Sex, T Cells, and the Microbiome in Natural ABO Antibody Production in Mice

Abstract

Background.

“Natural” ABO antibodies (Abs) are produced without known exposure to A/B carbohydrate antigens, posing significant risks for hyperacute rejection during ABO-incompatible transplantation. We investigated anti-A "natural" ABO antibodies versus intentionally induced Abs with regard to the need for T-cell help, the impact of sex, and stimulation by the microbiome.

Methods.

Anti-A was measured by hemagglutination assay of sera from untreated C57BL/6 wild-type (WT) or T cell–deficient mice of both sexes. Human ABO-A reagent blood cell membranes were injected intraperitoneally to induce anti-A Abs. The gut microbiome was eliminated by maintenance of mice in germ-free housing.

Results.

Compared with WT mice, CD4⁺ T-cell knockout (KO), major histocompability complex–II KO, and αβ/γδ T-cell receptor KO mice produced much higher levels of anti-A nAbs; females produced dramatically more anti-A nAbs than males, rising substantially with puberty. Sensitization with human ABO-A reagent blood cell membranes did not induce additional anti-A in KO mice, unlike WT. Sex-matched CD4⁺ T-cell transfer significantly suppressed anti-A nAbs in KO mice and rendered mice responsive to A-sensitization. Even under germ-free conditions, WT mice of several strains produced anti-A nAbs, with significantly higher anti-A nAbs levels in females than males.

Conclusions.

Anti-A nAbs were produced without T-cell help, without microbiome stimulation, in a sex- and age-dependent manner, suggestive of a role for sex hormones in regulating anti-A nAbs. Although CD4⁺ T cells were not required for anti-A nAbs, our findings indicate that T cells regulate anti-A nAb production. In contrast to anti-A nAbs, induced anti-A production was T-cell dependent without a sex bias.

INTRODUCTION

ABO-incompatible (ABOi) organ transplant recipients are at high risk of rapid rejection mediated by circulating ABO antibodies (Abs) following interaction with their cognate ABH(O) blood group antigens (A/B-Ags) expressed on graft endothelium.^1,2 We showed >20 y ago that ABOi heart transplantation is safe in young children because of a developmental lag in the production of ABO “natural” antibodies (nAbs), allowing expansion of the donor pool, leading to lifesaving transplants,³ and, furthermore, resulting in immunologic tolerance to donor A/B-Ags by mechanisms related to donor-specific B-cell elimination.⁴ Beyond infancy, ABOi Tx of other organs is performed with various strategies to remove preformed ABO Abs and diminish their recurrence^5,6; however, for optimal management, it is necessary to understand the mechanisms by which ABO Abs develop.

Some studies in mice have reported the presence of the ABO blood group A/B cis gene encoding for glycosyltransferase activity,^7,8 which suggests that mice express A/B-Ags. However, we⁹ and others¹⁰ have demonstrated the absence of ABH structures in several tissues (lung, liver, kidney, ileum, pancreas, and spleen) of wild-type C57BL/6 (WT B6) mice.¹¹ Congruent with this finding, mice produce anti-A and anti-B nAbs^11-14 and can be sensitized to produce ABO Abs by human ABO-A and -B erythrocytes,^11,13,15,16 suggesting that mice are useful to model the production of ABO Abs.

nAbs (or natural hemagglutinins) are defined as Abs produced in the absence of known stimulation, in contrast to Abs induced by recognized stimuli (iAbs).¹⁷ nAbs are generally described as broadly reactive, mostly IgM isotype, and not requiring T-cell help.¹⁸ In mice, nAbs are mainly produced by B1 B cells located in peritoneal and pleural cavities^18-21; however, mechanisms leading to nAb production are not fully understood.²² For ABO nAbs, the requirement for T-cell help remains unclear, with conflicting reports^13,15,16 possibly because of study designs that failed to distinguish ABO nAbs from iAbs additionally induced by intentional immunization. We hypothesize that T-cell involvement differs in production of ABO nAbs versus iAbs.

