Introduction Anti-tumor necrosis factor (TNF) agents were the first biologic agents to demonstrate efficacy in achieving and maintaining steroid-free remission in moderate to severe Crohn's Disease (CD) and Ulcerative Colitis (UC). Combination therapy with an anti-TNF agent and an immunomodulator is often used to prevent the development of neutralizing anti-drug antibodies, raise trough levels of the biologic, and maintain remission1–3 4–11 12–14 15,16
While thiopurines or anti-TNF agents are safe and effective during pregnancy, combination therapy with an anti-TNF agent and an immunomodulator has been associated with an increased risk of common viral and bacterial infections in infants16,17 16 17 7,18
Thiopurine metabolites and some anti-TNF agents are able to cross the placenta17,19,20 17,19 17,19 19 17,21–23 24
Since IFX and ADA are typically cleared within 6–12 months after birth, and since only trace amounts of these agents are transferred in breast milk, we hypothesized that the increased risk of infection at 1 year is due to aberrations in the developing newborn immune system. To discover potential differences in infant immunophenotypes, we performed multiparameter flow cytometry on infants at 1 year of age and stratified by drug exposure. For B-cell populations that appeared altered after exposure to combination therapy, we also analyzed WT and TNF-deficient mice to determine if these differences could be explained by TNF-deficiency during development.
Materials and methods Study design, participants, and procedures The study patients were recruited from the Crohn's and Colitis Foundation PIANO registry, a prospective, observational, multicenter registry of pregnant patients with IBD initiated in 20076 6 TM Plastic Blood Collection Tubes with K2 EDTA. Blood was shipped at room temperature to the UCSF Core Immunology Laboratory. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood using SepMate Lymphoprep (StemCell Technologies) density centrifugation. PBMCs were removed, washed once with phosphate-buffered saline (PBS) then treated with ACK (Ammonium-Chloride-Potassium) Lysing Buffer (Lonza Walkersville, Inc.) to remove red blood cell contamination. PBMCs were then viably frozen in heat-inactivated fetal bovine serum containing 10% dimethylsulfoxide and maintained at -190°C. PBMC were later thawed, counted and analyzed in a single experiment by multiparameter flow cytometry as described below.
We recruited 22 infants and divided them into five groups according to drug exposure between conception and delivery: no exposure to anti-TNF or thiopurines (unexposed, n = 1), CZP (CZP monotherapy, n = 4), IFX/ADA (IFX or ADA monotherapy, n = 11), IFX/ADA + IM (infliximab or adalimumab plus an immunomodulator, n = 4), and CZP + IM (CZP plus an immunomodulator, n = 2) (Table 1 ). The one “unexposed” infant and two CZP + IM infants are shown for reference but were not included in any of the analyses. For each cell subset, our primary analysis was a three-way comparison (CZP versus IFX/ADA versus IFX/ADA + IM). Since acute otitis media (AOM) is common, we reported episodes of infection as “Any” for any infection and “Not AOM”. Other common infections included upper respiratory infections including respiratory syncytial virus and pneumonia. The study was approved by the Institutional Review Board at each participating institution.
Table 1: Demographic and basic clinical data for maternal and infant study participants stratified by drug exposure
Mice TNF-/- and wild-type C57BL/6 J mice were obtained from Jackson Laboratory and have been previously described. All mice used in this study were housed and bred in a specific pathogen-free facility according to the IACUC guidelines of UCSF.
Antibodies and reagents Supplementary Table 1 summarizes the fluorochromeconjugated antibodies used for human immunophenotyping. The following fluorochrome-conjugated antibodies were used for immunophenotyping mouse splenocytes and lymph node mononuclear cells: Phycoerythrin (PE)-Cy7-conjugated anti-B220 (clone RA3–6B2), allophycocyanin (APC)-conjugated anti-CD138 (clone 281–2), peridinin chlorophyll (PerCP)-Cy5.5-conjugated anti-Gr-1 (clone RB6–8C5), fluorescein isothiocyanate (FITC)-conjugated anti-IgD (clone AMS9.1), and PE-Cy7-conjugated anti-CD95 (clone Jo2) were from BD; PE-conjugated anti-CD93 (clone AA4.1) and PerCP-Cy5.5-conjuagted anti-CD3 (clone 145–2C11) were from eBioscience; Pacific Blue-conjugated anti-I-A/I-E (clone M5/114.15.2), Pacific Blue-conjugated anti-B220 (clone RA3–6B2), Alexa®647-conjugated anti-CD73 (clone TY/11.8), and PE-conjugated anti-CD38 (clone 90) were from Biolegend; and LIVE/DEADTM Fixable Yellow Dead Cell Stain was from Invitrogen.
