MDR1 expression is associated to Th1.17 and Th17 cells and induced on polarization with TGF-β1

Using PBMC from HD, MDR1 was detected on 75% of CD8⁺ T cells while only 20% of CD4⁺ T cells expressed MDR1 (online supplemental figure 1, figure 1A). Among CD8⁺ T cells, MDR1 was expressed by most naive cells but with lower mean fluorescence intensity (MFI) compared with memory cells positive at 75% (online supplemental figure 1). In contrast, on CD4, MDR1 was mostly detected on Th cells but not on FOXP3^high Treg cells or naïve (CD45RA⁺ CCR7⁺) T cells (figure 1B).

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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

Based on CD45RA and CCR7 expression,27 MDR1 was equally distributed among memory CD4⁺ (figure 1C) and CD8⁺ (online supplemental figure 1D) subsets with a slightly higher MFI on the effector memory T cell (T_EM, CD45RA^neg CCR7^neg) population (figure 1D, online supplemental figure 1D). In vitro, the efflux of Rh123, a fluorescent substrate of MDR1, was highly correlated to the expression of MDR1 on CD4⁺ Th cells (figure 1E) and CD8⁺ memory T cells (online supplemental figure 1E) and blocked by the selective MDR1 inhibitor zosuquidar, but not by other ABC transporter blockers (MK571 (MRP-1) and KO143 (BCRP-1))8 28 29 (figure 1F), demonstrating MDR1 functionality on both CD4⁺ and CD8⁺ T cells (online supplemental figure 1F).

Based on chemokine receptors CXCR3 and CCR6,30 we observed MDR1 expression on 58.5±14% of Th1.17 (CXCR3⁺CCR6⁺) and 33.8±10% of Th17 (CXCR3^negCCR6⁺) cells whereas lower proportions were found among Th1 (CXCR3⁺CCR6^neg) (13.5±3%) and other Th (CXCR3^negCCR6^neg) (5.8±2 %) subsets (figure 2A, online supplemental figure 2A). Based on RORγt and T-bet expression, we confirmed the enrichment of MDR1⁺ cells within Th1.17 (RORγt⁺T-bet⁺) and Th17 (RORγt⁺T-bet^neg) cells (figure 2B). Analyzing cytokine production pattern by FC (after PMA/ionomycin reactivation) we confirmed preferential MDR1 expression on IFN-γ⁺IL-17A⁺ (59.5±12 %) and IFN-γ^neg IL-17A⁺ (45.7±9 %) cells. MDR1 was also detected on 26±7% of IFN-γ⁺IL-17A^neg and 16±5% of IFN-γ^negIL-17A^neg cells (figure 2C).

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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.

Boolean cytokine analysis highlighted the higher polyfunctionality of MDR1⁺ CD4⁺ Th cells (figure 2D). Indeed, 50% of MDR1⁺ Th cells produced at least two cytokines among IFN-γ, IL-17A, IL-22 and Tumor necrosis factor (TNF)-α, compared with 32% for the MDR1^neg counterpart. Moreover, MDR1⁺ Th cells produced higher levels of IL-22 (online supplemental figure 2D), very low levels of IL-4 and no IL-13 (online supplemental figure 2B). Consistent with these results, MDR1⁺ Th cells secreted higher levels of Th1.17 cytokines and decreased levels of Th2 cytokines following in vitro T cell receptor (TCR) stimulation (online supplemental figure 2C).

Using in vitro Th polarization assays, we found that one round of polarization of naïve CD4⁺ T cells under Th17 condition (online supplemental material) strongly induced MDR1 expression, whereas the Th1.17 cocktail induced only low MDR1 expression and Th1 and Th0 cocktails did not (figure 2E). Interestingly, Th17 polarized cells maintained MDR1 expression irrespective of the second polarization cocktail used (Th17, Th1 or Th0). This suggests that soluble factors common to Th17 and Th1.17 differentiation cocktails (IL-6, TGF-β1) might participate to MDR1 induction on CD4⁺ T cells but are dispensable for its sustained expression (figure 2E). Blocking TGF-β1 signaling but not IL-6 signaling, completely abolished MDR1 expression (figure 2F), thereby demonstrating the essential role of TGF-β1 during naive CD4⁺ T-cell polarization into MDR1⁺ Th cells. However, exposure to TGF-β1 did not induce MDR1 expression on sorted MDR1^neg Th cells (online supplemental figure 2D), suggesting that the induction of MDR1 expression might happen early after naïve CD4⁺ T-cell priming. It has to be noticed that induced Treg (iTreg) polarization cocktail (TGF-β1, IL-2) also induced the expression of MDR1 on these iTreg cells (online supplemental figure 2E).