It has long been proposed that ABO nAbs are produced in response to exposure to “A/B-like” Ags in nonpathogenic bacterial flora;^23-25 Khasbiullina et al²⁶ showed that oral inoculation of germ-free Swiss Webster mice (males, 3–4 mo old) with B longum or L reuteri stimulated ABO Ab production. However, there may not be a direct link between the Ab response and enteral antigenic stimulation because the specificity of murine ABO nAbs did not always correspond with the chemical structures of bacterial carbohydrates,²⁶ suggesting that exposure to gut bacteria may not be an absolute requirement for ABO nAbs.²⁷ Moreover, it was also reported that the overall repertoire of IgM antibodies specific for bacteria and abundance of total IgM are generally stable in both germ-free and conventionally housed mice.^28,29 We further hypothesize that production of ABO nAbs occurs spontaneously in the absence of bacterial flora.

Sex is increasingly recognized as a biologic variable in many immune responses.^30-32 Although WT mice produce variable amounts of ABO nAbs in adult life,^11,13,14 in a manner similar to humans,^33,34 whether sex plays a role in ABO nAb production has not been studied. Herein, we investigated the contribution of T cells, the microbiome, and sex in production of anti-A nAbs in mice.

MATERIALS AND METHODS

Mice

WT B6 (H-2^b) mice of both sexes were purchased from Charles River Laboratories (Quebec City, QC, Canada) and were used at 6 to 12 wk of age unless otherwise noted. Sera from germ-free mice were kindly provided by the International Microbiome Centre (University of Calgary; B6 and BALB/c mice, 4–12 wk old and T-cell receptor [TCR] βδ [TCRKO] mice, 9–10 wk old), the Gnotobiotic Research Animal Facility (University of Chicago; B6 mice, 17–21 wk old), and Dr Ben Willing (Gnotobiotic Mouse Facility, University of Alberta; B6 and Swiss Webster mice, 16–30 wk old). Mice homozygous for B6.129S2-Cd4^tm1Mak (CD4KO, stock No. 002663) and B6.129P2-TCRβ^tm1Mom/TCRδ^tm1Mom (TCRKO, stock No. 002122) targeted mutations were purchased from Jackson Laboratory (Bar Harbor, ME). Major histocompability complex (MHC) class II gene–targeted mutation B6.129-H2-Ab1^tm1GruN12 II KO (model ABBN12-F) mice were purchased from Taconic Biosciences (Rensselaer, NY). A-transgenic (A-Tg) mice (C57BL/6) expressing human A-Ag on erythrocytes and vascular endothelium were developed with our collaborators.¹¹

Antibiotic Treatment

Drinking water for groups of mice was replaced twice weekly and supplemented with 1 g/L each of neomycin, ampicillin, streptomycin, and metronidazole and 0.5 g/L of vancomycin.^35,36 Antibiotic administration was started just before the delivery of pups and discontinued at experimental end point (10 wk of age). All drugs except vancomycin (from Alfa Aesar, Tewksbury, MA) were purchased from Sigma-Aldrich (Oakville, ON, Canada).

Blood Cell Membrane Preparation

Blood cell membranes (BCMs) were prepared from pooled human reagent ABO-A₁ erythrocytes (Immucor Inc, Dartmouth, NS, Canada) described in detail elsewhere.³⁷ (Note that although not recorded in commercial product information, pooled reagent human erythrocytes are assumed to include cells from donors of both sexes.) Briefly, cells were washed in PBS and lysed in hypotonic buffer, and membranes were isolated after multiple centrifugations at 20 000g. Membranes were suspended in PBS at 10% (v/v) and stored at –30 °C until the time of injection.

Immunization

As described elsewhere,¹¹ mice were injected intraperitoneally with 100 to 150 µL of 10% v/v human type ABO-A₁ BCM (Hu-A BCM) and incomplete Freund’s adjuvant (1:1 mixture). Mice received 3 weekly injections beginning at the age of 7 wk.

CD4⁺ T-cell Isolation and Adoptive Transfer

CD4⁺ T cells were isolated from spleens of WT mice using mouse CD4⁺ T-cell Easy-Sep Stem Cells Technologies isolation kits (Vancouver, BC, Canada); purity of CD4⁺ T cells was analyzed by flow cytometry. Using a modified protocol established in our laboratory, 8 to 12 × 10⁶ CD4⁺ T cells per mouse in 150 μL of 0.9% PBS were injected via tail vein into 4-wk-old sex-matched CD4KO mice. Twenty-four hours after adoptive transfer, CD4⁺ T-cell reconstitution in peripheral blood was assessed by flow cytometry. At the end of this experiment (11 mo postinjection), we reexamined the presence of CD4⁺ T cells in peripheral blood.

