Low CD4 + T cell count is related to specific anti-nuclear antibodies, IFNα protein positivity and disease activity in systemic lupus erythematosus pregnancy

This Swedish multicenter study enrolled pregnant women with SLE (n = 80) meeting the 1997 American College of Rheumatology (ACR) and/or the 2012 Systemic Lupus International Collaborating Clinics (SLICC) classification criteria [26, 27] between November 2018 and June 2022 at Rheumatology clinics in: Gothenburg (Sahlgrenska University hospital, n = 24), Stockholm (Karolinska University Hospital, n = 38), Uppsala (Uppsala University Hospital, n = 3), Linköping (Linköping University Hospital, n = 6) and Lund (Skåne University Hospital, n = 9). Healthy pregnant women (HC, n = 51) were enrolled at one antenatal clinic in Gothenburg (Regionhälsan, Gothenburg) between October 2018 and December 2022. Most pregnant women with SLE and HC were included at 10–12 weeks of gestation and followed in the second (week 18–20) and third (week 32–34) trimester. Disease activity was evaluated according to the SLE Disease Activity Index 2000 (SLEDAI-2 K) [28], and measured at least once between week 10 and week 34 and if the disease activity was assessed more than once, the highest score was used in analyses. The number of pregnant women with SLE for whom SLEDAI-2 K assessments were obtained from in each trimester is shown in Supplementary Table 1. Clinical data including disease duration, medication, ever autoantibody positivity, gestational age at birth and giving birth to a small for gestational age (SGA) infant were retrieved from medical records. SGA (n = 13) was defined as birth weight less than the 10th percentile for expected birth weight [29]. Exclusion criteria were inability to understand the study-related patient information and informed consent form, presence of other serious disease, including active cancer and other rheumatic autoimmune diseases, or treatment with anti-BAFF or anti-CD20 antibodies within 12 months before inclusion. Women who had miscarriage during trimester one were excluded. None of the women with SLE were treated with anifrolumab before or during their pregnancy. All participants have given their written informed consent and the Ethics board in Gothenburg (Dnr 404–18) and The Swedish Ethical Review Authority (amendments Dnr 2020–05101 and Dnr 2022–01158-02) have approved the study.

Peripheral blood samples were collected in heparinized tubes from pregnant women with SLE and HC in the first, second and third trimester. One additional blood sample was collected late postpartum from 19 of the women with SLE (at least six months after delivery (median 10 [6,–36] months). Information about the number of blood samples collected for each trimester is presented in Supplementary Table 2. All blood samples were kept at ambient temperature until processed the day after, within 24 h after venipuncture at our laboratory in Gothenburg. Whole blood was used for flow cytometry analysis of total lymphocyte subset counts. Density centrifugation of whole blood was performed to isolate plasma that was kept frozen (-80 °C) until further analysis.

Analysis of positivity for immunofluorescence (IF)-ANA, for ANA fine specificities including antibodies to dsDNA, Sm, RNP, SSA, SSB, chromatin and ribosomal P protein, and for anti-phospholipid antibodies including anti-CL and anti-β₂GPI in plasma collected during pregnancy was performed at the accredited laboratory of Clinical Immunology, Sahlgrenska University Hospital. Most of these samples were collected in trimester three and plasma was diluted 1:1 in PBS. IF-ANA was analyzed using Hep-2 cells according to routine, and 66 out of 80 (83%) women with SLE were positive. ANA fine specificities were analyzed using multiplexed bead technology by the BioPlex™ 2200 System (BioRad Laboratories, Hercules, USA). The cut-off for most ANA-specificities was 1.0 AI (antibody index) except for anti-chromatin (1.5 AI), anti-RNP (3.0 AI) and anti-dsDNA (10 IU/mL). Positive ANA-specificities were confirmed with another method according to the manufacturers recommendation: Crithidia luciliae test for anti-dsDNA (ImmunoConcept, Sacramento, CA), automated ELISA-based test system Alegria® (Orgentec Diagnostics, Mainz, Germany) for anti-SSA52 and line blot ANA Profile 5 IgG for all other ANA-specificities (Euroimmun, Lübeck, Germany). Anti-CL and anti-β₂GPI of IgG isotype were examined using the BioPlex™ 2200 multiplex immunoassay system and APLS reagents. Cut-off values for positivity were 20 GPL for aCL IgG and 20 AU/mL for anti-β2GPI IgG as recommended by the manufacturer. Ever autoantibody status was obtained from medical records and included positivity for anti-dsDNA/Sm/SSA/SSB, anti-CL/β₂GPI and lupus anticoagulant (LAC). The method for analysis of ever antibody positivity differed between the study sites and dsDNA was confirmed by either Crithidia luciliae test or ELISA. Positivity was determined according to cut-off levels at the local laboratories.

