CD4+ T cells are required to improve the efficacy of CIK therapy in non-small cell lung cancer

Abstract

As a widely studied adoptive treatment method, CIK (cytokine-induced killer cells) treatment has shown clinical benefits in many clinical trials on non-small cell lung cancer. As a heterogeneous cell population, however, CIK cells have a strong instability and individual differences in their efficacies, which are collaboratively regulated by the tumor microenvironment and CIK subpopulations. Among them, CD4⁺ T cells belong to a crucial subgroup of the CIK cell population, and their influence on CIK therapy is still unclear. Herein, we show how CD4⁺ T cells positively regulate the functions of CD3⁺CD56⁺ T and CD3⁺CD8⁺ T cells. During this process, we found that Th1/Th17 CD4⁺ subgroups can induce the phosphorylation of the AKT pathway by secreting IL-17A, and upregulate the expression of T-bet/Eomes transcription factors, thereby restoring the function of CD8⁺/CD3⁺CD56⁺ T cells and reversing the exhaustion of PD-1⁺Tim-3⁺ T cells. These findings will provide guidance for the clinical screening of suitable populations for CIK treatment and formulation of strategies for CIK therapy plus immune checkpoint treatment. Based on these findings, we are conducting an open-label phase II study (NCT04836728) is to evaluate the effects of autologous CIKs in combination with PD-1 inhibitor in the first-line treatment of IV NSCLC, and hope to observe patients' benefits in this clinical trial.

Fig. 1. The percentage of CD4⁺ T cells correlated with clinical prognosis and function of CIK cells.

A Kaplan–Meier estimates of progression-free survival in the three (high, intermediate, and low) groups. B Kaplan–Meier estimates of overall survival in the three (high, intermediate, and low) groups. C Flow cytometric quality assessment of CIK cells after 14 days of ex vivo expansion before adoptive transfer to tumor-burdened NSG mice. D–F Subcutaneous growth of tumor cells (A549) in each group of mice (n = 4) treated with CD4⁺ T, CD4^-CIK, and CIK cells. G Bioluminescent imaging of NSG mice from three groups treated with T-cell tracer. H Flow cytometric analysis of IFN-γ, GzmB, PD-1, and Tim-3 expression in CD3⁺CD8⁺ T cells. Error bars indicate SEM, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 (Kaplan–Meier, one-way ANOVA or Student's t test).

Fig. 2. CD4⁺ T cells improved the migration of CD4⁻CIK cells through IFN-γ/CXCL9,10,11/CXCR3.

A Illustration of chemotaxis assay of CD4⁻CIK cells toward supernatants derived from mixed tumor co-cultures. B Quantification of CD4⁻CIK cell migration in the tumor-conditioned medium in the presence or absence of CD4⁺ T cells. C Flow cytometric quantification of CD4⁻CIK migration in the tumor-conditioned medium before and after transmigration. D Quantification of chemokine-related mRNA expression in tumor cells and macrophages (left); flow cytometric analysis of IFN-γ expression in supernatants from tumor-conditioned medium (right). E Quantification of chemokine-related mRNA expression in tumor cells and macrophages in the presence of IFN-γ or anti-IFN-γ. F Quantification of CD4^-CIK cell migration in tumor (A549 or H520)-conditioned medium in the presence of different neutralizing antibodies, IFN-γ or CD4⁺ T cells at 24 h. G Schematic diagram depicting the dosing schedule and timing for a subcutaneous transplantation tumor model. H, I Subcutaneous growth of tumor cells (A549) in each group of mice (n = 5) treated with PBS, CD4^-CIK cells, CIK cells, and CIK + AMG487. J Flow cytometric examination of the number of CD3⁺CD8⁺ T cells infiltration in tumor. K Specific imaging of immunohistochemistry from CIK + AMG487 and CIK groups, scale bars: 100 µm. Error bars indicate SEM, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 (one-way ANOVA or Student's t test).

Fig. 3. CD4⁺ T cells enhanced the function of CIK cells during ex vivo expansion.

A Schematic diagram depicting the ex vivo expansion of different CIK cells (left); flow cytometric quality assessment of CIK cells when ex vivo expansion at day 14 (right). B Flow cytometric examination of immune checkpoint receptors expression in CD3⁺CD8⁺ T cells. C Flow cytometric analysis of IFN-γ and GzmB production in CD3⁺CD8⁺ T cells. D Flow cytometric examination of the percentage of CD3⁺CD8⁺CD56⁺ T cells in CD3⁺CD8⁺ T cells. E Flow cytometric analysis of IFN-γ, GzmB, and Tim-3 expression in CD8⁺CD56⁺ T cells. F The cytotoxicity of CIK, CD4⁺ T, or CD4⁻CIK cells against A549, H520, and K562. Error bars indicate SEM, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 (one-way ANOVA or Student's t test).