Flow cytometry Infant PBMC samples were analyzed by the UCSF Core Immunology Laboratory in batch experiments using cryopreserved PBMCs. PBMCs were plated in a 96-well v-bottom plate and stained for 30 min at 4°C, then washed once with FACS buffer (PBS containing 0.5% bovine serum albumin and 1 mM Ethylenediaminetetraacetic Acid). Cells were then fixed in 0.5% formaldehyde and data was acquired on a customized 4-laser BD LSR II Flow cytometer (BD Biosciences), with ≥50,000 lymphocytes collected for each sample. CPT beads (BD Bioscience) were used for instrument set up. CD19+ B cells, CD3+CD4+ T cells and CD3+CD8+ T cells were defined after standard lymphocyte, singlet, and dead cell exclusion gates were applied to the data. FMO controls were used to define positive gates for expression of IgD, IgM, CD38 and HLA-DR on B cells, CCR7, CCR5, CD38, CD57, HLA-DR, and PD-1 on CD3+CD4+ and CD3+CD8+ T-cell populations and CD127, CD25, and FoxP3 on Tregs . To assess T-cell functional responses after thawing, PBMCs were initially rested in complete Roswell Park Memorial Institute (RPMI) medium containing 10% heat-inactivated fetal bovine serum and 10 ug/mL of DNase I (Sigma-Aldrich) for 2 h at 37 °C in a 5% CO2 incubator. PBMCs were then counted using Guava ViaCount Reagent on a BD Accuri flow cytometer. One million PBMCs were plated per well into a 96-well U-bottom plate and either cultured in complete RPMI alone (unstimulated control) or stimulated using plate-bound anti-CD3 (clone UCHT1) plus soluble anti-CD28. PBMCs were stimulated overnight at 37 °C in a 5% CO2 incubator in the presence of brefeldin A and monensin to inhibit cytokine secretion. Approximately 16 h after stimulation, PBMC were harvested, washed once in FACS buffer and stained for cell surface markers at room temperature for 15 min in a 96-well V-bottom plate. Cells were then washed twice, fixed and permeabilized with BD Cytofix/Cytoperm buffer for 20 min at 4 °C. Cells were washed twice with the BD Perm/Wash Buffer prior to intracellular staining for cytokines at 4 °C for 30 min. Cells were washed twice more with BD Perm/Wash Buffer and fixed a final time in 1% formaldehyde and data was acquired on a customized 4-laser BD LSR II Flow cytometer (BD Biosciences). A FMO control was used to define the positive gate for expression of TNF. Mouse splenocytes and mesenteric lymph nodes were analyzed on an LSR II flow cytometer. Mouse memory B cells were defined as B220+IgD-CD38+CD95+CD73+ and plasmablasts were defined as B220loCD138+CD93+I-A/I-E+25–27
Statistical analysis Demographic and clinical characteristics were summarized by descriptive statistics. Categorical variables were analyzed by Fisher's exact test to determine if there was statistical association between the categorical variable and the groups (excluding the unexposed mother and infant). Non-normally distributed continuous variables were summarized using median and interquartile ranges and were compared using Kruskal-Wallis one-way ANOVA test (excluding the unexposed mother and infant). For the primary three-way analysis comparing CZP versus IFX/ADA versus IFX/ADA + IM, cell population frequencies for each subset were compared using Kruskal-Wallis one-way ANOVA test. Multiple comparisons were then adjusted by controlling the False Discovery Rate (FDR) <10% as previously described by Benjamini and Hochberg28 Tables 2–4 were analyzed simultaneously, but displayed separately for convenience. Spearman Principal Component Analysis (PCA) was performed using XLSTAT. All other statistical analyses were performed using Prism 7.
