Shift toward more naïve phenotype of the T cell compartment in patients with aggressive B cell non-Hodgkin lymphoma undergoing high-dose methotrexate therapy

Abstract

Abstract Introduction: Immunotherapies such as bispecific antibodies (BsAbs) and chimeric antigen receptor T cell therapy (CAR-T) harness the immune system, particularly T cells, to target malignant cells. Currently approved only for second- and later-line treatment, holding and bridging therapies are frequently used during CAR-T cell preparation. Holding therapy is administered before apheresis for CAR-T production, while bridging therapy is administered after apheresis, prior to CAR-T reinfusion. However, the long-term effects of commonly used chemotherapeutics on T cell function – especially regarding CAR-T cell manufacturing and BsAb efficacy – remain poorly understood. More specifically, effects of high-dose methotrexate (MTX) have not been investigated yet. Because MTX is frequently used as holding or bridging therapy before CAR-T in CNS lymphoma, we aimed to characterize the effects of high dose MTX on the T cell compartment. Methods: Peripheral blood samples were collected from patients with newly diagnosed, relapsed, or progressive aggressive B-NHL undergoing either MTX-based or non-MTX-based therapy. Sampling was performed as baseline before therapy start and on day 10 – 17 after cycle 1 (c1) and cycle 2 (c2). At baseline, the MTX-group included n = 6 patients (median age: 70.5, male (m): 4) and the non-MTX group n = 5 (median age: 72, m: 3). At c1, MTX: n = 9 (69, m: 4), non-MTX: n = 14 (70, m: 11). At c2, MTX: n = 7 (63, m: 5), non-MTX: n = 7 (72, m: 3). The CD3+CD4+ and CD3+CD8+ T cell phenotypes were determined by flow cytometry. Maturation stages were analyzed using CD45RA and CCR7 antibody staining to distinguish between naïve and stem cell memory (naïve&SCM, CCR7+CD45RA+), central memory (CM, CCR7+CD45RA-), effector memory (EM, CCR7-CD45RA-) and terminal effector (Eff, CCR7-CD45RA+) T cells; CD27 and CD28 were used to determine the differentiation stages of naïve, undifferentiated T cells (CD27+CD28+) and senescent, cytotoxic effector cells (CD27-CD28-). CD3+ T cell subset distributions were then compared between both treatment groups. Statistical analysis was performed using Mann-Whitney U test. Results: Flow cytometry revealed no significant differences in the CD4+ and CD8+ T cell subsets at baseline for patients treated with high-dose MTX compared to those receiving non-MTX-based therapy. In comparison to this, MTX-based treatment was associated with a higher proportion of CD4+ and a lower proportion of CD8+ T cells in c1 (MTX: 86.90%/6.62% vs. non-MTX: 70.49%/21.43%; p = 0.015/0.018, all for CD4+/CD8+ cells, respectively). There was also a trend toward a more immature CCR7+ naïve&SCM and CM phenotype, increased naïve CD27+CD28+ T cells, and reduced EM and senescent CD27-CD28- cells in both CD4+ and CD8+ subsets. This shift was significant in CCR7+ CD4+ and CD8+ compartments in c1 and c2 (c1: MTX: 67.50%/22.28% vs. non-MTX: 40.72%/5.58%; p = 0.047/0.018; c2: MTX: 74.14%/32.61% vs. non-MTX: 49.12%/6.73%; p = 0.026/0.037). In c1, this shift was also present in CD27+CD28+ CD4+ T cells (MTX: 60.87% vs. non-MTX: 29.80%; p = 0.034), in naïve&SCM CD8+ T cells (MTX: 19.09% vs. non-MTX: 3.57%; p = 0.018) and in both, CD4+ and CD8+ CM T cells (MTX: 26.62%/3.73% vs. non-MTX: 21.90%/1.03%; p = 0.015/0.025). Reductions in CD4+ EM (MTX: 20.32% vs. non-MTX: 46.23%; p = 0.034) and in CD8+CD27-CD28- compartments (MTX: 39.35% vs. non-MTX: 70.66%; p = 0.029) were also significant. Outlook: Our data indicate a shift toward a more naïve T cell phenotype following MTX-based therapy, which may enhance the efficacy of CAR-T cells after MTX-based holding therapy. This supports the potential use of MTX as a holding therapy prior to apheresis in CNS lymphoma patients intended for CAR-T treatment. Further validation in larger cohorts, including CAR-T product and expansion data, is warranted. An updated dataset will be presented at the meeting.