MDR1-EXPRESSING CD4+ T CELLS WITH TH1.17 FEATURES RESIST TO NEOADJUVANT CHEMOTHERAPY AND ARE ASSOCIATED WITH BREAST CANCER CLINICAL RESPONSE

Abstract

Background: Multidrug resistance-1 (MDR1) transporter limits the intracellular accumulation of chemotherapies (paclitaxel, anthracyclines) used in breast cancer (BC) treatment. In addition to tumor cells, MDR1 is expressed on immune cell subsets in which it confers chemoresistance. Among human T cells, MDR1 is expressed by most CD8⁺ T cells, and a subset of CD4⁺ T helper (Th) cells. Here we explored the expression, function and regulation of MDR1 on CD4⁺ T cells and investigated the role of this population in response to neoadjuvant chemotherapy (NAC) in BC.

Methods: Phenotypic and functional characteristics of MDR1⁺ CD4 Th cells were assessed on blood from healthy donors and patients with BC by flow cytometry. These features were extended to CD4⁺ Th cells from untreated breast tumor by flow cytometry and RNA-sequencing (RNA-seq). We performed in vitro polarization assays to decipher MDR1 regulation on CD4 Th cells. We evaluated in vitro the impact of chemotherapy agents on MDR1⁺ CD4⁺ Th cells. We analyzed the impact of NAC treatment on MDR1⁺ CD4⁺ Th cells from blood and tumors and their association with treatment efficacy in two independent BC cohorts and in a public RNA-seq data set of BC tumor biopsies before and after NAC. Finally, we performed single cell (sc) RNAseq of blood CD4⁺ memory T cells from NAC-treated patients and combined them with an scRNAseq public data set.

Results: MDR1⁺ CD4 Th cells were strongly enriched in Th1.17 polyfunctional cells but also in Th17 cells, both in blood and untreated breast tumor tissues. Mechanistically, Tumor growth factor (TGF)-β1 was required for MDR1 induction during in vitro Th17 or Th1.17 polarization. MDR1 expression conferred a selective advantage to Th1.17 and Th17 cells following paclitaxel treatment in vitro and in vivo in NAC-treated patients. scRNAseq demonstrated MDR1 association with tumor Th1.17 and Th with features of cytotoxic cells. Enrichment in MDR1⁺ CD4⁺ Th1.17 and Th17 cells, in blood and tumors positively correlated with pathological response. Absence of early modulation of Th1.17 and Th17 in NAC-resistant patients, argue for its use as a biomarker for chemotherapy regimen adjustment.

Conclusion: MDR1 favored the enrichment of Th1.17 and Th17 in blood and tumor after NAC that correlated to clinical response.

Keywords: Adaptive Immunity; CD4-Positive T-Lymphocytes; Lymphocytes, Tumor-Infiltrating; Tumor Biomarkers; Tumor Microenvironment.

Figure 1

MDR1 is expressed by a subset of non-regulatory memory CD4⁺ T cells and is functional. (A–D) Proportions of MDR1⁺ cells in HD blood (n=17) among CD4⁺ and CD8⁺ T cells compartment: (A) CD4⁺ T cells subsets of naive, Th and Treg cells (B) memory CD4⁺ T cells subsets (T central memory (_CM), T effector memory (_EM), Teffector memory RA+ (_EMRA) based on CD45RA and CCR7 expression, in frequency (C) and expression intensity (MFI) (D) E) Correlation between proportions of MDR1⁺ cells and rhodamine 123 effluxing cells (Rh123^neg cells) among all memory CD4⁺ T cells of HD blood (n=10) (F) Representative dot plot of Rh123 efflux capacity of memory CD4⁺ T cells, according to ABC transporter inhibitors (zosuquidar: MDR1; MK571: MRP-1; KO143: BCRP-1). Statistical analyses: Wilcoxon (A) Friedman (B, C and D) and Spearman (E) (*p<0.05, **p<0.005, ***p<0.0005, ****p<0.0001). HD, healthy donors’; MDR1, multidrug resistance-1; Th, T helper cells; Treg, regulatory T cells. T_CM: T central memory; T_EM:T effector memory; T_EMRA : T effector memory RA⁺; FSC : Forward side scatter