MDR1⁺ Th cells present in the breast TME are enriched in Th1.17 cells

Next, we examined the expression of MDR1 on CD4⁺ Th cells in the blood and paired tumors of patients with untreated BC. While the proportions of MDR1⁺ Th cells were similar in the blood and TME (online supplemental figure 3A), MDR1 was expressed at higher levels in the latter (online supplemental figure 3B). The frequency of Th1.17 cells was unchanged between both sites but the frequency of Th1 cells was significantly higher in the TME. In contrast, the proportions of Th17 and other Th subsets were reduced in the TME (online supplemental figure 3C).

To better define the differences between MDR1⁺ and MDR1^neg Th cells in the TME, we performed a transcriptome analysis of those two subsets isolated from untreated (UT) BC tumors (online supplemental figure 4A,B). Our analysis identified 157 DEG segregating MDR1⁺ from MDR1^neg Th cells (online supplemental figure 4C). Among the 104 upregulated genes in MDR1⁺ subset were genes such as CCR6, CTSH, CCL20, RORC and IL-17A previously described in the core (Th17-Th1.17) common transcriptomic signature9 and others such as SLC4A10, ADAM23, LTK, IL4I1, KIT, IL23R, IL18RAP and DPP4 characteristics of a Th1.17 gene signature,9 31 further supporting the Th1.17 polarization of MDR1⁺ Th cells in the TME. We confirmed by real-time (RT)-qPCR the specific enrichment of RORC, IL4I1, CEBPD and LTK in sorted MDR1⁺ Th1.17 (online supplemental figure 4D). Accordingly, an enrichment in Th1.17 specific as well as core (Th17-Th1.17) common transcriptomic signatures but not Th17-specific,9 31 Th1 and Th2 signatures32 was also identified by ssGSEA in MDR1⁺ TA-Th cells (online supplemental figure 4E).

We also investigated the expression of ABCB1 gene transcripts within CD4⁺ T-cell meta-clusters, using the recently published “pan-cancer single-cell landscape of tumor-infiltrating T cells” data set by Zheng et al in breast tumor samples from UT patients.24–26 ABCB1 was detected on meta-clusters annotated as c14-Th17-SLC4A10” and “c15-Th17-IL23R”, sharing Th17 and Th1.17 specific genes confirming their Th17 and Th1.17 identity (online supplemental figure 4F) but also on “c13-TEMRA CX3CR1” meta-cluster enriched in characteristic cytotoxic genes previously reported on human CD4⁺ cytotoxic cells, including hallmark features such as granzymes and granulysin (for review33) (online supplemental figure 4G).

MDR1⁺ Th cells resist to paclitaxel treatment in vitro leading to Th17 and Th1.17 cell enrichment

To investigate whether MDR1 expression conferred CD4⁺ Th cells with resistance to chemotherapy, HD blood CD4⁺ Th cells were FACS-sorted based on their Rh123 efflux capacity. Purified Rh123^neg (MDR1⁺) and Rh123⁺ (MDR1^neg) Th cells were stimulated with TCR/CD28 signal and cultured 4 days with increasing doses of chemotherapy drugs. Paclitaxel (MDR1 substrate) treatment, did not alter the viability of MDR1⁺ Th cells whereas it strongly reduced, in a dose-dependent manner, that of MDR1^neg Th cells (figure 3A). As a result, when MDR1^neg and MDR1⁺ Th cells were mixed together at a physiological ratio (mix 80/20), paclitaxel treatment led to a dose-dependent enrichment of MDR1⁺ cells with higher levels of MDR1 at the end of the culture (figure 3B,C). In contrast, cisplatin (not MDR1 substrate) treatment similarly reduced the viability of both populations (figure 3D) and did not modulate their respective ratio in the mix 80/20 (figure 3E,F). MDR1⁺ Th cells maintained their proliferative capacity in the presence of paclitaxel, in contrast to the MDR1^neg fraction (figure 3G), whereas the proliferation of both subsets was inhibited by cisplatin (figure 3H). Moreover, the addition of zosuquidar restored sensitivity of MDR1⁺ Th cells to paclitaxel, impeding their proliferation as well as their enrichment in the mix 80/20 (figure 3B,C and G). Interestingly, the addition of paclitaxel in a mix (25/75) of purified naïve and memory CD4⁺ T cells blocked the proliferation of the naive compartment devoid in MDR1 whereas it allows that of purified memory T cells containing MDR1⁺ cells (online supplemental figure 5A). Importantly, paclitaxel treatment favored a dose-dependent selection of CXCR3⁺CCR6⁺ Th1.17 population (figure 3I) associated with an increase of cells producing IFN-γ, IL-17A and IL-22 (10-fold increase in IFN-γ⁺IL-17A⁺ cells) (figure 3J). Together, this demonstrates that MDR1 expression provides a survival and proliferation advantage to Th1.17 cells and Th17 cells following exposure to chemotherapy substrate of MDR1.

