Background
Kidney transplantation is the most effective form of therapy for end-stage renal disease (ESRD) in terms of mortality, quality of life and health care savings [
1]. Human leukocyte antigen (HLA) sensitization is a major barrier to successful kidney transplantation, especially amongst the highly sensitized. Sensitized kidney transplant candidates comprise approximately 30% of the deceased donor waiting list and have the longest wait times because of difficulty in finding a compatible donor [
2]. HLA sensitization refers to pre-existing antibodies against human HLA proteins that are produced in the transplant candidate after contact with non-self HLA antigens, commonly from previous transplants, pregnancies and blood transfusions [
3‐
5]. Antigen-specific B cells recognize, bind, internalize and process the antigens through the B cell receptor (BCR) [
6]. When a co-stimulatory signal is present from CD4
+ T cells, B cells undergo clonal expansion, producing plasmablasts, which secrete high affinity HLA antibodies, and memory B cells, which can provide a source of HLA antibodies long after the sensitizing event [
6].
Desensitization therapies include plasmapheresis, which physically removes proteins from sera, intravenous immunoglobulin (IVIG), which can decrease circulating HLA antibody proteins, anti-CD20 monoclonal antibody treatment (rituximab), which depletes most B cells with the notable exception of antibody-producing plasma cells, and proteasome inhibitors (bortezomib), which target plasma cells. Candidates who respond to desensitization therapy with a decrease in HLA antibodies and undergo successful transplantation show a survival benefit compared to remaining on dialysis [
7,
8]. However, for unknown reasons, circulating HLA antibody levels do not decrease in a significant number of sensitized kidney transplant candidates following desensitization therapy, and potential toxicity from medications could lead to unwarranted risk and poor outcomes. The degree of sensitization to individual donor HLA proteins is measured using single antigen bead assays. Defining HLA matching at the epitope level may allow for a more detailed assessment of HLA compatibility by addressing the immunogenicity of a particular HLA antigen mismatch [
9]. This information is crucial for matching donated organs with transplant candidates, but it is not informative as to which sensitized candidates will respond to desensitization therapy [
10,
11].
Genomic rearrangement of variable (V), diversity (D) and joining (J) segments of immunoglobulin genes generates a diverse set of BCRs that recognize different antigens [
12,
13]. Most of the diversity in the antibody repertoire is in the hypervariable complementarity-determining region 3 (CDR3) of the heavy chain of the BCR and largely accounts for antibody specificity [
14]. The constant region determines the antibody isotype. Naïve B cells produce both IgM and IgD isotypes. After encountering antigen, B cells undergo clonal expansion and somatic hypermutation, affinity maturation, and class-switch recombination to produce IgG, IgA or IgE antibodies [
15]. High-throughput DNA sequencing of immunoglobulin genes has been used to detect and follow residual disease in B cell malignancies, determine vaccine response, and perhaps has a role in autoimmune disease diagnostics [
16‐
20]. We reported that repertoire sequencing is correlated with immune activity during rejection episodes in heart transplant recipients [
21].
Investigations of HLA sensitization may benefit from B cell immune repertoire sequencing because the antigen is known and several therapies that target B cells and/or decrease HLA antibodies are available. We developed an assay utilizing high-throughput DNA sequencing of the variable domain of the antibody heavy chain of immunoglobulin genes to measure changes in B cell repertoires before and after desensitization therapy and transplantation. Our objective was to survey a small cohort of highly sensitized kidney transplant candidates to determine whether measuring baseline and longitudinal B cell repertoires could serve as early biomarkers to help select candidates that will respond to therapy.
Discussion
In this study we developed a high-throughput DNA sequencing assay to measure circulating B cell repertoires in highly HLA-sensitized kidney transplant candidates undergoing desensitization therapy to lower HLA antibodies and enable transplantation. We hypothesized that measuring circulating B cell repertoires could help identify which candidates will respond to desensitization therapy. cPRA is a measure of sensitization based on pre-existing HLA antibody specificities and strength in combination with the frequency of HLA antigens in the donor population. Since cPRA also represents the probability of receiving a compatible transplant, this measurement is often used to determine success of desensitization therapy [
23,
31]. We assessed “response” to therapy as a clinically meaningful and durable decrease in cPRA of 5% points or greater based on literature and transplant outcomes data [
2,
26].
We first compared B cell repertoires in pre-treatment samples in three groups: sensitized candidates who responded to desensitization therapy, sensitized candidates who did not respond to desensitization therapy, and control candidates with low to moderate levels of HLA sensitization. Our results do not identify a single dominant B cell clone circulating in highly sensitized candidates compared to controls, but we cannot rule out the presence of one or more smaller clones that may be responsible for producing HLA antibodies. The analysis of several different metrics including the fraction of unique sequences, CDR3 length, V gene and V-J gene abundances, and isotype mutation frequencies argues against a common feature of the repertoire that predisposes candidates to high levels of HLA antibody production. Possible explanations are that the B cell clones circulate at low levels in the peripheral blood or the presence of more dominant B cells makes it difficult to detect by our assay. It has been suggested that no more than 2% of all B cells are present in the peripheral blood compared to the lymph nodes, spleen and bone marrow [
32], and the relevant HLA-specific B cell clones may be sequestered in another compartment and only rarely present in circulation.
