High-Throughput Immunoglobulin Heavy Chain Variable Region Repertoire Analysis Of CD27+ B Cell Subsets From Patients With Chronic Gvhd

Patients with chronic graft versus host disease (cGVHD) have persistently altered B cell homeostasis and loss of B cell tolerance, even years after allogeneic hematopoietic stem cell transplantation (HSCT). Chronic GVHD patients have been shown to have a diverse group of autoantibodies. In cGVHD patients, antigen-experienced CD27+ B cells, unlike CD27- B cells, are capable of constitutive IgG secretion without the need for ex vivo stimulation [Sarantopoulos et al., Blood. 2009]. We previously showed that potentially allo- or auto-reactive B cells in human cGVHD signal via BAFF-associated pathways [Allen et al., Blood. 2012]. B cell receptor (BCR) repertoire composition in cGVHD B cell subsets remains unknown. We hypothesized that potentially pathologic CD27+ B cell subsets could be identified in cGVHD patients by sequencing of the immunoglobulin heavy chain (IGH).

In our study, high-throughput sequencing of the IGH was performed after preparation of single molecule real time (SMRT) B-cell amplicon libraries from RT-PCR products per Pacific Biosciences 500bp protocol, using framework region 2 (FR2) IGH variable (V) gene family primers and a common IGH Joining (J) gene primer [Boyd et al., Science Translational Medicine. 2009]. Sufficient numbers of purified B cell subsets, only available from large volume cGVHD leukapheresis samples, were obtained from three cGVHD patients and three healthy donors (HDs). In each cGVHD patient and in HDs, we examined the following CD27+ and CD27- B cell populations: naïve/transitional (CD27- IgD+ CD38+), double negative (CD27- IgD- CD38+), CD27+ IgD+ memory (CD27+ IgD+ CD38+) and CD27+ IgD- memory (CD27+ IgD- CD38+).

The IGH complementarity-determining region 3 (CDR3) is crucial for BCR antigen-specificity, and CDR3 characteristics have been previously shown to associate with autoreactivity [Wardemann et al., Science. 2003]. Thus, we assessed the CDR3 characteristics (length, charge, amino acid composition, hydrophobicity) in each B cell subset in HDs and cGVHD patients. We found that cGVHD CD27+ IgD+ B cells had overall CDR3 amino acid charge and length similar to naïve/transitional B cells. Since the CDR3 sequence is a result of V and J segment joining and may associate with capacity for autoreactivity, we also assessed relative V and J gene family usage. Gene usage analyses showed that the IGHV3 gene contributes to the majority of IGHV in all subsets of both cGVHD patients and HD groups, validating a previous report of three healthy individuals [Wu et al., Blood. 2010]. Notably, we found a 10-fold increase in frequency of IGHV7 usage in cGVHD patients compared to HD B cell subsets (1.13% vs. 0.09 in naïve/ transitional, 1.1% vs. 0.06 in double negative, 1.05 vs. 0.8 in CD27+ IgD+ memory and 0.8 vs. 0.06 in CD27+ IgD- memory). Additionally, the previously well-described autoreactive gene IGHV4-34 was more frequent in cGVHD patients compared to HD in all B cell subsets except in double negative cells. Interestingly, IGHV4-34 usage was particularly frequent in the CD27+ IgD+ cells, with a mean value of 33.1% compared to 23.3% in healthy donors. Consistent with autoreactivity, plasma IgG from these cGVHD patients had positive HEp-2 cell staining. Of the four B cell subsets examined, the CD27+ IgD+ memory in cGVHD had other distinct IGH characteristics. Additionally, IgD+ CD27+ memory B cells from cGVHD patients displayed a relative increase in IGHJ6 usage, with 37.1% compared to the 26.7% found in HD. Higher usage of tyrosine in CD27+ IgD+ population corroborated this finding.

Taken together, the IgD+ CD27+ B cell subset possessed an IGH repertoire with three features unique to cGVHD: 1) similarity to the naïve/transitional cells in the CDR3 length and total charge, 2) increased IGHJ6 usage, and 3) increased IGHV4-34 gene usage. We previously demonstrated increased cell size in CD27+ B cells, in particular in the pre-Germinal Center (GC) subset that was included in the IgD+ CD27+ gate in the current analysis. Of note, pre-GC cells were previously found to uniquely circulate in cGVHD and express high levels of the BAFF receptors TACI and BCMA [Sarantopoulos et al., Blood. 2009], corroborating the potential autoreactive capacity of this cell population. Thus, our current data suggest that IgD+CD27+ B cells in cGVHD patients display distinctly autoreactive features, and are a potentially pathologic B-cell subset in cGVHD.