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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.

NAC treatment selects functional MDR1⁺ Th cells enriched in Th1.17 and Th17 cells in blood and tumor of patients with BC

To evaluate the translation of our observations in vivo, we analyzed blood samples from patients with NAC-treated BC (NAC) at time of surgery (online supplemental table 1). As a control, we analyzed samples from patients with UT BC (online supplemental table 1) with SBR 2/3 grade, as well as from age-matched and sex-matched HD. While the proportion of CD3⁺ T cells was stable (online supplemental figure 6A), NAC treatment reduced the proportion of CD4⁺ T cells (figure 4A), accompanied by an increased frequency of memory CD4⁺ T cells compared with UT patients and HD (figure 4B) and a higher proportion of MDR1⁺ cells (figure 4C). Proportions of CD8⁺ T cells among total T cells were increased in NAC-treated patients (online supplemental figure 6B), in accordance with the higher frequency of MDR1⁺ cells in CD8⁺ T cells. We also observed a slight decrease in Treg cells frequency (figure 4D) and an increase in MDR1⁺ Th cells proportion (figure 4D), with a particular increase of central memory T cells (T_CM) (online supplemental figure 6C). Within the memory CD4⁺ Th cells, the proportions of Th1.17 and Th17 cells were significantly increased in NAC-treated patients whereas Th1 and other Th cells decreased (figure 4E,F, online supplemental figure 6D,E).

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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.

To ascertain the direct impact of NAC treatment on these observations, we performed similar analyses on paired frozen PBMC samples obtained before and after the second cycle of chemotherapy with anthracyclines in patients enrolled in the Breast-Immun cohort (online supplemental table 1). We confirmed the selective enrichment of MDR1⁺ Th cells post-chemotherapy (figure 4G,H), associated with a decrease in Treg cell frequency (figure 4I). Moreover, we confirmed the increase in Th1.17 and Th17 cells proportions among MDR1⁺CD4⁺ Th cells using both chemokine receptors expression (figure 4I) and cytokine production (figure 4J) whereas the proportion of Th1 cells was reduced. All other major modulations observed in the prospective cohort were confirmed (online supplemental figure 6F–H). Altogether, these results demonstrate that NAC treatment of patients with BC with an MDR1 substrate reshapes the blood CD4⁺ T-cell compartment with an enrichment in Th1.17 and Th17 cells.

To determine if the alterations of the CD4⁺ Th cell compartment in the periphery could also be detected at the tumor site, we analyzed the tumor infiltrate of UT-treated and NAC-treated patients. Similar to the blood, there were no changes in the proportions of total T cell between UT-treated and NAC-treated patients (figure 5A). However, while there was a 25% reduction in the proportion of CD4⁺ among tumorassociated (TA)-T cells in NAC-treated tumors (figure 5B), the frequency of MDR1⁺ TA-Th cells as well as its MFI were increased by 1.8-fold in NAC-treated tumors (figure 5C). We also observed a reduction in the frequency of TA-Treg cells (figure 5D). This correlated with a strong enrichment in Th1.17 cells (75% increase) and in cells co-producing IFN-γ and IL-17A or producing IL-17A only (respectively, 5.24-fold and 6.62-fold) and a decrease in Th1 cells (25% reduction) (figure 5E,F).

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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).21 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.

Using a recent public RNA-seq data set21 of BC tumor paired biopsies collected before (T1) and after one cycle (T2) of treatment with anthracycline/CTX, we further investigated the direct impact of NAC treatment on the enrichment in the different Th signatures in the TME. NAC treatment induced an enrichment in Th1.17 and Th17 selective and core (Th17-Th1.17) signatures but not Th1 and Th2 ones (figure 5G) in adequacy with results in the Breast-Immun cohort (figure 3I,J).

