3 and LAG-3 signaling was able to reverse the exhausted phenotype of CD8⁺ T cells resulting in increased IFN-γ and IL-2 production. This effect was further enhanced when CD8⁺ T cells were treated with both anti-TIM-3 and anti-LAG-3 Abs. Taken together, these results suggest that PD-1, TIM-3 and LAG-3 were involved in the induction of exhaustion of T cells in B-cell NHL. We find that PD-1, TIM-3 and LAG-3 are expressed on the same T cells and that blocking TIM-3 and LAG-3 can reverse T-cell exhaustion signaling. These results suggest that PD-1, TIM-3 and LAG-3 play a synergistic role in the development of T cell exhaustion in NHL.