ABO, alemtuzumab and allogeneic transplantation

ABO antigens are widely distributed carbohydrate histo-blood group antigens on red cells, platelets, vascular endothelium and epithelial tissues. Although ABO incompatibility is a major barrier to solid organ transplantation, 30%–50% of all allogeneic hematopoietic stem cell transplants (allo-HSCTs) are ABO mismatched.¹ ABO incompatibility has no impact on engraftment of stem cells or myeloid cells, which lack A/B antigens; however, ABO mismatching has been associated with delayed red cell engraftment, pure RBC aplasia, hemolysis, increased transfusion requirements, venous occlusive disease, severe GvHD and decreased survival.^{1, 2, 3, 4, 5, 6, 7, 8, 9}

Early studies have suggested that ABO incompatibility may be a particular risk factor in reduced-intensity conditioning peripheral blood HSCT due to the higher number of T- and B cells in those products.^{1, 2} Most large studies examining ABO and HSCT outcomes have consisted predominantly of marrow allo-HSCT (Table 1).^{1, 7, 8, 9} In this issue, Brierley et al.² report their 18 years of experience, using alemtuzumab-based reduced-intensity conditioning, including 504 (92%) peripheral blood HSCT. As expected, ABO mismatching had no impact on myeloid or platelet engraftment. Nor did the authors find evidence of clinically significant delays in red cell engraftment, using RBC transfusion during the first 100 days as a surrogate indicator. RBC utilization was essentially equivalent in matched and ABO-mismatched transplants, although there was a trend toward increased RBC transfusion in ABO major-incompatible HSCTs (Supplementary Table 2; P=0.06),² especially in patients requiring>35 units, and in patients with GvHD. This differs from marrow transplants, which have reported a 10- to 20-day delay in erythroid engraftment in ABO major-incompatible allo-HSCT.^{5, 6, 7, 8, 9} Unfortunately, the authors did not measure either the reticulocyte count, isoagglutinin titer or the appearance of donor red cells—more valid metrics of red cell engraftment. They also did not report the incidence of pure RBC aplasia, which is reported to occur in up to 24% of ABO major-incompatible HSCTs.^{1, 4}

Table 1 Comparison of ABO-mismatched transplants among selected published studies

There is ongoing debate regarding the impact of ABO mismatching on GvHD and transplant outcomes (Table 1).¹ Consistent with many other European studies,^{1, 5, 7} Brierley et al.² found no significant difference in relapse-free or overall survival among ABO-mismatched HSCT. The authors also did not find any significant increase in stage II–IV acute GvHD in ABO-mismatched HSCT. Interestingly, the authors did find a novel association between ABO major incompatibility and the development of extensive chronic GvHD (P=0.0036), which has not been reported by others.^{1, 6, 7}

A major difference between prior studies and the results reported by Brierley et al.² is the routine use of alemtuzumab in the conditioning regimen (Table 1). Alemtuzumab is a humanized murine monoclonal against CD52, a relatively small glycophosphatidylinositol-linked glycoprotein and a ligand for SIGLEC10. CD52 is widely distributed on both B and T cells, natural killer (NK) cells, NKT cells and dendritic cells, but is absent on hematopoietic stem cells.¹⁰ Alemtuzumab administration rapidly depletes B and T cells for several months due to a long circulating half-life, whereas NK cells are relatively spared with recovery within 3–4 weeks.¹¹ Alemtuzumab-based regimens have been shown to be highly effective in reducing acute GvHD and graft failure, even in haploidentical transplants.¹⁰ In early studies, the major drawback of alemtuzumab-based regimens was the potential for autoimmune cytopenias and a high risk of CMV and other infections due to delayed B- and T-cell recovery.^{10, 12} Subsequent protocols have reduced alemtuzumab dosage without compromising engraftment, GvL effect or GvHD prophylaxis.^{2, 10, 11} In the study by Brierley et al.,² the incidence of autoimmune cytopenias was 7.5%, which is comparable to other reports (2%–15%).¹² One major oversight was the absence of data regarding CMV and other infections. Over 60% of study patients were at potential risk for CMV infection. In addition, patients received different doses of alemtuzumab over the 18-year study period, including many patients who had received high-dose alemtuzumab.

Alemtuzumab-based reduced-intensity conditioning regimens may be particularly effective in ABO-mismatched transplants due to their effects on B cells and invariant NKT cells (iNKT), which strongly express CD52.^{13, 14} iNKT cells are a small but critical T-cell population involved in the B-cell response to glycolipid antigens via CD1d, a major histocompatibility complex-like molecule.¹⁴ iNKT cells promote B-cell proliferation, B memory responsiveness, plasma cell generation and antibody production.^{14, 15} Their relative absence in common variable immunodeficiency may contribute to both B-cell defects and hypogammaglobulinemia. Recent studies suggest that iNKT can stimulate B-cell proliferation and antibody production through cell–cell contact, probably due to the expression of stimulatory glycolipid self-antigens on B cells.^{14, 15}