Fig. 4. The supernatants of CD4⁺ T cell culture improved the functions of CIK cells in vitro.

A Illustration of co-culture of tumor cells (A549 and H520) and CIK cells. B Flow cytometric analysis of PD-1, CTLA-4, Tim-3, and LAG-3 expression in CD3⁺CD8⁺ T cells in a co-culture system (A549). C Flow cytometric examination of GzmB and IFN-γ production in CD3⁺CD8⁺ T cells in a co-culture system (A549). D The cytotoxicity of CIK or CD4⁻CIK cells against A549. E, F Flow cytometric assessment of Tim-3, IFN-γ, and GzmB expression in CD3⁺CD8⁺ T cells treated with tumor-conditioned medium in the presence (SCD4⁻CIK group) or absence (CD4⁻CIK group) of CD4⁺ T cells. G Flow cytometric or ELISA assessment of the concentration or MFI of 12 human inflammatory cytokines in tumor-conditioned medium in the presence or absence of CD4⁺ T cells. H–J Flow cytometric analysis of Tim-3, IFN-γ, and GzmB expression in CD3⁺CD8⁺ T or CD3⁺CD8⁺CD56⁺ T cells treated with either PBS, anti-IL-10 (200 ng/ml), or anti-IL-17 (100 ng/ml). K Western blot analysis of STAT3, AKT, and ERK phosphorylation in CD4⁻CIK cells treated with tumor-conditioned medium for 2 h. Error bars indicate SEM, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 (one-way ANOVA or Student's t test).

Fig. 5. CD4⁺ T cells improved the functions of CIK cells through IL-17A.

A Western blot analysis of STAT3, AKT, and ERK phosphorylation in CD4^-CIK cells stimulated with rIL-17 (100 ng/ml) protein for 2 h. B The cytotoxicity of CD4^-CIK cells treated with or without rIL-17 against A549. C, D Flow cytometric analysis of Tim-3 and GzmB expression in CD3⁺CD8⁺ T and CD3⁺CD8⁺CD56⁺ T cells treated with AKT inhibitor (Ly294002, 10 μM), rIL-17 or PBS. E Relative mRNA quantification of Eomes and T-bet in CD4⁻CIK cells treated with rIL-17, inhAKT, or PBS for 72 h. F Correlation analysis of IL-17A and CD8⁺ T cells infiltration or NK cells activated infiltration in LUAD and LUSC in TIMER public database. G Correlation analysis of IL-17A and EOMES or TBX21 in LUAD and LUSC in TIMER public database. H Flow cytometric examination of IL-17 and IFN-γ production in CIK cells before and after ex vivo expansion. I, J The relationship of Th1 and Th17 cell groups (n = 8). K Flow cytometric analysis of Tim-3 expression in CD3⁺CD8⁺ T and CD3⁺CD8⁺CD56⁺ T cells treated with tumor-conditioned medium in the presence of Th17 cells, CD4⁺ T cells or absence of these cells. L Flow cytometric determination of GzmB production in CD3⁺CD8⁺ T and CD3⁺CD8⁺CD56⁺ T cells. M Relative mRNA quantification of Tox, Eomes, and T-bet in CD4⁻CIK cells treated with tumor-conditioned medium for 72 h. Error bars indicate SEM, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 (one-way ANOVA or Student's t test).

Fig. 6. CIK cell therapy plus IL-17A or anti-PD-1 treatment reversed the functions of exhausted CIK cells in vivo.

A, B Subcutaneous growth of tumor cells (A549) in each group of NSG mice treated with CD4^-CIK cells (n = 5), CD4^-CIK cells+anti-PD-1 (n = 5), CD4^-CIK cells+rIl-17 (n = 5), or CIK cells (n = 5). C Flow cytometric examination of the percentage of CD3⁺CD8⁺CD56⁺ T cells in CD3⁺CD8⁺ T cells. D Flow cytometric analysis of the percentage of PD-1⁺Tim-3⁺CD8⁺ T cells in CD3⁺CD8⁺ T cells. E Flow cytometric determination of the percentage of GzmB⁺IFN-γ⁺CD8⁺ T cells in CD3⁺CD8⁺ T cells. Error bars indicate SEM, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 (one-way ANOVA or Student's t test).