Before desensitization therapy, responders had a slightly higher fraction of switched (IgG and IgA) isotypes compared to non-responders, but without a corresponding increase in mutation frequency. This shift may represent more memory B cells or plasmablasts in the circulation prior to therapy although the relatively small magnitude of the change would need to be validated in a larger study. Memory B cells are thought to be important in HLA sensitization as they can quickly transform into antibody-secreting plasma cells upon re-exposure to alloantigen [
33]. Thus, depleting these circulating memory B cells may reduce the number of plasma cells, and ultimately, the production of high affinity HLA antibodies.
We next analyzed changes in the B cell repertoires after desensitization therapy. After multiple doses of IVIG, we did not detect any changes in the B cell repertoire. IVIG is purified immunoglobulin products derived from pooled human plasma from thousands of donors and typically contains more than 95% unmodified IgG and trace amounts of IgA or IgM [
34]. IVIG has been used in transplantation to decrease HLA antibodies in order to help enable transplantation in highly sensitized patients [
23]. The mechanism is largely unknown but may involve binding to Fc receptors on immune cells, inhibiting IgG production or inducing B cell apoptosis [
35]. Our results suggest that IVIG inhibits IgG without reducing the number of B cells, although modest depletion of B cells may not be detected by our assay.
After rituximab, the changes in B cell repertoires were pronounced with significant decreases in the fraction of unique sequences, which is consistent with studies showing a long-lasting reduction in B cells after rituximab [
36]. We also observed an increase in switched isotypes after rituximab, with more than half of candidates’ repertoires containing 90% switched molecules. The mutation frequency of the switched molecules increased slightly after rituximab, but the mutation frequency in the remaining non-switched molecules increased substantially to levels comparable to the switched molecules. Our study was not large enough to determine the precise temporal dynamics of the repertoire change in each candidate following rituximab therapy. Other work reported that B cell depletion persists for more than 180 days following treatment [
36]. All but one of the post-rituximab samples was collected within 60 days following therapy with no significant differences in class-switched abundance or mutation frequency between samples collected within this interval. If the peak response following rituximab occurs after 60 days or later, our findings, which include many samples collected in the first 30 days, may underestimate the magnitude of the response. Larger studies are required to further characterize the individual variability in response after rituximab.
Rituximab, a chimeric CD20 monoclonal antibody, is postulated to decrease the production of HLA antibodies through targeting memory and naïve B cells without having any known effect on plasmablasts or plasma B cells, which do not express CD20 [
36,
37]. Our findings may suggest an increase or a persistence of memory B cells or plasmablasts after rituximab. These results are consistent with other work showing that rituximab can effectively decrease memory B cells, but the relative fraction of memory B cells in both peripheral blood and lymph nodes can also increase [
36,
38]. In addition, studies show that rituximab preferentially targets unmutated naïve B cells over class-switched mutated cells of the IgG and IgA isotypes [
36‐
38]. However, we did not find any significant differences in B cell repertoires between responders and non-responders after rituximab therapy. Similar work in a B cell mediated neuropathy was able to identify differences in abundance, mutation frequencies and persistent clones in B cell repertoires between patients that did and did not respond to rituximab [
39].
In the two candidates who received bortezomib close to rituximab, one showed changes similar to the other candidates receiving rituximab, whereas the other candidate showed little change. Bortezomib, a proteasome inhibitor, results in apoptosis of plasma cells although the effect on memory B cells is unknown [
24,
40]. In this limited analysis, we were unable to detect any additional effect of bortezomib in these two candidates, but larger studies are needed to confirm these preliminary findings.
Kidney transplantation and the accompanying immunosuppression resulted in the opposite effect on the B cell repertoires as rituximab. We interpret the increasing fraction of unique sequences, reduction in the abundance of switched isotypes and strong decrease in mutation frequency within non-switched isotypes as representing the repopulation of naïve B cells after transplantation and immunosuppression. It is possible that we are underestimating the magnitude of these effects since the four candidates that did not receive rituximab experienced larger changes than those that received rituximab. The sampling interval does not appear to affect the change in class-switched mutation frequency following transplantation with the exception of two post-transplant samples, showing a small increase in class-switched isotype abundance, which were collected approximately 10 days earlier than the others. At the time of transplantation, all candidates receive ATG, which depletes T cells and leads to apoptosis of many B cell lineages without a measurable effect on peripheral B cell counts [
30,
41,
42]. Reducing T cells may indirectly lower the mutation frequency since helper T cells are required for activating naïve B cells and triggering affinity maturation. Although MMF inhibits B cell proliferation with therapeutic uses in autoimmune disease, the exact effect on different B cell populations after transplantation is unknown [
43].
Strengths of this study include the single study design that allows uniform and longitudinal desensitization protocols and sample processing as well as prospective and consistent HLA antibody monitoring. Another strength is our assay, which can detect ample B cell transcripts even after depletion by rituximab and can measure isotype classes after IVIG infusions, which interfere in serum immunoglobulin measurements. The assay implements bulk repertoire sequencing from total RNA, which yields isotype information and a robust signal due to multiple RNA copies per cell. Variability in RNA expression levels between cells, however, has the negative effect of complicating estimates of cellular clonality. Using total RNA simplifies the workflow, is faster, and reduces cost compared with sorted cells, but some cell types are not distinguished, such as plasmablasts and memory B cells. Both of these cell types are class-switched with elevated mutation frequencies, but plasmablasts secrete antibodies whereas memory B cells do not.
Authors’ contributions
JFB, HCF, MUH, JMY, SRQ conceived and designed the experiments. RS, KJ, RC, MUH performed the experiments. JFB, HCF, EH, JMY, SRQ analyzed the data. JMY, JFB wrote the manuscript. All authors read and approved the final manuscript.