Flow Cytometry

Peripheral blood mononuclear cells or splenocytes were labeled with rat monoclonal fluorescein isothiocyanate anti-CD3, AlexaFluor 647 anti-CD4, and Pacific Blue anti-CD8, or PE-anti-CD19 eBioscience (San Jose, CA). Antibody-labeled cells were incubated for 30 to 60 min at 4 °C and analyzed with BD LSR Fortessa Cell Analyzer (Franklin Lakes, NJ). Acquired data were analyzed with FlowJo version 7.6.4 software (San Carlos, CA).

Hemagglutination Assay

Tail blood was obtained from untreated mice or after injection with Hu-A BCM to assess anti-A Ab titer by incubating serially diluted serum samples (starting at 1:2–1:8) in a 96-well plate with 1% to 2% (volume/volume) A-expressing erythrocytes from A-Tg B6 male mice.¹¹ Plates were read with ImmunoSpot Cellular Technology (Shaker Heights, OH) after incubation at room temperature for 1 h and reread for confirmation after incubation at 4 °C for 18 h. The Ab titer was assessed as the highest dilution at which agglutination was detected.

Statistics

Data were analyzed with GraphPad Prism version 9 software (San Diego, CA). Data are presented as mean + standard error of mean. The student t test or 2-way analysis of variance post hoc analysis was used to compare groups for ABO Ab production.

Study Approval

Animal protocols were approved by institutional animal use committees according to the guidelines of the Canadian Council on Animal Care.

RESULTS

In the Absence of T Cells, Anti-A nAb Production Is Higher in Females Than Males and WT Mice

We first investigated spontaneous anti-A production in WT B6 mice and found that without intentional stimulation, anti-A nAbs were minimal at the age of 4 wk and generally increased with time (Figure 1A). We further found that at 9 to 10 wk of age, females tend to produce more anti-A nAbs than males, but titers generally remained low (Figure 1B), indicating that sex does not play an important role in anti-A nAb production in WT mice of this age in conventional housing. (As presented below, however, a sex-specific impact is seen with aging.)

FIGURE 1.

In CD4⁺ T cell–deficient mice, anti-A nAb production was higher in both sexes than WT mice and to a much greater extent in females than males. A, Sera collected from untreated WT B6 mice of different ages and both sexes and assessed for anti-A nAbs showed that WT mice produced low nAbs in early life and widely variable Ab range in later life. B, In serial sera from untreated WT B6 mice, anti-A nAb titers were roughly similar in 7- to 10-wk-old females and males. In contrast, CD4, MHC-II, and TCR KO females produced significantly higher anti-A nAbs than male counterparts and WT mice. C, In 2- to 8-wk-old cohoused KO mice, CD4KO and MHC-IIKO females produced higher anti-A levels than males and WT mice. Anti-A Abs were measured by hemagglutination using reagent RBCs from A-transgenic mice. Anti-A Abs are presented using standard error of mean (mean + SEM) in t test (A), and 2-way ANOVA (B and C) were used to compare groups for anti-A. ns, nonsignificant (P ≥ 0.05); *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. Ab, antibody; ANOVA, analysis of variance; CD4KO, CD4 knockout; KO, knockout; MHC, major histocompatibility complex; nAb, natural antibody; RBC, red blood cell; SEM, standard error of the mean; TCR, T-cell receptor; WT, wild type.

