Fig 2: Absolute numbers of total CD3+, CD3+CD4+, and CD3+CD8+ T cells in wild-type (WT), TLR4 humanized transgenic (huTLR4), and muTLR4Lps-del infected with L. interrogans. Splenocytes were prepared and total cells were counted on a LUNA cell counter. Cells were stained for flow cytometric analysis of T lymphocyte profiles and a total of 100,000 events were captured in a ZE5 cell analyzer (Bio-Rad, Hercules, CA, USA). CD3, CD4, CD8 cell counts were obtained from single cell populations. Data are the mean ± SD of cell counts for three or four mice per group. Statistics by Unpaired t test with Welch’s correction were not significant between any of the uninfected and infected groups. Data for huTLR4 represents one of two experiments and data for WT/muTLR4Lps-del represents one experiment.

Fig 3: Dex induces immunosuppression. (A). Lymphocytes were isolated with a lymphocyte separation kit (P8740). CD4-FITC, CD8-PE and CD3-SPRD monoclonal antibodies were added and reacted at room temperature in the dark for 30 minutes. Resuspended cells were detected with a flow cytometer (Bio-Rad, ZE5, USA). (B). The percentage of peripheral blood lymphocytes, analyzed with Cell Quest software. Plasma TNF-a (C), IL-1ß (D), IFN-? (E), IFN-ß (F) and IL-6 (G) levels were assessed with an ELISA kit and standard curve ( Supplementary Materials Figure S2 ).

Fig 4: Effect of VERU-111 on cell cycle distribution and apoptosis in PanCa cells. (A) Effect of VERU-111 on cell cycle distribution of Panc-1 and AsPC-1 cells. Briefly, Cells were treated with VERU-111 for 24 h and analyzed by flow cytometric analysis using Propidium Iodide. Bar graph showing cell cycle distribution at different phases in G0-G1, S, and G2M as analyzed by MOTFIT Analysis Software (i-ii). (B) Effect of VERU-111 on protein levels of cell cycle regulatory proteins (Cyclin B1, Cdc25c, Cdc2, and pCdc2Tyr15) in Panc-1(i) and AsPC-1(ii) cells as determined by Western blot analysis. (C) Effect of VERU-111 on apoptosis induction. Briefly, cells were treated with indicated concentrations of VERU-111 for 24 h and apoptosis induction was analyzed by flow cytometry using Annexin V-7AAD Apoptosis kit. Bar graphs showing dose-dependent increase of apoptotic cells in VERU-111 treatment. (D) Effect of Protein levels of Bax, Bcl-2, Bad and Bcl-xl in Panc-1(i) and AsPC-1(ii) cells in response to VERU-111 treatment for 24 h as determined by Western blot analysis. (E) Effect of VERU-111 on protein levels of pro-caspase-3 and 9, cleaved caspase-3 and 9 and PARP cleavage in Panc-1(i) and AsPC-1(ii) cells as determined by Western blot analysis. (F) Effect of VERU-111 alone or in combination with caspase inhibitor Z-VAD-FMK on protein level of pro-caspase-3 and 9, cleaved caspase-3 and 9 and PARP cleavage in Panc-1(i) and AsPC-1(ii) cells as determined by Western blot analysis. (G) Effect of VERU-111 alone or in combination with caspase inhibitor Z-VAD-FMK on apoptosis induction analyzed by flow cytometry using Annexin V-7AAD Apoptosis kit. Data was acquired by using the Bio-RAD ZE5/Evererst Software v2.1 and analyzed using FlowJo v.10.3. Asterisk (*) denotes the significant value p < 0.05

Fig 5: Antigen specific T cell responses to common antigens in HC and PD patient’s PBMCs. a-d) AIM responses (OX40+ CD137+) in CD4 T cells and e) CD8 T cells (CD69+ CD137+) after stimulation to previously defined antigenic peptide pools. PBMCs from HC (blue circles) and PD (red circles) were stimulated with 1 µg/mL of NL63, 228E, HKU1, OC43, Flu-HA, Flu-Other, Rhinovirus, RSV, CMV, EBV, VZV, PT, or TT for 24 h, stained, and run on a Bio-Rad ZE5 flow cytometer. e) AIM responses (CD69+ CD137+) in CD8 T cells. Each dot represents an individual subject, HC n = 19, PD n = 20. Two-tailed Mann-Whitney test; geometric mean with standard deviation is displayed. Dashed line indicates threshold of positivity (TP). Numbers above groups indicate the test values. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)