Table 2: Frequency (%) of infant B-cell populations stratified by drug exposure
Table 3: Frequency (%) of infant CD4+ T-cell populations stratified by drug exposure
Table 4: Frequency (%) of infant CD8+ T-cell populations stratified by drug exposure
Results Study population The study population included 22 women and their infants from the PIANO registry who consented to an infant blood draw at 1 year of age. Baseline demographic and clinical data are shown in Table 1 for both maternal and infant study participants, stratified by drug exposure. Infants exposed to CZP monotherapy provided a “negative control” or “reference” population since CZP is not actively transported across the placenta, it is not present at biologically meaningful levels in breast milk, and CZP exposure is not associated with an increased risk of infection in infants16 Table 1 . None of the infants were preterm, although there were 4 early term infants born between 37 and 38 weeks. There were no statistically significant differences in gestational age, birth weight, or length (Table 1 ). There were no congenital anomalies. There was a statistically significant difference in mode of delivery. C-sections were more frequent in infants exposed to combination therapy, partly due to active perianal disease in some of the mothers. As described above, in the PIANO registry as a whole there was a statistically significant increase in infections at 1 year of age among infants exposed to combination therapy as compared to unexposed infants (Relative Risk (RR) 1.50, CI 1.08–2.09). We did not observe a statistically significant difference in this sub-study in the number of infections in the infants when stratified by exposure, likely due to the small sample size (Table 1 ). With regards to vaccination adherence, 17 infants received the recommended vaccinations except for rotavirus, which is not recommended for babies exposed to anti-TNF in utero, and one infant did not receive the Hepatitis B vaccine. Vaccination history was not available for 5 of the infants. No mothers received prednisone during pregnancy, and only two mothers in the IFX/ADA group were treated with a prednisone taper during the infant's first year of life. Additionally, 17 of 19 infants were breastfed. Data on breastfeeding were not available for 3 infants, one of which was in the IFX/ADA+ IM group.
Globally preserved T-cell and B-cell immune profiles among infants with different drug exposures To generate a more detailed assessment of the adaptive immune system in the infants, we collected blood at 1 year of age and performed multiparameter flow cytometry on B and T lymphocytes. Viability of lymphocytes after freeze/thaw was excellent, ranging from 87 to 99% with a median viability of 97%. Gating strategies are depicted in Supplementary Figure 1 & 2. We analyzed 39 immune cell subsets in total (Tables 2, 3, 4 ). Although the frequencies were analyzed together, they are reported separately as 12 B-cell subsets (Table 2 ), fourteen CD4+ T-cell subsets (Table 3 ), and thirteen CD8+ T-cell subsets (Table 4 ). As shown in Tables 2–4 , the B and T-cell immune profiles for most subsets did not vary significantly among the different drug exposures. Principal component analysis revealed largely overlapping datasets, consistent with overall similarity of the immune profiles despite different drug exposures (Fig. 1a ). Similarly, hierarchical clustering did not clearly cluster infants exposed to combination therapy separate from anti-TNF monotherapy (data not shown). Looking at individual subsets, overall frequencies of total CD19+ B cells, CD4+ T cells, and CD8+ T cells were not significantly altered based on drug exposure (Fig. 1b-e ). CD4+CD25+Foxp3+CD127- Tregs are an important subset of CD4+ T cells that suppress T-cell responses to antigen29 Fig. 1e ). The frequencies of CCR7+CD45RA+ naïve, CCR7-CD45RA+ T effector memory RA (TEMRA), CCR7+CD45RA- central memory (CM), and CCR7-CD45RA- effector memory (EM) CD4+ and CD8+ T cells did not vary significantly among the groups (Tables 3, 4 ). Moreover, markers of terminally differentiated T cells (i.e., PD-1 and CD57) were similar among the groups (Tables 3, 4 ). Similarly, percentages of naïve, switched memory, non-switched memory, and memory effector B cells were preserved regardless of therapeutic exposure (Table 2 ). Overall, lymphocyte subsets appeared to be relatively preserved among infants exposed to anti-TNF monotherapy and combination therapy with anti-TNF agents and immunomodulators.
