Eomesodermin-expressing CD4+ Th cells and association with pregnancy in multiple sclerosis

Abstract

Background: Pregnancy in patients with multiple sclerosis (MS) is accompanied by a decline of relapse activity with increased risk of relapses 3 months post-partum, for unknown reasons. Eomesodermin⁺ T-helper cells (Eomes⁺ Th cells) are known to mediate neuroinflammation and disease progression in MS and are induced by prolactin-secreting cells.

Objectives: Here, investigated immune cell alterations and the pathophysiological role of Eomes⁺ Th cells for disease activity during pregnancy and post-partum in MS.

Methods: We enrolled n = 81 pregnant patients with relapsing-remitting MS (RRMS), n = 27 post-partum RRMS and n = 26 female RRMS control patients under the umbrella of the German Multiple Sclerosis and Pregnancy Registry. Clinical data were collected and immune cell alterations were analysed using flow cytometry.

Results: While CD3⁺CD4⁺ Th cells were unaffected, CD3⁺CD8⁺ cytotoxic T-cells were elevated post-partum (p = 0.02) with reduced B-cell frequencies (p = 0.01) compared to non-pregnant RRMS patients. NK cells were elevated during first trimester (p = 0.02) compared to the third trimester. Frequencies of Eomes⁺ Th and Eomes⁺ Tc cells did not differ. There was no correlation of prolactin release and expression of Eomes⁺ Th cells. However, Eomes⁺ Th cells correlated with lower frequencies of regulatory T-cells during second (r = -0.42; p < 0.05) and third trimester (r = -0.37; p < 0.05). Moreover, Eomes⁺ Th cells correlated with frequencies of B-cells during third trimester (r = 0.54; p = 0.02). Frequencies of Eomes⁺ Th cells were not associated with the number of relapses before pregnancy, during pregnancy or post-partum. However, Eomes⁺ Th cells strongly correlated with disability post-partum as assessed using the EDSS (r = 0.52; p = 0.009).

Discussion: Pregnancy in MS is associated with robust immunological alterations. Eomes⁺ Th cells are capable of inducing immune cell alterations during the course of pregnancy, most evident during the second and third trimester as shown with a correlation of reduced Treg cells and a significant increase of B-cells. Importantly, Eomes⁺ Th cells correlate with disability post-partum. In summary, during late pregnancy in MS an inflammatory, cytotoxic and dysregulated immunological environment is primed gaining function post-delivery. This may be responsible for post-partum disability accumulation.

Keywords: Eomesodermin+ Th cells; multiple sclerosis; pregnancy; progression independent of relapse activity.

Visual Abstract

Figure 1.

Immune cell subsets during pregnancy in MS. (a) RRMS patients were included at different time points before, during or after pregnancy. After written informed consent, demographic clinical data were collected. Blood was drawn in order to analyse immune cell alterations via flow cytometry and concentration of prolactin in serum. Patients were clinically assessed during the course of pregnancy and post-partum. (b) CD4⁺ Th cells did not differ in pregnant women while (c) CD8⁺ Tc cells were elevated during post-partum period compared with non-pregnant RRMS patients. (d) Proportions of CD4⁺CD25⁺Foxp3⁺ regulatory T-cells amongst lymphocytes did not differ. (b–d) Data are derived from pregnant RRMS patients (first trimester: n = 25, second trimester: n = 24, third trimester: n = 32), RRMS patients during post-partum period (n = 24) and RRMS patients with childbearing preferences (n = 26). (e) Patients during first trimester expressed higher levels of NK cells than patients during third trimester (first trimester: n = 21, second trimester: n = 18, third trimester: n = 29, post-partum period: n = 17, non-pregnant RRMS patients: n = 11). (f–j) Immune cell subsets were shown for patients who had not received B-cell depleting therapy during the last 15 months [first trimester (n = 17), second trimester (n = 17), third trimester (n = 18), post-partum period (n = 14), non-pregnant RRMS patients (n = 21)]. (j) B-cells were reduced during post-partum period compared to non-pregnant RRMS patients (p < 0.05). Shown is a mean ± SD for each patient group. Data were analysed using non-parametric Kruskal-Wallis test with post hoc Dunn’s multiple comparison test. *p < 0.05. Source: (a) Created with BioRender.com. MS, multiple sclerosis; RRMS, relapsing-remitting MS.

Figure 2.