Figure 2

MDR1 expression delineates highly polyfunctional population enriched in Th1.17 and Th17 cells. (A–B) MDR1 expression among Th cell subsets from HD blood according to their expression of CCR6 and CXCR3 (n=11) (A) and RORγT and T-bet (n=12) (B). (C) MDR1 expression among IFN-γ and IL-17A expressing cells after PMA/ionomycin reactivation in HD blood (n=10) (D) SPICE representation of MDR1⁺ and MDR1^neg Th cell cytokines polyfunctionality (n=10) after PMA/ionomycin reactivation. (E) Representative dot plots of MDR1 expression on purified naive CD4⁺ T cells successively polarized with different Th polarizing cocktails (n=3). (F) MDR1 expression on Th0 and Th17 polarized naive CD4⁺ T cells with more or less TGF-β and IL-6 and in the presence of anti-IL-6R (RoActemra) or TGF-βR inhibitor (galunisertib). Statistical analyses: analysis of ANOVA-2 (A, B and C) and Wilcoxon (D). (**p<0.005, ***p<0.0005, ****p<0.0001). HD, healthy donors’; IFN, interferon; IL, interleukin; MDR1, multidrug resistance-1; Th, T helper cells.

Figure 3

MDR1⁺ Th cells resist to paclitaxel treatment and proliferate favoring an enrichment in Th1.17 and Th17 cells. (A and D) Percentage of viable cells among purified MDR1⁺ and MDR1^neg Th cells according to paclitaxel (A) or cisplatin (D) concentrations and zosuquidar. (B, C, E and F) Percentage (B and E) and MFI (C and F) of MDR1⁺ cells after treatment of a physiological mixed Th population (80% MDR1^neg 20% MDR1⁺) with different concentrations of paclitaxel (B and C) or cisplatin (E and F) and zosuquidar. (G–H) Proliferation (assessed by CTY dilution) of purified MDR1⁺ and MDR1^neg Th cells according to paclitaxel (G) or cisplatin (H) concentrations and zosuquidar. (I–J) Phenotypic (I) and functional (J) Th cell subset enrichment in a mixed Th population (80% MDR1^neg 20% MDR1⁺) after paclitaxel treatment (n=3 for each experiment). Statistical analyses: analysis of ANOVA-2 (A and D) and Friedman (B, E and J) (*p<0.05, **p<0.005, ***p<0.0005, ****p<0.0001). IFN, interferon; IL, interleukin; MDR1, multidrug resistance-1; Th, T helper cells.

Figure 4

NAC selects functional MDR1⁺ Th enriched in Th1.17 cells and increases IFN-γ⁺+IL-17A⁺ and IL-17A⁺ cells in patients with BC’s blood. (A–F) Comparison of blood samples from HD (n=13), untreated (UT; n=38) or patients with NAC-treated BC (NAC) (n=28): (A–E) Percentages of CD4⁺ T cells (A) memory CD4⁺ T cells (B) MDR1⁺ Th cells (C) Treg (D) and Th cell subsets (E). Proportion of IFN-γ⁺IL-17A^neg, IFN-γ⁺IL-17A⁺, IFN-γ^negIL-17A⁺ and IL-22⁺ cells after PMA/ionomycin reactivation (F). (G–J) Analysis of patients’ blood from Breast-Immun cohort before (T1) and after (T2) NAC. Proportions of memory CD4⁺ T cells (G) MDR1⁺ Th cells (H) Treg and Th cell subsets based on phenotype (I) and IFN-γ⁺IL-17A^neg, IFN-γ⁺IL-17A⁺, IFN-γ^negIL-17A⁺ and IL-22⁺ cells after PMA/ionomycin reactivation (J). Statistical analyses: Kruskal-Wallis (A to D and F) analysis of ANOVA-2 (E) Wilcoxon (G to J). (*p<0.05, **p<0.005, ***p<0.0005, ****p<0.0001). BC, breast cancer; HD, healthy donors’; IFN, interferon; IL, interleukin; MDR1, multidrug resistance-1; NAC, neoadjuvant chemotherapy; Th, T helper cells; Treg, regulatory T cells; UT, untreated.