T-cell depletion has been reported to enhance production of nAbs with specificity for Galα1,3Galβ1,4GlcNAc-R (α-gal), a carbohydrate with a similar structure to ABO-B antigen³⁸ often studied for its role as a major “xenoantigen.” This suggests that T cells may reduce or suppress nAb production of polysaccharides. We investigated spontaneous anti-A nAb production in various T cell–deficient mice longitudinally from 7 to 10 wk of age. In CD4⁺ knockout (CD4KO) mice (B6 background), we found not only significantly higher anti-A nAb levels than in WT mice but also a striking sex difference, with much higher anti-A levels in females than in males (Figure 1B). We additionally investigated anti-A nAb production in MHC-IIKO mice because CD4 is expressed also by non–T cells^39-41 and MHC-II is required for normal CD4⁺ T-cell development.⁴² Similar to CD4KO mice, we observed significantly higher anti-A nAb levels in MHC-IIKO mice than in WT mice, again with markedly higher anti-A titers in females than males (Figure 1B). Assessing also whether other T cells, such as CD8⁺ and γδ T cells, participate in anti-A nAb production, we measured anti-A nAbs in the complete absence of T cells in αβ/γδ TCR KO (TCRKO) mice. As above, we found that TCRKO females produced not only significantly higher anti-A nAb levels than TCRKO males but also more than WT females (Figure 1B).

To exclude environmental variations between cages, we cohoused CD4KO and MHC-IIKO males and females (from 0 to 8 wk; mothers removed after weaning); the sex difference remained of similar magnitude as observed in cages separated by sex (Figure 1C). Thus, the dramatically higher anti-A nAb levels in CD4KO, MHC-IIKO, and TCRKO females than in males beyond juvenile age indicates that sex is an important biological variable influencing the role of T cells in anti-A nAb production.

Historically, nAbs have been described as broadly cross-reactive. To determine whether anti-A nAbs cross-react with other Ags, we reacted sera of the KO strains with human and mouse reagent erythrocytes. We found that murine anti-A nAbs, similar to human anti-A nAbs, agglutinate human ABO-A₁, but not ABO-O reagent erythrocytes and agglutinate murine A-Tg but not WT erythrocytes (Figure S1, SDC, http://links.lww.com/TP/C774), indicating that anti-A nAbs produced in mice are specific and reactive only with A-Ag.

Anti-A nAb Production Is Downregulated by CD4⁺ T Cells

We next studied whether the presence of CD4⁺ T cells would have an impact on anti-A nAb production in CD4KO mice. We speculated that although CD4⁺ T cells are not required for anti-A nAb production, nonetheless, they may play a regulatory role. We isolated and adoptively transferred CD4⁺ T cells from adult WT mice into sex-matched 4-wk-old CD4KO mice (Figure 2A); CD4⁺ T cells were detected in peripheral blood 24 h later (Figure 2B), but CD4⁺ T cells were no longer detectable 11 mo after injection.

FIGURE 2.

Anti-A nAb production was downregulated in CD4KO mice injected with CD4⁺ T cells. A, CD4⁺ T cells were isolated from adult WT B6, and purity was evaluated by flow cytometry. Isolated CD4⁺ T cells were adoptively transferred into sex-matched 4-wk-old CD4KO mice. B, One day later, CD4⁺ T cells were detected in the peripheral blood of CD4KO mice. C, Blood samples collected from untreated WT and CD4KO mice and from CD4⁺ T cell–injected female and male mice were longitudinally assessed for anti-A nAbs. Untreated CD4KO (open red squares) and WT females (open black squares) produced substantially higher anti-A nAbs over time than untreated CD4KO (open blue circles) and WT males (open black circles), respectively. After CD4⁺ T-cell injection, CD4KO mice of both sexes produced significantly less anti-A nAb than untreated CD4KO mice. Anti-A Abs were measured by hemagglutination using reagent RBCs from A-transgenic mice. Anti-A Abs are presented using standard error of mean (mean + SEM) in 2-way ANOVA. ****P ≤ 0.0001. A-Ag, A-antigen; Ab, antibody; ANOVA, analysis of variance; CD4KO, CD4 knockout; nAb, natural antibody; RBC, red blood cell; SEM, standard error of the mean; WT, wild type.

Although anti-A nAb production over time in CD4KO females remained substantially higher than in aging males, the introduction of CD4⁺ T cells significantly reduced anti-A nAb levels in both sexes (Figure 2C), indicating that CD4⁺ T cells downregulate anti-A nAb production. In CD4KO males, anti-A nAbs were reduced to roughly WT levels by CD4⁺ T-cell transfer. In CD4KO females, having much higher anti-A nAb levels than CD4KO males, the magnitude of reduction of anti-A nAbs by CD4⁺ T-cell transfer was similar; nonetheless, anti-A nAbs remained much higher than WT levels. We also found that aging WT female controls produced significantly higher anti-A nAbs than WT males (Figure 2C).

