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. 2015 Nov 10;5(3):e1100791.
doi: 10.1080/2162402X.2015.1100791. eCollection 2016 Mar.

Immune cell dysfunctions in breast cancer patients detected through whole blood multi-parametric flow cytometry assay

E Verronèse  1 , A Delgado  1 , J Valladeau-Guilemond  2 , G Garin  3 , S Guillemaut  3 , O Tredan  4 , I Ray-Coquard  4 , T Bachelot  5 , A N'Kodia  1 , C Bardin-Dit-Courageot  1 , C Rigal  1 , D Pérol  3 , C Caux  6 , C Ménétrier-Caux  6
Affiliations

V体育官网入口 - Immune cell dysfunctions in breast cancer patients detected through whole blood multi-parametric flow cytometry assay

"V体育2025版" E Verronèse et al. Oncoimmunology. .

V体育2025版 - Abstract

Monitoring functional competence of immune cell populations in clinical routine represents a major challenge. We developed a whole-blood assay to monitor functional competence of peripheral innate immune cells including NK cells, dendritic and monocyte cell subsets through their ability to produce specific cytokines after short-term stimulation, detected through intra-cytoplasmic staining and multi-parametric flow-cytometry. A PMA/ionomycin T cell activation assay complemented this analysis. Comparing cohorts of healthy women and breast cancer (BC) patients at different stages, we identified significant functional alteration of circulating immune cells during BC progression prior to initiation of treatment. Of upmost importance, as early as the localized primary tumor (PT) stage, we observed functional alterations in several innate immune populations and T cells i. e. (i) reduced TNFα production by BDCA-1+ DC and non-classical monocytes in response to Type-I IFN, (ii) a strong drop in IFNγ production by NK cells in response to either Type-I IFN or TLR7/8 ligand, and (iii) a coordinated impairment of cytokine (IL-2, IFNγ, IL-21) production by T cell subpopulations. Overall, these alterations are further accentuated according to the stage of the disease in first-line metastatic patients. Finally, whereas we did not detect functional modification of DC subsets in response to TLR7/8 ligand, we highlighted increased IL-12p40 production by monocytes specifically at first relapse (FR). Our results reinforce the importance of monitoring both innate and adaptive immunity to better evaluate dysfunctions in cancer patients and suggest that our whole-blood assay will be useful to monitor response to treatment, particularly for immunotherapeutic strategies. VSports手机版.

Keywords: Breast cancer; immune system; monitoring; multi-parametric flow cytometry; whole blood assay V体育安卓版. .