Figure 5

MDR1⁺ Th are present in BC tumors and selected by NAC. (A–F) Comparison of tumor associated (TA) T-cell subsets on UT or patients with NAC-treated (NAC) BC: Percentage of TA-T cells (A) CD4⁺ TA-T cells (B) MDR1 expression TA-Th cells in frequency and MFI (C) TA-Treg (D) and TA-Th cell subsets (E). Proportion of IFN-γ⁺IL-17A^neg, IFN-γ⁺IL-17A⁺, IFN-γ^negIL-17A⁺ and IL-22⁺ TA-CD4⁺ T cells after PMA/ionomycin reactivation (F). (G) ssGSEA analyses of Th17-selective, Th1.17-selective, (Th17-Th1.17) core, Th1 and Th2 signatures on RNA-sequencing data set on 46 paired tumor samples from Park et al data set (GSE123845). Statistical analyses: Mann-Whitney (A to D and F), ANOVA-2 (E), Kruskal-Wallis (G) (*p<0.05, **p<0.005, ***p<0.0005, ****p<0.0001). BC, breast cancer; HD, healthy donors’; IFN, interferon; IL, interleukin; MDR1, multidrug resistance-1; NAC, neoadjuvant chemotherapy; ssGSEA, single sample gene set enrichment analysis; Th, T helper cells; Treg, regulatory T cells; UT, untreated.

Figure 6

Analysis of ICP and proliferation capacity of MDR1⁺ CD4⁺ Th cells in the NAC-treated breast tumor environment (A) Expression of ICP on MDR1⁺ and MDR1^neg Th cells from NAC-treated breast tumors (n=5). (B) Compared Ki67 expression of Th cells, Tregregulatory T cells and CD8⁺ T cells in the tumor environment of NAC-treated tumors (n=4). (C) CD73 expression on MDR1⁺ and MDR1^neg Th cells from NAC-treated tumors (n=4). (D) Differential expression of CD161 on MDR1⁺ and MDR1^neg Th cells in blood (n=4) and tumor environment (n=5) of patients with NAC-treated BC. BC, breast cancer; MDR1, multidrug resistance-1; NAC, neoadjuvant chemotherapy; Th, T helper cells.

Figure 7

Response to chemotherapy is associated with an increase in MDR1⁺ Th, Th1.17 and Th17 cells in the TMEtumor microenvironment and an increase in IFN-γ⁺+IL-17A⁺ and IL-17A⁺ producing cells in the blood. (A–E) Comparison of TA-T cell subsets from patients with NAC-treated BC according to their response to chemotherapy (response: RCB I-II; no response: RCB III): proportions of TA-T cells (A) CD4⁺ TA-T cells (B) MDR1⁺ cells (C) TA-Treg (D) and TA-Th subsets (phenotypically) (E). F–I) Evolution of blood cell proportions under treatment (ratio (T2/T1) in patients with BC from Breast-Immun cohort, according to their response to NAC: blood sample collection scheme in the Breast-Immun cohort (F). Evolution of the ratio (T2/T1) of MDR1⁺ cells (G) Th1, Th1.17 and Th17 cells (H) (G) and IFN-γ⁺IL-17A^neg, IFN-γ⁺IL-17A⁺, IFN-γ^negIL-17A⁺ after PMA/ionomycin reactivation (I). Statistical analyses: Mann-Whitney (A to E); Kruskal-Wallis (G to I) (*p<0.05, **p<0.005). BC, breast cancer; IFN, interferon; IL, interleukin; MDR1, multidrug resistance-1; NAC, neoadjuvant chemotherapy; PBMC, peripheral blood cells; pCR, pathological complete response; RCB, residual cancer burden; TA, tumor associated; Th, T helper cells.