Humoral Immunity Induced By Viral Infection Provides a Major Barrier To Hematopoietic Cell Transplantation
Authors: Ruhil Ravi , Huang Daniel , Bulut Ozlem P , Knechtle Stuart J , Kean Leslie S. , Iwakoshi Neal N 
Published: October 2019

Abstract

Background
Sensitization to MHC antigens because of transfusion, pregnancy, and previous grafts is among the most critical challenges to clinical transplantation. The presence of preformed donor-specific antibody increases the risk for bone marrow and solid organ graft rejection. It is well documented that some microbial molecules and microorganisms can directly induce the proliferation and differentiation of antibody-secreting cells from different B cells, regardless of their antigen specificity. The presence of a strong polyclonal B cell response resulting in hypergammaglobulinemia secretion has been described in several animal models of infection with viruses, parasites, and bacteria. Recent experiments in our lab showed that pathogen infection in naïve animals induces activation of naïve allospecific B cell and formation of de novo alloantibody. This prompted us to analyze the effect of pathogen infection induced alloantibody on hematopoietic cell transplantation. Herein, these studies show that preformed donor specific antibody induced by pathogen infection in the absence of donor-antigen is a major barrier to bone marrow engraftment.

Methods
/results: LCMV infection is a model system of viral infection and also an established system to examine virus nonspecific, polyclonal hypergammaglobulinemia. Naïve C57BL/6 (H2b) mice were infected with acute and chronic strain of LCMV (Armstrong and clone 13, n=10 each). Virally infected mice were bled weekly to analyze total IgG and formation of de novo alloantibody in the serum. Viral infection induced both hypergammaglobulinemia and the formation of de novo alloantibody (anti-H2d). Peak levels of donor-specific antibody in acute and chronically infected mice were increased to approximate 3 fold when compared to uninfected controls as assessed by flow cytometric crossmatch. The results were consistent with formation of anti-H2d antibody when measured by anti-H2d ELISA with peak level of approximately 336.3 ± 51.84 units/ml when compared to 2.179 ± 0.4597 units/ml in uninfected mice (p<0.0001, Figure 1). Interestingly, the response generated by chronic infection was relatively sustained (up to 75 days) when compared to the transient response induced by acute infection.
Next we analyzed if virally induced preformed de novo alloantibody would represent a barrier to bone marrow engraftment. Naïve C57Bl/6 mice were irradiated with 950 rads and received 15x106BALB/c bone marrow. BALB/c bone marrow cells were either incubated ex vivo with 1:6 diluted sera from naïve B6 mice or with the sera from clone 13 infected mice (d28 post-infection). After incubation, bone marrow was washed and complement inactivated prior to infusion. All of the 10 mice receiving bone marrow incubated with naïve mice serum engrafted and survived, while only 1/10 mice survived receiving bone marrow incubated with serum from infected mice (Mean survival = >100 vs 24.5 days respectively, p < 0.0067, figure 2).
We also examined the effects of virally induced preformed de novo alloantibody in a costimulation blockade based regimen to induce mixed allogeneic chimerism and transplant tolerance. Naïve C57BL/6 mice received 30x106 BALB/c bone marrow infusion along with costimulation blockade treatment (250ug each of CTLA4-Ig and anti-CD40L (MR1) at 0,2,4,6 days post transplant) after nonirradiation-based conditioning with busulfan (-3,-2 days. 20mg/kg IP injection). BALB/c bone marrow cells were either incubated ex vivo with 1:6 diluted sera from naïve B6 mice or with sera from clone 13 infected mice (d28 post-infection) as described above. Consistent with the experiments using the irradiation based conditioning regimen, mice receiving bone marrow incubated with naïve mice serum engrafted and established stable donor chimerism while none of mice receiving bone marrow incubated with serum from infected mice showed any donor cell engraftment

Discussion:
Allogeneic hematopoetic cell is used as therapy for hematologic malignancies and for non-malignant hematologic diseases. Our results indicate that preformed donor specific antibody induced by viral infection in the absence of previous exposure to donor-antigen is a major barrier to bone marrow engraftment. These results suggest that an individual’s prior immune history to pathogens may significantly impact subsequent humoral responses upon bone marrow transplantation.

Disclosures:
No relevant conflicts of interest to declare.