Fig. 1 Infants exposed to anti-TNF monotherapy or combination therapy with an anti-TNF agent and a thiopurine immunomodulator exhibited similar immune profiles. a:
Spearman's PCA of immunophenotyping and study subjects by drug exposure. Principal components F1, F2, and F3 are shown. Each individual is represented by one data point. The percentage of the variance explained by each principal component is shown on the axis. b -e Median, interquartile ranges, and ranges of frequencies of each cell subset, expressed as a percent of the parent population, are shown. The p -value and q -value displayed in b -e were calculated in the three-group analysis (CZP vs IFX/ADA vs IFX/ADA+ IM)
CD27+ B cells, IgD-CD27+ switched memory B cells, and plasmablasts in infants exposed to combination therapy None of the B-cell subsets reached statistical significance after adjusting for multiple comparisons, suggesting that B-cell development in infants at 1 year is largely unaltered by these maternal immunosuppressive regimens. While there were no statistically significant differences, three B-cell subsets exhibited trends toward reduction with exposure to combination therapy including plasmablasts, CD27+ B cells, and IgD-CD27+ switched memory B cells (Fig. 2a-d ). The CD27+CD38hi plasmablast frequency appeared reduced in the IFX/ADA + IM exposure group as compared to the CZP or the IFX/ADA monotherapy groups, but this was not significant after correcting for multiple comparisons (p = 0.037, q = 0.55; Table 2 , Fig. 2b ). The median frequency of CD27+ B cells in the combination therapy group was approximately half of the CZP monotherapy group, although again not statistically significant (p = 0.04, q = 0.55; Table 1 and Fig. 2c ). Similarly, the switched memory IgD-CD27+ B-cell subset exhibited a trend toward reduction with combination IFX/ADA+ IM exposure as compared to the CZP and IFX/ADA groups (p = 0.099, q = 0.65; Table 1 , Fig. 2d ). For all of these B-cell subsets, the trends were most apparent between the CZP and the IFX/ADA+ IM groups, while the IFX/ADA group exhibited intermediate values.
Fig. 2 Infants exposed to combination therapy with an anti-TNF agent and an immunomodulator exhibited a trend toward reduced CD27+ B cells, switched memory B cells, plasmablasts, IFNγ-producing CD4+ and CD8+ T cells, and CD4+CCR5+ T cells which did not reach statistical significance.:
(a ) Ranked p-values for cell subsets in the three-group analysis comparing CZP vs IFX/ADA vs IFX/ADA+IM. (b -g ) Median, interquartile ranges, and ranges of frequencies of each cell subset, expressed as a percent of the parent population, are shown. The p-value and q-value displayed in (b -g ) were calculated in the three-group analysis. KW-Kruskal Wallis
The possibility of reductions in switched memory B cells and plasmablasts in infants exposed to IFX/ADA with an immunomodulator suggested that it was not an effect of TNF neutralization alone. In mice, TNF is required for splenic primary B-cell follicles, although immunoglobulin class switching can occur in the absence of TNF30 -/- mice25–27 -/- mice (Supplementary Fig 3). These data are consistent with the human data in that TNF neutralization alone does not reduce baseline memory B cells and plasmablast frequencies.
CD4+IFNγ+, CD8+IFNã+, and CCR5+CD4+ T cells in infants exposed to combination therapy With regard to alterations in T cell-mediated immunity, we observed a trend toward lower frequencies of CD4+IFNγ+, CD8+IFNγ+, and CCR5+CD4+ T cells in infants exposed to combination therapy, though these did not reach statistical significance (Tables 3, 4 and Fig. 2e-g ). CD8+IFNγ+ cells appeared slightly reduced in infants exposed to combination IFX/ADA + IM therapy as compared to CZP and IFX/ADA monotherapy (p = 0.056, q = 0.55; Fig. 2e ). CD4+IFNã+ cells showed a similar trend (p = 0.21, q = 0.74; Fig. 2f ). IL-2 and TNF were similar in the CD4+ and CD8+ T cells across the different groups (Tables 3, 4 ). CD107a or lysosomalassociated membrane protein-1 (LAMP-1), a marker of degranulation, was also similar in stimulated T cells independent of drug exposure (Tables 3, 4 ). Finally, we also observed a trend toward reduction in CD4+CCR5+ cells in infants compared to anti-TNF monotherapy (p = 0.055, q = 0.55; Fig. 2g ), although this again did not reach statistical significance. Overall, exposure to combination therapy did not significantly alter the frequencies of CD4+ and CD8+ T-cell subsets, although some subsets exhibited trends which could be consistent with an increased susceptibility to infection.
