Figure 3: Supplementation with IL2 restores TCE activity in tumors with low antigen density.

(A) Procedure to determine T cell cytokine responses after activation via the TCR or TCE. OT-I splenocytes were pulsed with low amounts (0.13 nM) of either Ova N4, resulting in stimulation via the TCR, or TRP2 peptide, resulting in no stimulation (without TRP2 TCE) or stimulation via the TCE (in the presence of 1 nM TRP2 TCE).

(B) OT-I splenocytes were prepared as described in (A) and stimulated for 12 h, followed by fixation and intracellular cytokine staining. Bar graphs show the fraction of OT-I T cells positive for IFNγ.

(C) Fraction of IL2-positive OT-I T cells. Values depict means of intra-assay duplicates, means-comparison: Student‘s t-test; *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001, ****: p ≤ 0.0001.

(D) 0.5 x 10⁶ B16F10 (TRP2 low) were injected s.c. into C57BL/6 mice and treated with either HBS, TRP2 TCE (5 mg/kg, q6d) or murine IL2 (3 μg, q2d) monotherapy, or the combination of TCE and IL2. Arrow indicates treatment start. Linear mixed-effects model, pairwise comparisons: IL2 vs. TCE + IL2: p<0.0001, TCE vs. TCE + IL2: p<0.0001, HBS vs. TCE + IL2: p<0.0001, HBS vs. IL2: p < 0.05, HBS vs. TCE: p < 0.05, p-adjustment by Holm-method (HBS: n=4, IL2: n=3, TCE: n=6, TCE + IL2: n=6 mice).

(E) Representative flow cytometry plots showing IL2Rα/CD25 upregulation on tumor-infiltrating T cells during TCE treatment. T cells were isolated from tumors, tumor-draining lymph nodes (TdLN), or spleens from mice harboring KP1 SCLC tumors under DLL3 TCE monotherapy or HBS treatment. CD25 upregulation was most pronounced on CD8⁺ TILs (highlighted in green).

(F) Fraction of CD25-expressing CD8⁺ T cells in TdLNs, spleens, and tumors under DLL3 TCE treatment.

(G) Fraction of CD25-expressing CD4⁺ T cells in TdLNs, spleens, and tumors under DLL3 TCE treatment.

(H) 0.5 x 10⁶ B16F10 (TRP2 low) were injected s.c. into C57BL/6 mice and treated with either TRP2 TCE monotherapy (5 mg/kg, q6d) or the CD25 biased IL2 variant IL2_REH (3 μg, q2d), HBS or the combination of TCE and IL2_REH. Arrow indicates treatment start. Linear mixed-effects model, pairwise comparisons: TCE vs. TCE + IL2_REH: p<0.0001, HBS vs. TCE: p < 0.05, HBS vs. TCE + IL2_REH: p<0.0001, IL2_REH vs. TCE + IL2_REH: p = 0.0004, p-adjustment by Holm-method (HBS: n=5, IL2_REH: n=5, TCE: n=11, TCE + IL2_REH: n=11 mice).

(I) 0.5 x 10⁶ KP1 SCLC cells (DLL3 low) were injected s.c. into B6129SF1/J mice and treated with either DLL3 TCE monotherapy (5 mg/kg, q4d), IL2_REH (3 μg, q2d), HBS or the combination of TCE and IL2_REH. Arrow indicates treatment start. Linear mixed-effects model, pairwise comparisons: TCE vs. TCE + IL2_REH: p<0.05, IL2_REH vs. TCE + IL2_REH: p <0.0001, HBS vs. TCE: p <0.0001, TCE vs. TCE: p <0.0001, HBS vs. TCE + IL2_REH: p <0.0001, p-adjustment by Holm-method (n= 10 mice per group).

(J) Fraction of αβ T cells as a fraction of CD45⁺ hematopoietic cells in tumors from mice treated in (I).

(K) Fraction of CD8⁺ T cells as a fraction of all αβ T cells in tumors from mice treated in (I) Comparison of means in (F), (G), (J), (K): Student’s t-test; *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001, ****: p ≤ 0.0001.

See also Supplemental Figures S3 and S4.