We next adoptively transferred CD4⁺CD25⁻ T cells and CD4⁺CD25⁺ T cells into sex-matched 4-wk-old CD4KO mice. CD4⁺CD25⁺ T cells injected into CD4KO mice (both sexes) downregulated anti-A nAbs similar to mice injected with CD4⁺ T cells or CD4⁺CD25⁻ T cells (Figure S2, SDC, http://links.lww.com/TP/C774). Thus, anti-A nAb production in mice does not require T-cell help; rather, CD4⁺ T cells suppress anti-A nAbs.

T Cells Are Required for Intentional Induction of Anti-A Abs

Our findings above indicate that T-cell participation is not required for anti-A nAbs in mice, similar to anti-α-gal nAbs.³⁸ However, a requirement for T-cell help for the induction of anti-A and anti-α-gal iAbs (in α-gal KO mice) by intentional stimulation was suggested by previous studies, although these studies did not discriminate iAbs from nAbs.^15,16,43-46 To investigate whether mice can be sensitized to A-Ag in the absence of T cells, we immunized CD4KO mice in 3 weekly injections with human ABO-A BCM (Hu-A BCM) starting at week 7. In contrast to WT mice (Figure 3A), Hu-A BCM did not further augment anti-A levels in CD4KO mice beyond the production of nAbs (Figure 3B).

FIGURE 3.

Anti-A Abs could not be intentionally induced in CD4⁺ T cell–deficient mice. A, Injection (3 weekly injections beginning at the age of 7 wk) of Hu-A BCM in WT mice induced abundant anti-A Ab production. B–D, In contrast to WT mice, in T cell–deficient strains of both sexes, anti-A Ab production was not further augmented by a similar Hu-A BCM injection. E and F, CD4⁺ T cells were isolated from WT B6 spleen and adoptively transferred into 4-wk-old sex-matched CD4KO mice (8–12 × 10⁶ cells) in a similar procedure to Figure 2A–C. The presence of CD4⁺ T cells rendered CD4KO mice responsive to stimulation by Hu-A BCM. Anti-A Abs were measured by hemagglutination using reagent RBCs from A-transgenic mice. Two-way ANOVA analyses were used to compare groups for anti-A Abs. ns, nonsignificant (P ≥ 0.05); **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. A-Ag, A-antigen; Ab, antibody; ANOVA, analysis of variance; CD4KO, CD4 knockout; Hu-A BCM, human ABO-A blood cell membrane; MHC, major histocompatibility complex; TCR, T-cell receptor; WT, wild type.

Tyznik et al⁴⁷ showed that CD8⁺ T cells in CD4KO mice can respond to MHC-II-restricted epitopes; this suggests that CD8⁺ T cells could possibly be responsive in lieu of CD4⁺ T cells in our mice. Similarly, γδ-intraepithelial lymphocytes are produced in the complete absence of thymus and lymph nodes.⁴⁸ Therefore, we additionally tested MHC-IIKO (Figure 3C) and TCRKO mice (Figure 3D) and found that injection of Hu-A BCM did not stimulate additional anti-A Ab production higher than anti-A nAb levels.

As WT mice responded to stimulation by Hu-A BCM, whereas CD4KO mice did not, we tested whether the adoptive transfer of CD4⁺ T cells gave CD4KO mice the capacity to respond to Hu-A BCM stimulation. In CD4KO males, we found that reconstitution with CD4⁺ T cells not only reduced anti-A nAb production to WT levels as noted above (Figure 2C) but also restored the ability of Hu-A BCM injection to stimulate anti-A iAbs similar to WT males (Figure 3E). Reconstitution of CD4KO females with CD4⁺ T cells, although only partially reducing anti-A nAbs from the much higher levels than those in CD4KO males (Figure 2C), similarly restored the ability of Hu-A BCM injection to stimulate anti-A iAbs (Figure 3F).

