IgG Allotypes Reveal That Antimicrobial Humoral Immunity Persists after Reduced-Intensity Hematopoietic Cell Transplantation

Allogeneic hematopoietic cell transplantation (HCT) is an effective treatment for hematologic malignancies through graft-versus-leukemia/lymphoma (GVL) responses and replacement of the patient’s immune system. Conditioning intensity may influence hematopoietic reconstitution, persistent recipient immunity, and donor alloimmune responses. High-dose conditioning (HDC) causes rapid conversion to full donor T- and B-cell chimerism, while patients who undergo reduced-intensity conditioning (RIC) progress through a dynamic mixed chimerism extending weeks to months. We hypothesize that long-lived recipient plasma cells persist, providing beneficial antimicrobial serologic immunity, while donor B cells target allo-antigens, contributing to both GVL and GVHD. Furthermore, recipient humoral immunity persists longer following RIC than HDC, leading to fewer infections and decreased transplant-related mortality. Inherited polymorphisms in IgG heavy chain constant regions can be recognized by allotype-specific monoclonal antibodies and thus distinguish donor and recipient antibodies. We developed a quantitative ELISA by coating mouse monoclonal antibodies specific for the G1m(f) and G2m(n) allotypes. Serial dilutions of 63 patients and their donors were incubated and detected by alkaline phosphatase-conjugated anti-human IgG polyclonal antibody. Twenty-eight HDC patients were conditioned with VP-16 and total body irradiation, while 35 RIC patients received total lymphoid irradiation and anti-thymocyte globulin (TLI-ATG). One hundred fifteen of the total 126 subjects (91%) expressed G1m(f) (null allele frequency: 0.29), and 100 of 126 (79%) expressed G2m(n) (null allele frequency: 0.45). Twenty-six of the 63 pairs (41%) were informative because either the donor or recipient was homozygous null for an allotype. Following both RIC and HDC when the recipient was allotype null, the donor allotype was first detected six months post-HCT and reached 50–80% of donor levels by 12 months (n=8 pairs). Eighteen donors were homozygous allotype null, and recipient-specific antibody was prospectively determined. By 12 months after HDC, recipient antibody levels fell to 90% of pre-HCT levels through 12 months post-HCT (n=7 pairs), demonstrating a significant difference from HDC patients in one-year median recipient allotype levels (p=0.016). We confirmed RIC humoral immune persistence by DNA chimerism detection of CD38+CD138+ plasma cells from bone marrow aspirates collected 12 months post-HCT. Analysis revealed 30–58% donor levels of plasma cells, suggesting that RIC patients maintain long-lived plasma cells. Allotype-informative patients were measured by ELISA for total IgG and allotype-specific IgG against Epstein-Barr virus (EBV), varicella zoster virus (VZV), pneumococcus, and influenza. HDC patients displayed loss of recipient antimicrobial allotype between five and eight months in conjunction with loss of bulk allotype. RIC patients maintained >90% pre-HCT allotype levels through one year correlating to levels of total IgG against all four infectious agents when they were seropositive before HCT. In addition, new donorspecific responses to influenza, pneumococcus, and VZV were detected in both sets of patients within seven to 14 months. In the allotype-informative setting, H-Y antibody analysis confirmed that donor-derived allogeneic antibodies against UTY2 and DDX3Y developed after nine and 12 months, respectively, and persisted in both HDC and RIC male patients with female donors. In summary, RIC recipients benefit from a twofold effect: persistent recipient-derived antimicrobial humoral immunity and donor-derived allogeneic B-cell responses. Our studies may explain why RIC results in decreased infectious complications in the post-HCT setting.