Discussion In summary, infants exposed to combination therapy with anti-TNF agents and thiopurines have been shown to have an increased risk of infection during the first year of life, suggesting an underlying difference in immunity. We used multiparameter flow cytometry as a discovery tool to identify changes in the frequency of B and T lymphocyte subsets. On a global level, these data should be reassuring to providers and pregnant mothers with IBD that major B and T-cell subsets, including Tregs, are relatively unperturbed at 12 months of age after exposure to these treatment regimens.
Given the large number of comparisons and relatively small sample size, after adjusting for multiple comparisons, reductions in B-cell subsets did not meet statistical significance. However, several cell subsets demonstrated intriguing trends toward reduction after exposure to combination therapy. Reductions in CD27+ B cells, switched memory B cells, and plasmablasts in infants exposed to combination therapy as compared to CZP or IFX/ADA monotherapy could contribute to an increased susceptibility to infection. CD27 is a co-stimulatory TNF receptor superfamily member that plays critical roles in survival, function, and differentiation of T, B, NK, and plasma cells31,32 31–34 32 35
CD4+ and CD8+ subsets were largely similar, regardless of treatment. Production of IL-2 and TNF appeared largely preserved. IFNγ production exhibited a trend toward suppression in infants exposed to combination therapy. Interestingly, IFNγ responses to bacterial endotoxin in TNF-/- mice are reportedly normal36 37 38
Our data reinforce a recent analysis of vaccine responses in a subset of PIANO cohort infants39 39
A recent study looking at seven infants born to mothers exposed to anti-TNF agents and eight healthy control infants showed decreased Treg frequency at birth, as well as more immature B and CD4+ T-cell phenotypes, and a muted response to mycobacterial stimuli40 40 16,17
There are several important limitations to our study. The sample size is small due to the difficulty of recruiting to a study involving an infant blood draw without tangible results to the mother. We also do not have a group of infants exposed to immunomodulators alone. Infants exposed to thiopurine monotherapy have not been previously reported to have an increased risk of infection, so we would hypothesize that those infants would not exhibit reductions in any of the populations we analyzed. Finally, C-sections were more common in infants exposed to combination therapy, so this could account for some of the trends we observed in those infants. Delivery by C-section alters the microbiome, which could in turn alter infant immune development41
Future directions include replicating these results, as well as extending immune profiling to include additional components of the immune system. This study focused on B and T-cell subsets and the adaptive immune response. The innate immune response would also be important to evaluate. For example, monocytes, dendritic cells, NK cells, and neutrophils would all be of interest. Additionally, including Ki67 and CD45RA in our Treg staining panel would allow us to better characterize Treg activation status in these infant samples42 43
In conclusion, this analysis adds to a growing body of literature regarding the effect on infants after treating pregnant IBD patients with anti-TNF agents, as monotherapy or in combination with immunomodulators. Although we highlight trends toward subtle differences in B and T-cell subsets, none of the 39 that we analyzed are statistically significant, and the infant adaptive immune system appears to develop largely intact regardless of drug exposure.
Study Highlights What is current knowledge
Anti-TNF agents and thiopurines are low risk and effective during pregnancy and breastfeeding, but infants exposed to combination therapy may exhibit an increased risk of infection
What is new here
Overall frequencies of B cells, CD4+ T helper cells, CD8+ cytotoxic T cells, Tregs, and various subsets are similar among infants exposed to anti-TNF monotherapy versus combination therapy
Translational impact
Normal infant immune development will help counsel pregnant IBD patients regarding the favorable risk/benefit profile of continuing combination therapy during pregnancy
Acknowledgements The flow cytometry was made possible in the Core Immunology Laboratory with help from the University of California San Francisco-Gladstone Institute of Virology & Immunology Center for AIDS Research (CFAR), an NIH-funded program (P30 AI027763). The authors gratefully acknowledge invaluable cytometry support from Chris Baker and Valerie Girling. We thank Andrea Kattah and Li Zhang for assistance in statistical analysis and manuscript preparation.
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