OX40 costimulation by a chimeric antigen receptor abrogates CD28 and IL-2 induced IL-10 secretion by redirected CD4(+) T cells

Abstract

Adoptive therapy with chimeric antigen receptor (CAR) redirected T cells recently showed remarkable anti-tumor efficacy in early phase clinical trials; self-repression of the immune response by T-cell secreted cytokines, however, is still an issue raising interest to abrogate the secretion of repressive cytokines while preserving the panel of CAR induced pro-inflammatory cytokines. We here revealed that T-cell activation by a CD28-ζ signaling CAR induced IL-10 secretion, which compromises T cell based immunity, along with the release of pro-inflammatory IFN-γ and IL-2. T cells stimulated by a ζ CAR without costimulation did not secrete IL-2 or IL-10; the latter, however, could be induced by supplementation with IL-2. Abrogation of CD28-ζ CAR induced IL-2 release by CD28 mutation did not reduce IL-10 secretion indicating that IL-10 can be induced by both a CD28 and an IL-2 mediated pathway. In contrast to the CD28-ζ CAR, a CAR with OX40 (CD134) costimulation did not induce IL-10. OX40 cosignaling by a 3rd generation CD28-ζ-OX40 CAR repressed CD28 induced IL-10 secretion but did not affect the secretion of pro-inflammatory cytokines, T-cell amplification or T-cell mediated cytolysis. IL-2 induced IL-10 was also repressed by OX40 co-signaling. OX40 moreover repressed IL-10 secretion by regulatory T cells which are strong IL-10 producers upon activation. Taken together OX40 cosignaling in CAR redirected T cell activation effectively represses IL-10 secretion which contributes to counteract self-repression and provides a rationale to explore OX40 co-signaling CARs in order to prolong a redirected T cell response.

Figure 1. CAR expression by engineered T cells. (A) Schematic diagram of the expression cassettes for the anti-CEA chimeric antigen receptors (CARs) which harbor the same extracellular binding domains and different signaling endodomains. TM: transmembrane domain. (B) CD4⁺ T cells were retrovirally transduced to express the respective CAR on the cell surface. CAR expression was recorded by incubation with a FITC-conjugated anti-CD3 mAb and a PE-conjugated anti-human IgG1 mAb, which binds to the extracellular CAR spacer domain, and analyzed by flow cytometry.

Figure 2. CD28ζ CAR induces IL-10 secretion by engineered T cells. CD4⁺ T cells were engineered with the anti-CEA CAR and (A) coincubated (1.25 × 10³−1 × 10⁴ CAR T cells/well) for 48 h with CEA⁺ LS174T or CEA^- Colo320 tumor cells (each 2.5 x 10⁴ cells/well) or (B) incubated (2.5 x 10⁴/well) in microtiter plates that were coated with 10 µg/ml of the CAR-specific anti-idiotypic mAb BW2064 which binds to the CAR BW431/26 scFv domain to specifically stimulate CAR T cells, or an isotype control mAb (IgG1). The number of CAR expressing T cells was adjusted to equal numbers by adding non-transduced cells from the same donor. Cytokines in the supernatant were recorded by ELISA, proliferation was determined by BrdU incorporation. Data represent the mean of triplicates ± standard error of the mean (SEM).

Figure 3. Added IL-2 induced IL-10 secretion in ζ CAR, but not in ζ-OX40 CAR stimulated T cells. CAR engineered CD4⁺ T cells (2.5 × 10⁴ cells/well) were incubated for 48 h in the presence of added IL-2 (10–1,000 U/ml) in microtiter plates coated with the CAR-specific anti-idiotypic mAb BW2064 (2.5 µg/ml coating solution). Plates coated with an antibody of irrelevant specificity served as control (w/o). Culture supernatants were recorded for cytokines by ELISA. Data represent the mean of triplicates ± SEM. A representative experiment out of three is shown.

Figure 4. The modified CD28Δζ CAR which lacks IL-2 induction also mediated IL-10 secretion. The anti-idiotypic mAb BW2064 which binds to the scFv domain of the CAR and an isotype control mAb IgG1 were coated onto microtiter plates (10 µg/ml coating solution). CD4⁺ T cells were engineered to express CARs with ζ, CD28ζ and CD28Δζ signaling domains, respectively, and incubated for 48 h in coated microtiter plates (2.5 × 10⁴ CAR T cells/well). Culture supernatants were tested by ELISA for IFNγ, IL-2 and IL-10, respectively. The assays were performed three times in triplicates; data represent mean values ± SEM of a representative experiment.

Figure 5. OX40 costimulation repressed CD28 CAR mediated IL-10 secretion in redirected T cells. Serial dilutions of the anti-idiotypic mAb BW2064 and an isotype control mAb IgG1 were coated onto microtiter plates (0.01–10 µg/ml). CD4⁺ T cells were engineered with the CEA-specific CD28ζ, CD28Δζ, CD28OX40, and CD28ΔOX40 CAR, respectively, and incubated for 48 h in coated microtiter plates (2.5 × 10⁴ CAR T cells/well). Culture supernatants were recorded by ELISA for IFNγ, IL-2 and IL-10, respectively. The assays were performed three times; data represent mean values of triplicates ± SEM of a representative experiment.

Figure 6.

IL-10 secretion was repressed in CD28OX40 CAR redirected Treg cells. (A) Isolation of CD4⁺CD25⁺IL-7R^- Treg cells. CD4⁺CD25^-IL-7R⁺ and CD4⁺CD25⁺IL-7R^- T cells were isolated from peripheral blood by MACS procedures. Isolated T cells were incubated with anti-IL-7R-FITC, anti-CD25-PE and anti-CD4-APC mAbs, respectively, and analyzed by flow cytometry. (B) Isolated T reg cells were activated and expanded in the presence of IL-2 (1,000 U/ml) on solid phase bound agonistic anti-CD3 and anti-CD28 mAbs (each 10 µg/ml). Cells were rested for 48 h without stimuli and tested for suppressor activity. Allogeneic CD4⁺ responder cells (1 × 10⁴ cells/well) and Treg cells (1 × 10⁴ cells/well) were cocultivated for 5 d in the presence or absence of agonistic anti-CD3 (10 µg/ml) and anti-CD28 (1 µg/ml) antibodies, pulsed with BrdU and proliferation determined by a cell ELISA. (C) CD4⁺CD25⁺IL-7R^- regulatory T cells express CARs with high efficacy. Isolated CD4⁺CD25⁺IL-7R^- regulatory T cells were retrovirally transduced to express the anti-CEA CARs which harbor either the CD28ζ or CD28ΟX40 signaling domain. CAR engineered T cells were detected by a PE-conjugated anti-human IgG1 antibody and analyzed by flow cytometry. (D) Serial dilutions of the anti-idiotypic mAb BW2064 were coated on microtiter plates (0.01 - 10 µg/ml). CD4⁺CD25⁺IL7R^- T cells were engineered with the CD28ζ or CD28-CD3ζOX40 CAR, respectively, adjusted to equal numbers of CAR expressing cells and incubated for 48 h in coated microtiter plates (1 × 10⁴ CAR T cells/well). Culture supernatants were recorded by ELISA for IFNγ, IL-2 and IL-10, respectively. The assays were performed three times; data represent mean values of triplicates ± SEM of a representative experiment.