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. 2023 Mar 8;12:e85739. doi: 10.7554/eLife.85739

Figure 7. Loss of tumor-derived IL-6 drives changes in Treg numbers and function but is not sufficient to reverse clearance of Kin1-NULL tumors.

(A) Met-1 Kin1-NULL IL-6-CTRL or Kin1-NULL IL-6-NULL tumors were established via subcutaneous injection in FVB mice, and harvested at day 10 for immunophenotyping by flow cytometry. Gating of CD4+ T cell populations was conducted and quantified as percentage of total (alive) cells. (B) As in A with quantification of expression of markers of degranulation (CD107a) and cytotoxicity (Granzyme B) in tumor infiltrating CD8+ T cells. Example contour plots (left) and quantification of double positive cells (right). (C) Weights of tumors from mice shown in A-B. (D) Met-1 Kin1-NULL IL-6-CTRL or Kin1-NULL IL-6-NULL tumors were established via subcutaneous injection in FVB mice. Tumor size was recorded. (E) Met-1 Kin1-NULL tumors were established via subcutaneous injection in FVB mice, with 20 μg anti-IL-6 neutralising antibody administered on Day –1, 0, 4, 8, 12, and 16 post tumor cell injection. Tumor size was recorded. (F) Met-1 tumors were established in mice pre-treated with anti-CD25 to deplete Tregs. Tumor growth for individual mice (left and middle) and averages (right) are shown. Depletion demonstrated in Figure 7—figure supplement 1 (G) Tumor size at Day 17 from F. n=3–7 per group, error bars = SD. Unpaired t-test with * =< 0.05, ** =< 0.01, *** =< 0.001, **** =< 0.0001.

Figure 7.

Figure 7—figure supplement 1. Depletion of Tregs with anti-CD25 antibody treatment.

Figure 7—figure supplement 1.

(A) After 3 day pre-treatment with anti-CD25 or isotype control antibody, Met-1 Kin1-WT or Kin1-NULL tumors were established via subcutaneous injection in FVB mice. Antibody treatment was continued weekly for 21 days at which point tissue was harvested to assess Treg-specific deletion. Representative plots showing depletion of Treg cells in tumour (top), spleen (middle) and draining lymph node (dLN – bottom). (B) Quantification of tumour data for both FoxP3+ (Treg) and FoxP3- (non-Treg CD4+) cells. (C) Example histogram demonstrating CD25 expression on Treg cells. Representative example of two experiments. n=3 per group, error bars = SD. Unpaired t-test * =< 0.05, ** = <0.01.