Recently, iNKT cells were identified as necessary for the development of allo-anti-A formation in a murine mouse model.¹⁶ Tazawa et al.¹⁶ were able to induce high-titer anti-A in wild-type mice after repeated injections with group A human red cells, whereas little or no anti-A was observed in nude mice, which are deficient in iNKT cells. Similar results were observed in CD1d-knockout mice and in wild-type mice pretreated with an anti-CD1d monoclonal antibody. Finally, monoclonal anti-CD1d successfully prevented anti-A formation in a group O, humanized NOD/SCID/γc^null mouse model. These results suggest that depletion or inhibition of iNKT cells could be highly effective in ameriolating the potential adverse effects due to ABO incompatibility. NKT cells are highly susceptible to alemtuzumab even at low doses, with depletion of NKT cells for up to 3 months.^{11, 13}

In ABO major-mismatched transplants, loss of recipient ABO antibodies typically precedes the appearance of donor-type circulating red cells: a persistence of isoagglutinins can be associated with delayed red cell recovery and pure RBC aplasia.^{4, 5} Several studies have observed accelerated isoagglutinin loss and red cell recovery among MUD transplants, and in patients with grade II–IV GvHD, suggesting a possible graft-versus-plasma cell effect.^{5, 6, 9} A small study of alemtuzumab in allo-HSCT showed a low incidence of pure RBC aplasia (2%) with appearance of donor red cells and loss of isoagglutinins within 2–3 months—consistent with clearance of preformed antibodies and suppression of endogenous antibody production.⁹

In contrast, ATG appears to have little impact on either isoagglutinin titers or NKT cells. Blin et al.⁵ and Ozkurt et al.⁴ found no difference in isoagglutinin levels or red cell engraftment in patients who received ATG. Furthermore, Mielcarek et al.⁹ reported a rapid rebound and prolonged persistence of isoagglutinins in patients receiving pre-transplant plasma exchange (ABO titers >1:16), despite routine use of ATG and methotrexate. ATG does contain antibodies reactive with NKT cells, including iNKT cells, although activity appears to be weak based on antibody clearance.¹⁷ In mice and humans, ATG leads to only mild decreases in iNKT cells (9%) compared with other lymphocyte populations, leading to a 200-fold relative increase in circulating iNKT cells.¹⁸ As a consequence, there is increasing interest in the use of alemtuzumab over ATG in ABO-mismatched solid organ transplantation, with encouraging results. The results reported by Brierley et al.² suggest that alemtuzumab may also be superior to ATG in ABO-mismatched allo-HSCT, especially using peripheral blood stem cells. Transplant regimens using ATG in ABO-mismatched peripheral blood allo-HSCT showed an increase in non-transplant-related mortality and decreased overall survival, despite a significantly higher percentage of matched-related donor transplants (70%–91% versus 39%, Table 1) and myeloablative conditioning.^{3, 4}

ABO antibodies may also be cleared by adsorption via ABO-active substances present in the recipient or in transfused blood products. The latter could account for the apparent association between GvHD, isoagglutinin levels and red cell engraftment,as GvHD is often accompanied by increased blood utilization.^{2, 4} ABO-soluble substances could also theoretically protect against severe GvHD by preventing binding of donor-derived antibody to ABO antigens on endothelium and epithelium. Finally, soluble ABO may be necessary for long-term tolerance in minor-incompatible and bidirectional transplants. In mice, circulating A-antigen/antibody complexes lead to a specific deletion of B splenocytes secreting anti-A.¹⁹ Indirect evidence for a similar process in humans can be observed in a humanized NOD/SCID/γc^null mouse model.²⁰

A neglected area of study is the role of blood group genetics on transplant outcomes, which may account for some of the differences between published studies. Cellular and soluble ABO expression is determined by ABO, Secretor (FUT2) and Lewis (FUT3) genes, with several alleles showing enzymatic, ethnic and geographic variation. In particular, Caucasians have a high incidence of A₂ (20%), a weak A subtype characterized by little or no A-antigen in the plasma, platelets, endothelium or epithelial tissues. As a consequence, A₂ individuals are considered ‘group O’ compatible in solid organ transplantation. In the American and European studies, it is likely to be that a significant proportion of A–O mismatched HSCT included A₂–O transplants, thereby diluting the potential impact of ABO incompatibility. In contrast, A₂ is extremely rare in Japan and this could explain the higher morbidity among both ABO major- and minor-mismatched HSCT reported by Kimura et al.⁶

Soluble ABO is also dependent on Secretor/FUT2 and is influenced by Lewis/FUT3. The majority (70%–80%) of Americans and Europeans are ‘secretors’ with ABO-active glycolipids and proteins in the plasma and on mucosal epithelium. In Japan and other Asian populations, however, there is a high prevalence of weak FUT2 and FUT3 alleles. These individuals have diminished or absent soluble ABO (and Le^b) antigens in the plasma, especially among heterozygous individuals, although ABO expression is retained on gut mucosa (and hepatic endothelium). This might also contribute to the reported increase in severe hepatic and gastrointestinal GvHD among Japanese ABO-mismatched HSCT recipients.⁷ Similar to HLA, allelic variation among ABO and FUT2 could subtly influence allo-HSCT and could be the subject of future studies.