Strategies to inhibit alloantibody production in alloprimed murine recipients of hematopoietic stem cell grafts.

Alloantibody, not primed T cells, is the major barrier to bone marrow (BM) engraftment in allosensitized mice. We have shown that a single intravenous injection of donor splenocytes, to mimic a blood transfusion, results in high, sustained levels of serum alloantibody sufficient to eliminate donor BM within 3 h, resulting in uniform mortality in lethally irradiated allogeneic recipients. Current studies focused preventing and treating allopriming. Blockade of B cell survival signals with mTACI-Ig pre- and postpriming was ineffective, as was the B cell but not plasma cell depleting anti-CD20 mAb. Germinal center formation inhibition by lymphotoxin-beta receptor-Ig (LßR-Ig) diminished allosensitization, although conditional Prmd1 (Blimp-1) deletion in CD19+ cells was highly effective. By combining anti-CD20 mAb to reduce B cells and LTßR-Ig to diminish the frequency of B cells that could form germinal centers pre- and postpriming, allosensitization was precluded, permitting long-term survival in T- and NK-depleted, irradiated allogeneic recipients, whereas combined therapy postpriming alone was ineffective. As evidence of the critical role of B cells, the proteosomal inhibitor, bortezomib, given unencapsulated or encapsulated, proved ineffective in influencing allosensitization. These data extend our understanding of allopriming and provide a potential therapy for patients at risk for allosensitization and BM graft rejection.

In situ determination of B-cell heavy chain and kappa/lambda light chain expression patterns: methodology and clinical utility.

The authors describe a reverse transcriptase in situ polymerase chain reaction (RT in situ PCR) method that allows the determination of B-cell clonality as defined by expression of kappa and lambda mRNA as well as the different VH families that comprise the entire heavy chain (IgH) sequence using paraffin-embedded, formalin-fixed tissue. Polyclonal expression of B-cell light and heavy chains was documented in two reactive lymph nodes and a spleen in the same histologic distribution as the B-cell marker CD20. Monoclonal expression of kappa versus lambda mRNA was documented in 11 cases of B-cell lymphoma and was corroborated in 5 cases by flow cytometry; each case showed monoclonal expression of IgH. The authors analyzed seven additional tissues where a definitive diagnosis of B-cell lymphoma could not be rendered based on histologic, immunohistologic. and clinical analysis. RT in situ PCR for IgH and kappa versus lambda expression differentiated the B-cell lymphomas (n = 2) from reactive B-cell processes (n = 3) and from two cases of B-cell-rich T-cell lymphoma. The described RT in situ PCR methodology allows routine determination of the monoclonal versus multiclonal expression patterns of B cells, which will facilitate the diagnosis of B-cell lymphomas and aid in understanding the pathogenesis of B-cell malignancies using archival, paraffin-embedded tissues.