Using STM and a sensitive multiplex electrochemiluminescence method (online supplemental material), we detected both IFN-γ and IL-17A in a higher proportion (26.3%, 5/19) of NAC-treated tumors compared with UT ones (16.6%, 8/48), IFN-γ alone was also increased with, respectively, 47.36% and 37.5% whereas IL-17A alone was never detected (online supplemental figure 7A).

In NAC non-responding patients, tumor infiltrating MDR1⁺ Th cells do not present signs of high activation

Using frozen tumor cell suspensions of five NAC-treated patients with available fresh material at surgery but who did not respond to treatment, we highlighted, by FC, lower levels of Programmed cell Death protein1 (PD-1), CD39 and CD38 on MDR1⁺ Th cells compared with MDR1^neg Th cells whereas no significant expression of OX40 and ICOS was found independently of their MDR1 phenotype (figure 6A). Low proliferation was observed on both Th cell populations (figure 6A) that strongly contrasted with high proliferation observed on Treg cells (figure 6B). This is in adequacy with scRNAseq public data set analysis (online supplemental figure 4F) demonstrating that ABCB1-enriched meta-clusters did not express genes coding for proliferation or markers of activation or exhaustion except PDCD1 in contrast to Treg meta-clusters (c20 and c21) (online supplemental figure 4F). In contrast, the c13 meta-cluster, also associated to ABCB1 expression, contained a high number of features associated with cytotoxic CD4⁺ T cells33 (online supplemental figure 4G).

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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.

In addition, an scRNAseq analysis of patients with BC’s blood CD4⁺ memory T cells identified two clusters (c8, c9) enriched with cytotoxic features. Both clusters expressed PRF1, GNLY and NKG7. In addition, the cluster 9 expressed GZMK and KLRK1 but not GZMB, whereas the cluster 8 expressed GZMB and GZMH but low levels of GZMK and not KLRK1. ABCB1 was found selectively enriched in the cluster 9 (online supplemental figure 8A–D). Moreover, the comparison at the messenger RNA (mRNA) level of blood (scRNAseq) and tumor-associated (RNA-seq) MDR1⁺ Th cells (online supplemental figure 8E), highlighted the expression of KLRB1 gene (coding for CD161) specifically on blood MDR1⁺ Th cells. This was confirmed at the protein level, MDR1⁺ Th cells from either HD or patients with NAC-treated BC’s blood expressing high proportion of CD161 (online supplemental figure 2F, figure 7C), previously described on Th1.17 cells9 but this expression was sharply reduced in the TME from NAC-treated patients suggesting they received specific signal in the TME (figure 6C).

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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.

Only two genes were found shared by blood and tumor MDR1⁺ T cells, including PDE4D coding for the phosphodiesterase that degrades cyclic AMP.34 This could be connected to their higher expression levels of CD73 producing Ado that signals through AdoR and adenylate cyclase (figure 6D).

Clinical response to NAC treatment is associated with an increase in MDR1⁺ Th cells, Th1.17 and Th17 cells in both TME and blood

In light of these results, we investigated the possible association between TA-Th subsets composition and clinical response to NAC therapy. Using RCB, as a measurement of response to NAC treatment,35 patients were segregated into responders (RCB-I/RCB-II), and non-responders (RCB III). Of note, patients with pCR could not be included in this analysis due to the absence of remaining tumor tissue. The proportion of CD4⁺ TA-T cells among total TA-T cells was strongly reduced (by two-fold) in responding patients (figure 7A,B) and a reduction even though not significant was also observed for TA-Treg cells (figure 7C). Interestingly, MDR1⁺ TA-memory CD4⁺ T cells, Th1.17 and Th17 cells, but not Th1 cells, were significantly enriched in responding patients (figure 7D,E).

Finally, we assessed whether the treatment-induced changes in blood CD4⁺ Th cell composition were associated with response to chemotherapy. Within the Breast-Immun cohort (online supplemental table 1, figure 7F) we highlighted, after two cycles of chemotherapy, a significant T2/T1 fold-change increase in MDR1⁺ Th cells (figure 7G) but also in Th1.17 and Th17 cells (figure 7H) according to RCB. In contrast, no differences were observed for Th1 cells. Moreover T2/T1 fold-change of Th cells producing IFN-γ only, IFN-γ/IL-17A and IL-17A only (figure 7I) were also increased in patients with RCB-0/pCR compared with those with RCB-III, values for RCB-I/II being intermediate.