*Purpose: ABO-incompatible organ transplants (ABOi-Tx) are at high risk of rapid rejection mediated by preformed ABO antibodies (Ab) except during infancy when they are absent. ABO Ab are produced spontaneously in humans and mice without obvious exposure to ABO antigens (Ag) by mechanisms that remain poorly understood but are thought to be related to cross-reactive stimulation by epitopes in the gut microbiome. Despite descriptions of ABO Ag and other carbohydrates as ‘T-independent’ structures, our group and others showed that CD4 T cells are necessary to induce anti-A/B Ab production (T-dependent) following immunization of wild-type (WT) mice with human A red blood cells (Hu A-RBC). It remains unclear whether production of ‘natural’ ABO Ab is also T-dependent. Here we investigated the requirement for CD4 T cells and the role of the gut microbiome in the production of natural anti-A Ab.

*Methods: Six to eight weeks old WT, CD4, MHC-ii, and αβ/γδ T cell knock-out (KO) mice (C57BL/6 [B6] background, n=3-10 per group) received x3 weekly i.p. injection of Hu A-RBC (100ul of 10% v/v) or left untreated (n=3-10 per group). Serum anti-A Ab was measured by hemagglutination assay using blood group A erythrocytes from A-transgenic mice we previously generated. To study whether gut bacteria play a role in natural anti-A Ab production, antibiotics were added to drinking water (1g/L each of neomycin, ampicillin, streptomycin, metronidazole; 0.25 g/L of vancomycin); antibiotics were replaced twice weekly, from week 0 to the end of experiments at week12.

*Results: Although WT B6 mice produced anti-A Ab spontaneously with age (titer ranged from 1/8 to 1/16), CD4KO, MHC-iiKO and αβ/γδ T cellKO mice developed vastly higher titer (1/32 – 1/4096, by week 12 in both sexes). Analyzed by sex, female CD4KO, MHC-iiKO, and αβ/γδ T cellKO mice produced significantly higher natural anti-A Ab titer (1/512 – 1/4096) compared to male mice (1/8 – 1/32). Hu ARBC injection did not induce further increase in anti-A Ab production in both male and female mice. Treatment of CD4KO mice with antibiotics (duration of the experiment is from week 0 to 12) dramatically reduced the production of natural anti-A Ab titer in female (from 1/2048 to 1/32) compared to male mice (from 1/32 to 1/8).

*Conclusions: Our studies show that, whereas intentional induction of anti-A Ab is T-dependent, natural ABO Ab can develop in the absence of CD4 T cells. Additionally, we found an important sex difference in CD4-deficient mice with females producing much higher levels of anti-A Ab than males, suggesting differential regulation between the sexes. A role for the microbiome in natural ABO Ab production was suggested by decreased anti-A Ab following antibiotic treatment.

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