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Figures

Figure 1.
Figure 1.
11-color flow cytometry gating strategies to assess circulating immune cell functionality. Dot plots represent results obtained after a healthy donor WB stimulation. (A) After short term P/I activation, we analyzed by multi-parametric flow cytometry the ability of γδ T cells (CD3+TCRγ9+), LTCD8+CD45RA+ (CD3+TCRγ9negCD8+CD45RA+), memory LTCD8+ (CD3+TCRγ9negCD8+CD45RAneg), naive CD4+ (CD3+TCRγ9negCD4+CD45RA+) and memory CD4+ T cells (CD3+TCRγ9negCD4+CD45RAneg) to synthetize IFNγ, IL-2, TNFα, IL-21 or IL-17A. (B) The “Innate Immunity” panel allowed the simultaneous identification of NK cells (LINnegHLA-DRnegCD56+), DC subsets (LINnegHLA-DR+) including pDC (BDCA2+CD11cneg), BDCA-3+ mDC (CD11c+BDCA-3high), BDCA-1+ mDC (CD11c+BDCA-1+), monocytes (HLA-DR+Lin+CD11c+CD56neg) including CD14+CD16+/−monocytes and non-classical (nc-monocytes CD14lowCD16+).
Figure 2.
Figure 2.
Evaluation of the functional capacity of innate immune cells upon short-term stimulation by multi-parametric flow cytometry. After 5 h of culture in presence of medium or activators, brefeldin A being added after 1 h, we assessed the functional capacity of different innate immune cells characterized as described in Fig. 1B. Dot plots present cytokine intracytoplasmic staining to evaluate production of IFNγ/TNFα by NK cells or IFNα/TNFα by pDC as well as IL-12p40/TNFα by mDC (BDCA-3+, BDCA-1+) or monocyte (CD14+CD16+/−, CD14lowCD16+) subsets. (A) The efficiency of different activators (TLR7/8 ligand (R848), Type-I IFN (IFNα−2b), TLR3 ligand (poly(I:C)), TLR9 ligand (CpG-B)) was compared to medium condition in whole blood assay. (B) Comparison of results obtained after 5 h of stimulation with R848 (10µg/mL) on whole blood or PBMC from the same donor.
Figure 3.
Figure 3.
(See previous page) Innate immune cell subset functional alterations observed in periphery during breast tumor progression. The functionality of innate immune cells was assessed in WB after TLR7/8 ligand (R848, 10µg/mL) or IFNα2b (1000 IU/mL) stimulation in cohorts of patients with breast cancer at different stage of disease (PT (n = 46), FR (n = 34), SR (n = 20)) and compared to a HD cohort (n = 31) and presented as percentage of cell subset producing a specified cytokine in the different cohorts: (A) percentage of BDCA-3+DC and pDC subsets producing TNFα upon TLR7/8 ligand stimulation, (B) percentage of pDC producing IFNα upon TLR7/8 ligand stimulation, (C) percentage of nc-monocytes, monocytes (CD14+CD16+/−), BDCA-1+DC and BDCA-3+DCs producing IL12p40/70 upon TLR7/8 ligand stimulation, (D) percentage of NK cells producing IFNγ upon TLR7/8 ligand and IFNα-2b stimulations and (E) percentage of nc-monocytes, monocytes (CD14+CD16+/−) and BDCA-1+DC producing TNFα upon IFNα-2b stimulation. *: p value < 0.05, **: p value < 0.01, ***: p value < 0.001.
Figure 4.
Figure 4.
T cell subset functional alterations observed in periphery during breast tumor progression. The functionality of T cell subsets was assessed on WB after short-term polyclonal stimulation (P/I) in presence of brefeldin A in cohorts of patients with breast cancer at different stages of disease (PT (n = 46), FR (n = 34), SR (n = 20)) and compared to a HD cohort (=31) and presented as percentage of cell subsets producing a specified cytokine in the different cohorts: percentage of IL-2 production by CD4+ (A) and CD8+ (B) T cell subsets (CD45RA+ and CD45RAneg), percentage of IFNγ production by CD4+ (C) and CD8+ (D) T cell subsets (CD45RA+ and CD45RAneg) and (E) percentage of IL2 and IFNγ and IL2 and TNFα (F) co-production by CD45RAneg memory CD4+ and CD8+ T cells. *: p value <0.05, **: p value <0.01, ***: p value <0.001, ****: p value <10−4.
Figure 5.
Figure 5.
Modulation of IL-17A and IL-21 production by CD4+ CD45RAneg T cells detected in periphery during breast tumor progression. The capacity of CD4+ CD45RAneg T cells to produce IL-17A or IL-21 was assessed in WB after short-term polyclonal stimulation (P/I) in presence of brefeldin A on cohorts of patients with breast cancers at different stage of disease (PT (n = 46), FR (n = 34), SR (n = 20)) and compared to a HD cohort (n = 31) Results are presented as percentage of CD4+ CD45RAneg T cells producing IL-17A (A) or IL-21 (B) or co-producing IL-21 and TNFα, IL-21 and IFNγ or IL-21 and IL-2 (C). * p value< 0.05, **: p value < 0.01.
Figure 6.
Figure 6.
Characterization of γδ T cell functional alterations in periphery during breast tumor progression. The capacity of γδ T cells to produce IFNγ and TNFα was assessed in WB after short-term polyclonal stimulation (P/I) in presence of brefeldin A in cohorts of patients with breast cancer at different stages of disease (PT (n = 46), FR (n = 34), SR (n = 20)) and compared to a HD cohort (n = 31) and results are presented as percentage of γδ T cells producing IFNγ or TNFα. **:p value < 0.01.
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