(P-CB-5) CMV coinfection enhances RBC alloimmunization via a novel, Type I Interferon- independent mechanism

Background/Case Studies: A key question in the field of transfusion medicine is why some patients make anti-RBC alloantibodies while most do not. Previous reports have identified that the dsRNA viral mimetic polyI:C and the ssRNA virus influenza both can enhance antibody responses in mice in a Type I interferon-dependent manner. Though disseminated viral infections in general have been shown to be associated with increased alloimmunization risk, the majority of alloimmunization events in patients are not obviously associated with dsRNA or influenza viral infections. CMV is a dsDNA virus that can re-activate in HSCT patients at a time when these patients also require multiple RBC transfusions. Thus CMV coinfections represent a clinically relevant immune stimuli that may increase anti-RBC alloimmunization risk.

Study

Design/Methods: We used the HOD mouse model of RBC alloimmunization to study the impact of CMV coinfection on RBC alloimmunization. To study the underlying cellular and molecular mechanisms of CMV action, we used neutralizing antibodies against IFNAR1 and CD4 as well as KO mice for IFNAR1 and IL-21R. For human studies, we analyzed an incident new-user cohort comprised of patients who received their RBC transfusions in six hospitals between 2005 and 2016.

Results/Findings:

Murine CMV infection led to significant enhancement of the anti-RBC antibody responses compared to mice that received RBCs alone (Median IgG- 4742 vs 115, p=0.0016. Median IgM- 413 vs 64, p=0.0016). We further found that type I IFN signaling was largely not required for the generation of anti-RBC antibodies following CMV infection. CD4⁺ T cells were required for the CMV-driven enhancement of anti-RBC antibodies. Moreover, CMV infection led to increased expansion of RBC-specific CD4⁺ T cells and increased CD4⁺ T cell differentiation into “helper” cells expressing IL-4 and IL-21. Surprisingly, IL-21 deficiency only impacted IgM and IgG3 production, while the majority of IgG subclasses and total IgG levels were unaffected. Most importantly, patients who were CMV viremic at the time of their transfusion had a 1.91 fold (0.84-4.33) increased risk of RBC alloimmunization.

Conclusions:

Our data in both mice and humans show that CMV infection increases RBC alloimmunization. Unlike what has been observed for other viral infections and mimetics, CMV enhancement is largely type I IFN independent.  Collectively our data suggest that CMV coinfection induces multiple robust and redundant cytokine pathways that both enhance helper CD4⁺ T cell differentiation and drive class switching to a broad range of IgG subclasses. Thus our data have uncovered a unique and specific risk factor for RBC alloimmunization which stimulates the anti-RBC alloantibody response through novel immune mechanisms.

Importance of research: Viral induction of Type I interferons has been proposed as a major mechanism by which infections can enhance anti-RBC alloimmunization. Herein we study a virus (CMV) whose reactivation in leukopenic HSCT patients is almost always associated with recurrant transfusions. Our data in both mice and patients show that CMV infection increases RBC alloimmunization in a type I IFN independent manner, and therefore represents a clinically relevant and novel pathway for RBC alloimmunization enhancement.