TruCount™ assay was used to analyze total number of lymphocytes, CD4 + T cells, CD8 + T cells, B cells and NK cells in whole blood. In brief, blood and antibodies against CD45, CD3, CD4, CD8, CD19, CD20 and CD56 (Supplementary Table 3) were added to BD TruCount™ tubes (BD Bioscience) and incubated for 15 min. Red blood cells were then lysed using BD FACS™ Lysing solution (BD Sciences). All samples were acquired in a FACSVerse equipped with FACSuite Software (BD Biosciences) and analyzed with FlowJo Software (TreeStar, Ashland, Oregon, USA).

The concentration of IFNα protein levels in plasma diluted 1:1 in PBS was quantified with Single molecule array (Simoa) digital ELISA on a HD-X Analyzer (Quanterix, Billerica, MA). To prevent false positive results the Simoa assay contained an inhibitor for heterophilic antibodies. If the concentration in a sample was below the lower limit of quantification (70 fg/ml) its value was adjusted to 35 fg/ml. IFNα positivity was defined as protein levels ≥ 136 fg/ml, representing three standard deviations above mean IFNα protein concentration among healthy blood donors [30].

Demographic and clinical data of the cohort are shown in Table 1. The data presented, except for the cross-sectional analysis of autoantibody positivity, have been previously described for subsets of both patients and controls [24]. In brief, the age and percentage of nulliparous women were similar in SLE and HC. For pregnant women with SLE, the median disease activity according to SLEDAI-2 K was 2 during pregnancy. Disease activity did not vary much between the trimesters (Supplementary Fig. 1A-B). For all patients with moderate/high disease activity (SLEDAI-2 K ≥ 4), the components that contributed to the score were due to the disease per se and not to pregnancy, and the most common features were increase in anti-dsDNA, low complement, and arthritis. Historically, all except one were ever ANA-positive by immunofluorescence and the majority were ever anti-dsDNA positive. In the cross-sectional analysis of autoantibody positivity during pregnancy, 71% were positive for at least one of the ANA fine specificities assessed. In line with previous findings [19, 20], the percentage of positive women was mostly lower in cross-sectional compared to ever positive analysis: anti-dsDNA (36% vs. 84%), anti-Sm (15% vs 25%), anti-SSB (10% vs 14%), anti-CL (8% vs 16%) and anti-β2GPI (9% vs 20%). Moreover, 30% were positive for anti-chromatin, 23% for anti-Sm/RNP, 14% for anti-RNP, and 3% for anti-Ribosomal P during pregnancy. Most women with SLE were treated with hydroxychloroquine (93%) and acetylsalicylic acid (88%) during pregnancy, while one third or less were treated with prednisone (33%), azathioprine (29%) and/or low molecular weight heparin (24%).

We first examined total numbers of circulating lymphocytes and the lymphocyte subsets CD4 + T cells, CD8 + T cells, B cells and NK cells in pregnant women with SLE and HC. A representative gating strategy for the different lymphocyte subsets in SLE and HC is presented in Fig. 1A. Late postpartum samples from pregnant women with SLE were analyzed to determine if potential differences in lymphocyte subset numbers were an effect of SLE combined with pregnancy or to SLE per se. Neither total lymphocyte count, nor the subsets CD4 + T cells, CD8 + T cells, B cells and NK cells differed significantly between trimesters or compared to late postpartum in SLE (Fig. 1B-F). Similar results were observed when only including data of women from whom late postpartum samples were collected (Supplementary Fig. 2A-E). However, total lymphocyte count and all subsets were significantly lower in SLE compared to HC throughout pregnancy (Fig. 1G-K, combined data from trimester one to three). The comparisons of cell counts between pregnant women with SLE and pregnant HC for each trimester separately are shown in Supplementary Fig. 3A-E. We also examined whether treatment related to lymphocyte subset counts in pregnant women with SLE. OPLS analysis indicated an inverse relation between azathioprine treatment and numbers of NK cells and B cells in all trimesters (Fig. 2A). Prednisone and low molecular weight heparin related inversely to B cell counts in all trimesters (Fig. 2B-C). These associations were corroborated in univariate analysis. NK cell and B cell counts were significantly lower in women treated compared to not treated with azathioprine (Fig. 2D-E). B cell counts were also lower in women treated compared to not treated with prednisone or heparin, respectively (Fig. 2F-G). Still, significantly lower numbers of both NK cells and B cells were found in women without respective treatment compared to HC (Fig. 2D-G). Treatment with hydroxychloroquine and acetylsalicylic acid were unrelated to lymphocyte subset counts (Supplementary Fig. 4A-B). In summary, pregnant women with SLE present with lower number of lymphocyte subset counts in blood relative to pregnant HC, which seem to be a feature of SLE per se and in part by treatment with immunosuppressive drugs and heparin but not related to pregnancy.