We also investigated whether CD4⁺ regulatory T cells participate in induction of anti-A Abs. We adoptively transferred CD4⁺ CD25⁺ T cells isolated from WT B6 spleen into 4-wk-old sex-matched CD4KO mice (1.7–2.8 × 10⁶ cells) and then attempted sensitization with Hu-A BCM in a similar procedure to Figure 2A–C. In contrast to CD4KO mice reconstituted with total CD4⁺ T cells, Hu-BCM did not stimulate anti-A iAb production in CD4KO mice (both sexes) injected with the CD4⁺CD25⁺ T-cell fraction (Figure S3, SDC, http://links.lww.com/TP/C774).

Together these data show a previously unreported dual role for CD4⁺ T cells in anti-A Ab production: for ABO-A sensitization, CD4⁺ T-cell help is required to stimulate anti-A iAbs; in contrast, for anti-A nAbs, CD4⁺ T cells downregulate production, especially powerfully in females.

Disruption or absence of gut flora does not reduce anti-A nAb levels or eliminate sex differences, with females producing more abundant anti-A nAbs than males

The formation of anti-B Abs in germ-free chickens was reported by Springer et al²⁵ in 1959 to require exposure to “B-like” Ag expressed on Escherichia coli O86 that has structural similarity to ABO-B-Ag.^49,50 More recent reports, in contrast, suggested that germ-free animals produce ABO nAbs without exposure to “ABH” bacterial Ags.²⁶ To examine whether gut flora are required²⁵ for ABO nAb production, we supplemented the drinking water of mice (beginning just before the delivery of pups and discontinued at 10 wk old) with neomycin, ampicillin, streptomycin, metronidazole, and vancomycin, following standard protocols.^35,36 We found that oral antibiotics did not affect anti-A nAb production in adult WT, CD4KO, MHC-IIKO, or TCRKO mice of either sex (Figure S4, SDC, http://links.lww.com/TP/C774).

Disruption of normal gut flora with antibiotics is limited by emergence of drug-resistant bacterial strains;⁵¹ thus, we also examined anti-A nAbs in mice housed under germ-free conditions. We found that germ-free B6 adult males produced similar anti-A nAb levels as conventionally housed males (Figure S5A, SDC, http://links.lww.com/TP/C774), indicating that, in contrast to most historical reports,^25-28,49,50 the microbiome is not required for anti-A nAbs in mice.

We also examined whether the sex of germ-free mice (aged >16 wk) influenced anti-A nAbs. We found that germ-free outbred Swiss Webster females produced roughly comparable levels of anti-A nAbs to conventionally housed females but significantly higher levels than males (Figure 4A). To further examine whether this sex difference was related to age and housing conditions, we examined anti-A nAbs at an earlier age (cross-sectional study at the age of 4, 8, and 12 wk) and found that B6 females under germ-free conditions began to produce significantly higher levels of anti-A nAbs at ages 8 and 12 wk (ie, from puberty onward) than germ-free males or conventionally housed B6 males and females (Figure 4B; Figure S6, SDC, http://links.lww.com/TP/C774). The same pattern was also observed in germ-free BALB females but was significant only at the age of 12 wk (Figure 4C). We additionally found that germ-free TCRKO female mice produced abundant anti-A nAbs, which was significantly higher than germ-free TCRKO males (Figure S5B, SDC, http://links.lww.com/TP/C774).

FIGURE 4.

Germ-free mice produced abundant anti-A nAbs with significant sex differences. A, Outbred Swiss Webster mice housed in both germ-free and conventional conditions produced abundant anti-A nAbs, with significantly higher levels in females than males. B and C, Adult B6 and BALB mice housed under germ-free conditions produced abundant anti-A nAbs. At 8 wk (B6) and 12 wk (B6 and BALB) of age, female germ-free mice produced more anti-A nAbs than conventionally housed females of a similar age, whereas germ-free males produced similar anti-A nAbs as conventionally housed males. Anti-A nAbs were measured by hemagglutination using reagent RBCs from A-transgenic mice. Standard error of mean (mean + SEM) of anti-A Abs was used in the paired t test. ns, nonsignificant (P ≥ 0.05); *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. nAb, natural antibody; RBC, red blood cell; SEM, standard error of the mean.