Expression of Eomesodermin (Eomes) in Th cells and correlation with prolactin in serum. Frequencies of (a) Eomes⁺ Th cells amongst CD3⁺CD4⁺ Th cells and (b) Eomes⁺ Tc cells amongst CD3⁺CD8⁺ Tc cells did not differ. (c) Prolactin in serum rose continuously during the course of pregnancy and decreased after delivery. Breastfeeding patients are marked in green colour. There was no correlation between prolactin in serum and Eomes⁺ Th cells in (d) non-pregnant RRMS patients, (e) during first trimester, (f) second trimester, (g) third trimester, (h) in breastfeeding patients and (i) non-breastfeeding patients. (a, b) Data are derived from pregnant RRMS patients (first trimester: n = 25, second trimester: n = 24, third trimester: n = 32, RRMS patients during post-partum period: n = 24 and non-pregnant RRMS patients: n = 26). (c–i) Data are derived from pregnant RRMS patients [first trimester: n = 22, second trimester: n = 22, third trimester: n = 28, RRMS patients during post-partum period (breastfeeding: n = 8; no breastfeeding: n = 12) and non-pregnant RRMS patients (n = 17)]. Shown is (a–c) mean ± SD and (d–i) linear regression with 95% confidence interval. Data were analysed using (a–c) non-parametric Kruskal-Wallis test and (d–i) non-parametric Spearman test. *p < 0.05, **p < 0.01, ****p < 0.0001. RRMS, relapsing-remitting MS.

Figure 3.

Correlation of Eomes⁺ Th cells and regulatory T-cells and B-cells. The frequency of Eomes⁺ Th cells and frequencies of regulatory T-cells did neither correlate in (a) non-pregnant RRMS patients nor in (b) pregnant RRMS patients during first trimester and (e) patients during post-partum period. The frequency of Eomes⁺ Th cells and regulatory T-cells correlated negatively in pregnant RRMS patients during (c) second trimester (r = −0.42; p = 0.05) and (d) third trimester (r = −0.37; p = 0.05). Expression of Eomes in Th cells and frequencies of B-cells did not correlate in (f) non-pregnant RRMS patients, pregnant RRMS patients during (g) first trimester, (h) second trimester and (j) patients during post-partum period. Eomes⁺ Th cells and B-cells correlated positively in pregnant RRMS patients during (i) third trimester. (k) Pregnant and post-partum patients with B-cell depleting therapy before pregnancy (n = 32) showed a trend towards lower frequencies of Eomes⁺ Th cells compared to patients with no B-cell depleting therapy before pregnancy (n = 64); p = 0.12. (a–e) Data are derived from pregnant RRMS patients (first trimester: n = 25, second trimester: n = 22, third trimester: n = 30, RRMS patients during post-partum period: n = 20 and RRMS patients with childbearing preferences: n = 26). (f–k) Data are derived from pregnant RRMS patients (first trimester: n = 19, second trimester: n = 18, third trimester: n = 18), RRMS patients during post-partum period: n=15 and RRMS patients with childbearing preferences: n = 21. Patients did not receive B-cell depleting therapy within the last 12 months. Shown is (a–j) linear regression with 95% confidence interval and (k) mean ± SD. Data were analysed using (a–j) non-parametric Spearman test and (k) non-parametric Mann-Whitney U test. Eomes, Eomesodermin; RRMS, relapsing-remitting MS.

Figure 4.

Frequencies of Eomes⁺ Th cells correlate with disability post-partum. (a, b) Eomes⁺ Th cells and (c, d) regulatory T-cells did neither differ between pregnant patients with current relapse activity and pregnant patients without relapse activity nor between pregnant patients with relapse activity after pregnancy and pregnant patients with stable disease activity. Frequencies of Eomes⁺ Th cells and EDSS did neither correlate in (e) non-pregnant RRMS patients nor in pregnant RRMS patients during (f) first trimester, (g) second trimester or (h) third trimester. The frequency of Eomes⁺ Th cells and EDSS strongly positively correlated, however, in (i) post-partum patients (r = 0.52; p = 0.009). (a, c) Date are derived from n = 10 patients with relapse during pregnancy and n = 41 patients without relapse during pregnancy. (b, d) Data are derived from n = 7 patients with relapse post-partum and n = 52 patients without relapse post-partum. (e–i) Data are derived from pregnant RRMS patients [first trimester (n = 25), second trimester (n = 24), third trimester (n = 33)], RRMS patients during post-partum period (n = 24) and RRMS patients with childbearing preferences (n = 26). (a–d) Shown is a mean ± SD for each patient group and (e–i) linear regression with 95% confidence interval. Data were analysed using (a–d) non-parametric Mann-Whitney test and (e–i) non-parametric Spearman test.