We next performed PCA to investigate how CD4 + T cell, CD8 + T cell, B cell and NK cell counts relate to IFNα protein levels, autoantibody positivity during pregnancy, as well as to SLEDAI-2 K, gestational age at birth and SGA in SLE. We previously reported that plasma IFNα protein positivity is present in a subgroup (36%) of pregnant women with SLE [24]. As shown in Fig. 3, lymphocyte subset counts were projected on the opposite side to positivity for ANA specificities, IFNα protein levels and SLEDAI-2 K indicating on an inverse association. ANA positivity for anti-dsDNA/Sm/RNP/chromatin (cluster one) separated from anti-SSA/SSB positivity (cluster two), suggesting that these clusters relate differently to lymphocyte subset numbers. IFNα protein levels associated with cluster two, while SLEDAI-2 K projected close to anti-dsDNA in cluster one. Antiphospholipid antibody positivity (anti-CL/β2GPI, cluster three) separated from the other variables on the bottom right side of the plot.

Guided by the PCA analysis, we first investigated the association between lymphocyte subset counts and the two ANA clusters. As shown in Fig. 4A, CD4 + T cell counts were significantly lower in women positive to all ANA specificities in cluster one compared to those who were negative to respective ANA specificity. CD8 + T cell count was lower among women positive for anti-Sm, anti-RNP and anti-chromatin compared to those who were negative (Fig. 4B). B cell numbers were significantly lower in women positive for disease specific anti-dsDNA and anti-Sm compared to those who were negative (Fig. 4C). NK cell numbers were lower in women positive for all ANA in cluster one, except for anti-dsDNA, compared to those who were negative (Fig. 4D). Lymphocyte subset counts did not differ in women positive for anti-SSA or anti-SSA/SSB compared to those who were negative (Supplementary Fig. 5A-D), and none of the women were anti-SSB + /SSA-. Additionally, all lymphocyte subset counts were lower in women positive for three to five ANA compared to those who had one to two ANA and to those who were ANA negative (Fig. 4E-H). The CD4 + T cell counts were also lower in women who had one to two ANA compared to those who were negative (Fig. 4E). Few women were positive for aPL during pregnancy (anti-CL n = 6 and anti-β2GPI n = 7), and there were no significant differences in lymphocyte subset counts in women who were aPL-positive compared to negative except for NK cells counts that were higher in women with compared to without aPL (Supplementary Fig. 5E-H). In summary, lymphocyte subset counts are lower in women positive compared to negative for anti-dsDNA, anti-Sm, RNP, Sm/RNP and chromatin, while low counts are unrelated to positivity for anti-SSA/SSB and aPL in SLE pregnancies.

Next, we examined the association between lymphocyte subsets counts and disease activity in SLE pregnancies. Both CD4 + T cell and B cell counts were lower in women with moderate/high disease activity (SLEDAI-2 K ≥ 4) compared to those with no/low disease activity, while there was no difference in numbers of CD8 + T cells and NK cells between the two groups (Fig. 5A-D). Similar results were obtained when lymphocyte subset counts for each trimester were analyzed in relation to SLEDAI-2 K for the respective trimester (Supplementary Fig. 6A-C). As expected, SLEDAI-2 K projected close to anti-dsDNA positivity in the PCA analysis, and disease activity was higher in women positive for anti-dsDNA compared to those who were negative (Supplementary Fig. 7A). SLEDAI-2 K was unrelated to other ANA fine specificities in cluster one and to number of positive ANA (Supplementary Fig. 7B-F). We have previously reported that higher proportions of low-density granulocytes in late SLE pregnancy correlate to lower gestational age at birth [24], but lymphocyte subset counts were unrelated to pregnancy duration and to giving birth to an SGA infant in women with SLE (Supplementary Fig. 8A-B and 9A-D). To conclude, pregnant women with SLE who have a moderate/high disease activity have lower circulating numbers of CD4 + T cells and B cells compared to women with no/low disease activity, but low lymphocyte subset counts do not predict shorter pregnancy duration in SLE.

In PCA, there was an inverse association between lymphocyte subset numbers and IFNα protein levels. In univariate analysis, CD4 + T cell counts inversely correlated with IFNα protein levels during SLE pregnancy (Fig. 6A), and CD4 + T cell counts were significantly lower in IFNα protein-positive compared to negative women (Fig. 6B). Numbers of CD8 + T cells showed a weaker negative correlation to IFNα protein levels and there was no difference in CD8 + T cell count between IFNα protein-positive compared to negative women (Fig. 6C-D). B cell and NK cell counts were unrelated to IFNα protein levels (Supplementary Fig. 10A-B). As previously demonstrated by others [31‐33], IFNα protein levels related to number of positive ANA specificities (Supplementary Fig. 10C). Thus, pregnant women with SLE who are IFNα-positive present with lower numbers of CD4 + T cells in blood compared to those who are IFNα-negative.