Here we showed that anti-A nAbs (detected in 3 germ-free strains in 3 different germ-free facilities) are spontaneously produced even in the absence of a microbiome. Unlike anti–E coli nAb production in which germ-free WT females produced equivalent nAbs to conventionally housed females,⁵² here germ-free females produced significantly higher anti-A nAbs than conventionally housed females. Moreover, the female preponderance of higher anti-A nAbs was not limited to inbred B6 and BALB strains but was also observed in outbred Swiss Webster mice.

DISCUSSION

Mice and humans spontaneously produce ABO nAbs over time.^11-14 Historically, it has been proposed that ABO Abs are produced in response to exposure to bacterial flora.^23-25 Additionally, studies have variably reported that ABO Ab production in WT mice is T-independent,¹³ T-dependent,¹⁵ and natural killer T-cell dependent.¹⁶ However, previous studies did not examine the role of sex or distinguish natural ABO Abs from those induced by intentional stimulation.

Here we found that in the absence of functional CD4⁺ T cells, CD4KO, MHC-IIKO, and TCRKO mice not only produced significantly more anti-A nAbs than WT mice but that there was also a striking sex difference in these KO strains, with much higher anti-A nAb production in females. Although this sex difference was highly significant in T cell–deficient mice, young WT mice did not show a similar sex difference in production of anti-A nAbs. However, after puberty, aging WT females produced significantly higher anti-A nAbs than WT males. Furthermore, the predominance of anti-A nAbs in females was not impacted by cohousing with KO males, indicating that the sex difference was not because of environmental factors such as food and allergens.^53,54

Larkin et al⁵⁵ reported that mice produce anti-A nAbs with cross-reactive specificities in contrast to anti-A nAbs produced in humans. However, our finding that, similar to human anti-A nAbs, murine anti-A nAbs agglutinate human ABO-A₁ but not ABO-O reagent erythrocytes, and murine A-Tg, but not WT erythrocytes, indicates that anti-A nAbs produced in mice are specific and reactive only to A-Ag. Because immunization with Hu-A BCM will induce not only anti-A production but also Abs to xenogeneic proteins, using reagent A-Tg red blood cells in our hemagglutination assay is particularly important. This is consistent with our previous observation that A-Tg mice generated in our laboratory do not produce anti-A nAbs (ie, to self A-Ag).¹¹

Although production of anti-A/B and anti-α-gal Abs was reported in some studies to require T-cell help,^15,43-45 depletion of T cells was shown in other studies to enhance anti-α-gal nAbs.³⁸ In our experiments, adoptive transfer of WT CD4⁺ T cells indeed reduced the greatly elevated anti-A nAbs in CD4KO mice. Although detected 24 h after transfer, adoptively transferred CD4⁺ T cells were absent several months later, suggesting a long-lasting early impact on B cells despite short persistence in peripheral blood. The finding that the absence of T cells had a much greater impact on anti-A nAbs in females than in males suggests stronger mechanisms of suppression of anti-A nAb production normally exist in WT females compared with males. The mechanisms are currently unknown but may involve CD4⁺CD25⁺FoxP3⁺ regulatory cells T cells, in which there are noted sex differences.³² Additionally, estrogen has been shown to stimulate nAb production in female mice, with female mice producing higher anti–E coli nAbs than males.⁵² Accordingly, we speculate that estrogen could be a stimulant of the high anti-A nAbs produced in female mice. However, the precise interactions between CD4⁺ T cells and estrogen in anti-A nAb production remain unclear (depicted schematically in Figure 5).

The previously reported failure to stimulate anti-A Abs in MHC-IIKO mice,¹⁶ although not discriminating iAbs from nAbs, suggested that CD4⁺ T-cell participation is required to induce anti-A Ab production. This is consistent with our data showing that Hu-A BCM injection stimulated anti-A Abs in WT and CD4KO mice reconstituted with WT CD4⁺ T cells but not in CD4KO, MHC-IIKO, or TCRKO mice. As expected, CD4⁺CD25⁺FoxP3⁺ regulatory T cells did not participate in induction of anti-A iAbs in response to Hu-A BCM, consistent with the role of regulatory T cells in suppression of antibody production reported by others.⁵⁶

