Fig 2: Effector-to-memory transitioning CD4+ T cells are the primary targets for HIV-1 latency establishment.(A and B) Expression of activation markers (CD25, CD69, HLA-DR; panel A) and exhaustion markers (LAG-3, TIM-3, PD-1; panel B) of the DFV-B infected primary CD4+ T cells. Primary CD4+ T cells were infected as described in Figure 1B and the surface markers were measured by FACS at indicated time points. Data indicate mean ± SD for three different donors. p-values calculated using unpaired t-test, ns, not significant; **p<0.01; ***p<0.001. (C and D) Expression of activation markers and exhaustion markers in the DN, DP, and mC population from A and B of this figure. Panel C only shows the results of day 8 post-infection. Red dots represented marker-positive cells; blue dots represented negative cells. Panel D shows quantified values of markers expression. Data indicate mean ± SD for three different donors. (E) Infection profile of EMT CD4+ T cells when infected by DFV-B at indicated time points. Top panel depicts scheme of experimental procedure. Bottom panel shows the flow cytometric chart of infected EMT CD4+ T cells on day 5 post-infection. (F) Infected EMT CD4+ T cells are more likely to become latently infected cells. The ratios of DP cells or mC cells to total infected cells were calculated using data from panel E. Data represents the average of three donors.

Fig 3: Effect of Src, JAK and mTOR kinase inhibitors on T cell exhaustion induced by prolonged treatment with CD19-TCB. PBMCs co-cultured with SU-DHL-8 tumors cell were stimulated by CD19-TCB in the presence of 100 nM dasatinib (Src inhibitor), 100 nM sirolimus (mTOR inhibitor) or 100 nM ruxolitinib (JAK inhibitor) for 5 days. Cells from technical duplicates were pooled and the expression of PD1, TIM3 and LAG3 on CD4+ and CD8+ was measured by flow cytometry. (A) Histogram plots showing the effects of 100 nM dasatinib (Srci), 100 nM sirolimus (mTORi) and 100 nM ruxolitinib (JAKi) on the expression of PD1, TIM3 and LAG3 induced by 10 nM CD19-TCB for n=3 donors. (B) The dose-response curves show the effect of kinase inhibitors on PD1, TIM3 and LAG3 expression on CD4+ and CD8+ T cells, means of n=3 donors±SD with *p≤0.05 by one-way analysis of variance (Kruskal-Wallis test). LAG3, lymphocyte-activation gene 3; PBMCs, peripheral blood mononuclear cells; PD1, programmed cell death protein-1; TCB, T cell bispecific antibody; TIM3, T cell immunoglobulin and mucin-domain 3.

Fig 4: Treatment with RXR agonists resulted in increased T cell activity and decreased checkpoint markers. (A,B) Jurkat T cell was treated using multiple concentrations (1 µM, 2 µM, and 4 µM) in 24 h and 48 h. CD69, IFN-γ, Granzyme B, and T cell checkpoints, such as TIM3, CTLA-4, TIGIT, LAG3, and PD-1. Cell were measured using flow cytometry. (C) Primary human T cells were isolated from the bone marrow of three myeloma patients (P1, P2, P3), then treated with 2 µM LG100754 for 24 h. CD69, IFN-γ, and Granzyme B were measured using flow cytometry. (D,E) CRBN knockout reversed the effect of LG100754 on Jurkat T cell. Jurkat T cell was treated with 2 µM LG100754 for 24 h in CRBN-KO and wild-type cells. CD69, IFN-γ, Granzyme B, and T cell checkpoints (such as TIM3, CTLA-4, TIGIT, LAG3, and PD-1) were measured using flow cytometry. (F) The protein lysates acquired from CRBN knockout Jurkat T cells were analyzed by Western blotting using indicated antibodies. *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001.

Fig 5: Anti–LAG-3+anti–PD-1 enhances NK cell degranulation, cytokine secretion, and T cell proliferation.(A) Degranulation (CD107a/b) and IFN-γ/TNF-α production of primary NK cells after stimulation with the chronic myelogenous leukemia (CML) cell line K562 in patients before (up in pre) and after (up in post) anti–LAG-3+anti–PD-1 treatment and in healthy donors at different time points (n = 4, 12 samples). When samples were available, they were examined as 3 replicates, but here, values are shown as averages. P values were calculated with the Kruskal-Wallis test. (B) Relationship between extracellular LAG-3 and degranulation responses (CD107a/b) to K562 target cells in melanoma samples before and after anti–LAG-3+anti–PD-1 treatment (n = 10, 30 samples). P values and correlation coefficients were calculated with Spearman’s rank correlation. (C) Top: Proliferation of CD4+ and CD8+ T cells induced by anti-CD3 and -CD28 beads (n = 3, 9 samples). Bottom: Cell divisions in CD4+ and CD8+ T cells after anti-CD3 and -CD28 bead stimulation in a selected patient. Cell proliferation was traced with flow cytometry by dilution of CellTrace Violet dye. Cells from different time points (before and after therapy) are marked with different colors.