*Purpose: Interaction of ‘natural’ ABO antibodies (nAbs) with their cognate AB(H)-antigens (Ags) poses a high risk of rapid rejection of ABO-incompatible (ABOi) organ transplants. We previously demonstrated that a clear understanding of factors influencing ABO nAbs is crucial for successful ABOi heart transplantation. Here we investigated anti-A nAbs vs. intentionally-induced Abs (iAbs) with regard to role of sex and T cell requirement.

*Methods: Adult wild-type (WT) and CD4 T cell knock-out (CD4KO) mice (C57BL/6 (B6) background) received weekly i.p. injection x3 of human ABO-A blood cell membranes (Hu-A BCM; 100ul of 10% v/v) or left untreated. Serum anti-A Ab was measured by hemagglutination assay using ABO-A erythrocytes from our A-transgenic mouse line. To test for T cell help and/or suppression, sex-matched CD4⁺ T cells (8-12×10⁶/mouse) or CD4⁺CD25⁺ T cells (1.7-2.8×10⁶/mouse) from spleens of WT mice were transferred to CD4KO mice. After adoptive transfer, CD4⁺ T cell reconstitution in peripheral blood was confirmed and mice were left untreated or challenged with Hu-A BCM and assessed for anti-A Ab.

*Results: In contrast to WT mice, untreated CD4KO females produced dramatically more anti-A than males, rising substantially with puberty, and this was significantly suppressed in both sexes by adoptive transfer of sex-matched CD4⁺ T cells. Unlike WT mice, attempted sensitization of CD4KO mice with Hu-A BCM failed to induce additional anti-A beyond the already high levels in either sex; CD4⁺ T cell adoptive transfer rendered CD4KO mice responsive to A-sensitization. CD4⁺CD25⁺ T cell transfer into CD4KO mice neither suppressed anti-A nAbs nor rendered them responsive to A-sensitization (figure).

*Conclusions: When ABO ‘natural’ antibodies are discriminated from intentionally induced Abs, several important findings emerge: 1) Anti-A nAbs are produced without CD4⁺ T cell help in a sex- and age-dependent manner, suggestive of a role for sex hormones in regulating anti-A nAbs. 2) CD4⁺ T cells, but not CD4+CD25+ regulatory T cells, down-regulate anti-A nAb production. 3) In contrast to anti-A nAbs, production of anti-A iAbs was CD4⁺ T cell-dependent without a sex bias.

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