Early studies reported that formation of ABO Abs in germ-free animals required exposure to introduced bacterial flora,^25-27 in agreement with reports that the ingestion of E coli in humans was associated with increased ABO Ab titers.^57,58 However, we found that disruption of gut bacterial flora with antibiotics did not significantly reduce anti-A nAbs in CD4KO mice. The use of antibiotics to study the impact of gut bacterial exposure in mice is limited because it does not eliminate all bacterial flora. Furthermore, antibiotic treatment could differentially affect anti-A Abs by sex because the composition of bacterial flora in female mice is different than males.^59-61 Moreover, it is possible that elimination of some bacterial strains with antibiotics allows replacement with other antibiotic-resistant strains.⁶² Germ-free conditions allow a more precise assessment of the potential impact of the microbiome on nAbs. The presence of ABO nAbs in germ-free mice indicates that the microbiome is not required for ABO nAb production. Our finding of significantly higher anti-A nAb levels in germ-free TCRKO females than in males in T cell–independent manner suggests that production of nAbs may be “stimulated” or influenced by sex-related factors produced during puberty, such as estrogen⁵² or ovarian glycolipids.^63,64 Interestingly, rabbits produced high nAbs during puberty,⁶⁵ and as noted above, estrogen was recently reported to influence natural anti–E coli Ab production in germ-free mice.⁵² Here, our findings show that puberty is important for anti-A nAb production in females, not only under germ-free and conventional housing conditions for WT mice but also in T cell–deficient mice (ie, CD4KO and MHC-II KO mice). Although germ-free mice have no contact with bacterial-derived “A/B-like Ags,” traces of such structures may exist in food and allergens^53,54 and could stimulate nAbs under germ-free conditions. In addition, some reports have suggested that B cells can spontaneously produce nAbs without exposure to Ags.^29,66,67 Consistent with our finding that CD4⁺ T cells suppress anti-A nAb production, previous reports suggested that CD4⁺CD25⁺Foxp3⁺ regulatory T-cell function is impaired in germ-free mice,^68-71 offering a possible explanation for higher ABO nAbs in germ-free adult females than in conventionally housed mice.

In summary, when ABO nAbs are discriminated from iAbs, several important findings emerge. CD4⁺ T-cell help is not required for anti-A nAbs to develop, yet anti-A nAb production is not entirely “T-cell independent” as CD4⁺ T cells suppress anti-A nAbs. In contrast, sensitization to A-Ag is “T-cell dependent”; T-cell help is required for iAb production. Additionally, in the absence of T cells or a microbiome, sex emerges as an important biologic variable, with a strong female predominance of high nAb production. The precise mechanisms and relative contributions of T cell–mediated suppression of nAbs and T cell–mediated help to iAbs with specificities to ABH and other glycans remain under investigation. It is suggested that interferon-γ produced by CD4⁺ T cells downregulates B-cell proliferation and IgM production, which we speculate could play a role here (depicted schematically in Figure 5).^72,73

Future studies will provide greater insights into exact mechanisms by which sex and the microbiome influence ABO nAbs and their regulation by CD4⁺ T cells. For instance, studying the impact of male T cells in female KO mice (ie, adoptive transfer of sex-mismatched CD4⁺ T cells), the microbiota (ie, sex-matched and -mismatched fecal transfer), or estrogen (ie, estrogen receptor-deficient female mice or injection of estrogen in males) may reveal why KO females produced higher anti-A nAbs than males. In addition, discriminating antibodies induced through sensitization from those arising spontaneously is clearly essential. Understanding how nAbs are produced will not only help us develop clinical strategies^74,75 to manage ABOi organ transplantation but also inform the broader understanding of glycoimmunology. The historic nomenclature and the oversimplification of immune responses to ABH glycans and other carbohydrate structures pose a potential obstacle to clinical progress in ABOi transplantation and xenotransplantation, in which a more nuanced understanding of glycoimmunity in biologic systems is critical.

ACKNOWLEDGMENTS

The authors thank Dr Ben Willing and Ms Stephanie Tollenaar (University of Alberta) and Drs Kathy McCoy and Paul Kubes (International Microbiome Centre, University of Calgary) for providing serum from germ-free mice. In addition, the authors thank Drs Colin Anderson and Robert Ingham for their insightful discussion and constructive feedback on this work. Biostatistical expertise was kindly provided by the Women and Children’s Health Research Institute, University of Alberta.