Contributions
Abstract: EP697
Type: E-Poster Presentation
Session title: Enzymopathies, membranopathies and other anemias
Background
Warm autoimmune hemolytic anemia (wAIHA) is a rare acquired autoimmune disease mediated by antibodies targeting red blood cells. Until now, the involvement of T helper (Th) lymphocytes has been scarcely studied in humans, most of the conclusions being extrapolated from animal models. Although still debated, a skewing to a pro-inflammatory Th17 polarization seems to be associated with AIHA. On the other hand, a quantitative regulatory T cell (Treg) deficiency has been shown in one study, but their suppressive functions were not assessed. Importantly, the balance between the pro- and anti-inflammatory T cell responses has never been measured simultaneously.
Aims
Assuming that an imbalance between the pro- and the anti-inflammatory T cell responses could participate to AHAI pathogenesis, we quantified the different circulating helper T cell (Th) subsets, Tregs and assessed their functional capabilities and their transcriptomic profiles.
Methods
Newly diagnosed wAIHA patients (n=20) were compared to sex- and age-matched healthy donors (n=30). Cells were quantified at baseline by flow cytometry, and polarization was determined by intracellular cytokine staining after stimulation with PMA and ionomycin. Treg functions were evaluated by their capability to inhibit effector T cell proliferation. Cytokines were measured by luminex assays in culture supernatants and sera. RNA sequencing was performed on Treg isolated from 4 patients and 4 controls by fluorescence-activated cell sorting (CD4+CD25HiCD127-).
Results
AHAI patients have a decrease in circulating Treg (CD4+CD25HiFoxp3+, 3.20% vs. 4.45%, p=0.008), mostly affecting activated Treg (CD4+Foxp3hiCD45RA-), known to arbor high suppressive capabilities. Moreover, Treg have a functional deficiency in AIHA, as shown by their lower ability to inhibit T cell proliferation (51% vs. 73%, p=0.03) associated to a trend to a lower inhibition of the secretion of pro-inflammatory cytokines (TNF-α, IFN-γ and IL-2) measured in coculture supernatants. Treg transcriptomic revealed an activated profile, as supported by an engagement in transcriptional and translational processes, an activation of TCR and IL-2 signaling, and an increased expression of CTLA-4 mRNA. Interestingly, TNF signaling pathway was also engaged, probably due to the increase in TNF-α in the sera of patients (2.29 vs. 1.24 pg/ml, p=0.002).
Although, Th polarization assessed by flow cytometry did not differ between patients and controls, the production of IL-17 by effector T cells measured in supernatants was higher during AHAI. Considering the Th17/Treg ratio, we observed an unbalance (0.023 vs. 0.01, p=0.01) of the pro-inflammatory over the anti-inflammatory T cell response during AIHA.
Conclusion
This work is the first to provide a concomitant evaluation of effector and regulatory T cells in human AIHA, showing an imbalance in the Th17/Treg ratio, associated with a quantitative and functional deficiency of Treg. Most interestingly, our results pointed out TNF-α as an interesting cytokine to target as its concentration is elevated in the sera and could participate to the inhibition of Treg functions.
Keyword(s): Autoimmune hemolytic anemia (AIHA), Regulatory T cell, T cell response
Abstract: EP697
Type: E-Poster Presentation
Session title: Enzymopathies, membranopathies and other anemias
Background
Warm autoimmune hemolytic anemia (wAIHA) is a rare acquired autoimmune disease mediated by antibodies targeting red blood cells. Until now, the involvement of T helper (Th) lymphocytes has been scarcely studied in humans, most of the conclusions being extrapolated from animal models. Although still debated, a skewing to a pro-inflammatory Th17 polarization seems to be associated with AIHA. On the other hand, a quantitative regulatory T cell (Treg) deficiency has been shown in one study, but their suppressive functions were not assessed. Importantly, the balance between the pro- and anti-inflammatory T cell responses has never been measured simultaneously.
Aims
Assuming that an imbalance between the pro- and the anti-inflammatory T cell responses could participate to AHAI pathogenesis, we quantified the different circulating helper T cell (Th) subsets, Tregs and assessed their functional capabilities and their transcriptomic profiles.
Methods
Newly diagnosed wAIHA patients (n=20) were compared to sex- and age-matched healthy donors (n=30). Cells were quantified at baseline by flow cytometry, and polarization was determined by intracellular cytokine staining after stimulation with PMA and ionomycin. Treg functions were evaluated by their capability to inhibit effector T cell proliferation. Cytokines were measured by luminex assays in culture supernatants and sera. RNA sequencing was performed on Treg isolated from 4 patients and 4 controls by fluorescence-activated cell sorting (CD4+CD25HiCD127-).
Results
AHAI patients have a decrease in circulating Treg (CD4+CD25HiFoxp3+, 3.20% vs. 4.45%, p=0.008), mostly affecting activated Treg (CD4+Foxp3hiCD45RA-), known to arbor high suppressive capabilities. Moreover, Treg have a functional deficiency in AIHA, as shown by their lower ability to inhibit T cell proliferation (51% vs. 73%, p=0.03) associated to a trend to a lower inhibition of the secretion of pro-inflammatory cytokines (TNF-α, IFN-γ and IL-2) measured in coculture supernatants. Treg transcriptomic revealed an activated profile, as supported by an engagement in transcriptional and translational processes, an activation of TCR and IL-2 signaling, and an increased expression of CTLA-4 mRNA. Interestingly, TNF signaling pathway was also engaged, probably due to the increase in TNF-α in the sera of patients (2.29 vs. 1.24 pg/ml, p=0.002).
Although, Th polarization assessed by flow cytometry did not differ between patients and controls, the production of IL-17 by effector T cells measured in supernatants was higher during AHAI. Considering the Th17/Treg ratio, we observed an unbalance (0.023 vs. 0.01, p=0.01) of the pro-inflammatory over the anti-inflammatory T cell response during AIHA.
Conclusion
This work is the first to provide a concomitant evaluation of effector and regulatory T cells in human AIHA, showing an imbalance in the Th17/Treg ratio, associated with a quantitative and functional deficiency of Treg. Most interestingly, our results pointed out TNF-α as an interesting cytokine to target as its concentration is elevated in the sera and could participate to the inhibition of Treg functions.
Keyword(s): Autoimmune hemolytic anemia (AIHA), Regulatory T cell, T cell response