Fig 2: Maturation profile of peripheral blood T cells and TALs from ovarian cancer patients. The maturation profile of peripheral blood T cells, ex vivo TALs, young TALs and transduced REP TALs was assessed by the distribution of naïve, Tcm, Tem and Temra cells gated out of the live CD3+ T cell population. (A) Representative dot plots of gating strategy: CD45RA expression gated on the CD3+ T cells, and further subgated by CD62L and CD57 expression. (B-E) Frequencies of Tnaïve, Temra, Tem and Tcm, respectively, in OC PBMC (n = 12), ex vivo ascites (n = 12), young TAL cultures (n = 10) and REP TAL cultures (n = 8, 4 MOCK and 4 CXCR2 transduced). (F) Pie charts of maturation profile (mean) of Tnaïve, Temra, Tem and Tcm represented in B-E in addition to CD57+ Tem and “other”, as shown in gating strategy (A). Error bars designate standard deviation (SD), *P < 0.05, **P < 0.01 and ***P < 0.001.

Fig 3: BAFF-R expressing B cells at different maturational stages are present in decidua.To identify lymphocytes, in the first panel to the far left, live cells were gated using a viability dye that marks all dead cells and the lymphocyte gate was then set within CD45-expressing singlet leukocytes. (a–c) Within lymphocytes, CD56+ NK cells, CD3+ CD56neg T cells, CD3+ CD56+ NK-T-like cells and CD19+ CD20+ B cells were identified (second and third panels). B cell maturational stages were distinguished based on CD24 and CD38 expression (fourth panel). The BAFF-R expression on the different lymphocyte populations is depicted to the far right. (d) The proportion of different cell subsets with in the lymphocyte gate and (e) the proportion of B cells at different maturational stages isolated from decidua basalis, decidua parietalis and cord blood. (f) The mean fluorescence intensity of BAFF on B cells at different maturational stages. Floating bars show minimum to maximum values, and horizontal bar indicates the median (n = 3). Approximately 40.000 cells were collected in the lymphocyte gate for d. basalis and CBMC and 20.000 for d. parietalis.

Fig 4: (a) Temporal upregulation of mHsp90 compared to total Hsp90 levels in T cells stimulated with CD3/CD28 antibodies over a 96-h period. The data were analyzed by 2-way ANOVA with Sidak’s multiple comparison test, *p < 0.05, **p < 0.01, ***p < 0.001. Experiments were performed with 6 biological replicates. (b) Flow cytometric analysis of CD3, CD25, and CD69 T-cell populations relative mHsp90 expression compared to baseline. (c) Upregulation of total Hsp90 in activated T cell populations. Experiments were performed with 3 biological replicates for each timepoint (b, c). (d) Mean (± SD) CD25 expression on T cells 24–96 h post CD3/28 stimulation. Temporal upregulation of mHsp90 (green line) post T cell stimulation. CD25 expression; 6–8 biological replicates per time point. mHsp90 expression 6 biological replicates for each timepoint. (e) mHsp90 and total Hsp90 expression in CD3+ T cells isolated from PBMC’s were stimulated with IL-2 (60 IU/mL) for 24 h. Data represent mean ± SD. The data were analyzed by 2way ANOVA with Sidaks multiple comparison test. Experiments were performed once with 3 biological replicates. (f) Cytokine expression 24 h post Th1 or Th2 T cell stimulation treated with or without HS-131 (1 µM). Data represent mean ± SEM. The data were analyzed by One-way ANOVA with Sidaks multiple comparison test *p < 0.05,**p < 0.01, ***p < 0.001. Experiment represents 3–4 biological replicates per treatment.

Fig 5: Regulatory T cells accumulate in ovarian cancer ascites. Frequencies of regulatory T cells were analysed by flow cytometry of peripheral blood mononuclear cells (PBMC) and ascites from patients with ovarian cancer (OC) compared to PBMC from healthy donors (HD). Tregs were gated as CD25hi CD127low/neg cells out of total live CD3+ CD4+ T cells. A) Representative dot plots of CD25hi CD127low/neg gating from HD PBMC, OC PBMC and OC Ascites respectively. B) Representative histograms of FoxP3 and CCR4 MFI of Tregs from OC ascites show a higher expression of FoxP3 and CCR4 compared to Tregs from HD PBMC and OC PBMC. C) Percentages of FoxP3+ Tregs and D) percentage of CCR4+ FoxP3+ Tregs in HD, OC PBMC and OC ascites. HD PBMC n = 13, OC PBMC n = 14, OC Ascites n = 13, Unpaired t-test, error bars designate mean with SEM, *P < 0.05, **P < 0.01, *** P < 0.001.