B cell reconstitution following alemtuzumab induction under a belatacept-based maintenance regimen

Abstract

Lymphocyte depletion has been shown to control costimulation blockade-resistant rejection but, in some settings, to exacerbate antibody-mediated rejection (AMR). We have used alemtuzumab, which depletes T and B cells, combined with belatacept and rapamycin and previously reported control of both costimulation blockade-resistant rejection and AMR. To evaluate this regimen's effect on B cell signatures, we investigated 40 patients undergoing this therapy. B cell counts and phenotypes were interrogated using flow cytometry, and serum was analyzed for total IgG, IgM, and donor-specific alloantibody (DSA). Alemtuzumab induction produced pan-lymphocyte depletion; B cells repopulated faster and more completely than T cells. Reconstituting B cells were predominantly naïve, and memory B cells were significantly reduced (P = .001) post repopulation. Two B cell populations with potential immunomodulatory effects-regulatory (CD38^hi CD24^hi IgM^hi CD20^hi ) and transitional B cells (CD19⁺ CD27^- IgD⁺ CD38^hi )-were enriched posttransplant (P = .001). Total serum IgG decreased from baseline (P = .016) while IgM levels remained stable. Five patients developed DSAs within 36 months posttransplant, but none developed AMR. Baseline IgG levels in these patients were significantly higher than those in patients without DSAs. These findings suggest that belatacept and rapamycin together limit homeostatic B cell activation following B cell depletion and may lessen the risk of AMR. This regimen warrants prospective, comparative study. ClinicalTrials.gov NCT00565773.

Keywords: B cell biology; basic (laboratory) research/science; clinical research/practice; costimulation; immunosuppression/immune modulation; immunosuppressive regimens - induction; lymphocyte biology: differentiation/maturation.

Figure 1:. Patient demographics. Clinical trial conduct with regard to donors, donor bone marrow infusion, and maintenance immunosuppressive regimen.

All patients received alemtuzumab induction followed by belatacept maintenance therapy, plus sirolimus (or MMF in patients who were intolerant to sirolimus). The opportunity to wean off sirolimus (or MMF) followed by belatacept monotherapy was offered to patients who met prespecified criteria at 12 months posttransplantation. Unsuccessfully weaning was determined by evidence of rejection (clinical or subclinical) on biopsy. No patient had clinical rejection in the first year. * All episodes of CMV or EBV were transient low positive defined as values < 300 copies/mL.

Figure 2:. Repopulation of peripheral blood CD20⁺ B cells after renal allograft transplantation (n=40).

The absolute lymphocyte cell counts and subsets are analyzed by polychromatic flow cytometry, and a representative dot plots of gating strategy for B cell analysis (top). Profound T cell and B cell (CD3⁻CD20⁺) depletion is achieved with alemtuzumab induction followed by rapid repopulation to baseline level at 6 months, then significantly exceeding baseline values thereafter (bottom). The box borders indicate 75^th and 25^th percentile, and the line within box indicates median. Upper and lower whiskers represent 90^th and 10^th percentiles. The dots represent fifth and 95^th percentiles.

Figure 3.. Postdepletional reconstitution of naïve and memory B cell subsets after kidney transplantation (n=40).

(a) Absolute numbers and frequencies over time of CD27⁻IgD⁺ naïve B cells showing rapid repopulation post–alemtuzumab induction. (b) Absolute numbers and frequencies over time of memory B cells, including CD27⁺IgD⁻ (switched), CD27⁺IgD⁺ (unswitched), and CD27⁻IgD⁻ (exhausted) memory subsets. Alemtuzumab induction and belatacept-based immunotherapy significantly inhibits the repopulation of memory B cells. (c) Analysis of naïve and memory B cells using Bm1-Bm5 classification based on CD38/IgD expression shows that repopulating B cells are arrested at the Bm2 stage, consisting largely of naïve B cells. The Bm2’ subset, considering mainly regulatory/transitional cells, increases significantly over time during B cell repopulation. The box borders indicate 75^th and 25^th percentile, and the line within box indicates median. Upper and lower whiskers represent 90^th and 10^th percentiles. The dots represent fifth and 95^th percentiles.

Figure 3.. Postdepletional reconstitution of naïve and memory B cell subsets after kidney transplantation (n=40).

(a) Absolute numbers and frequencies over time of CD27⁻IgD⁺ naïve B cells showing rapid repopulation post–alemtuzumab induction. (b) Absolute numbers and frequencies over time of memory B cells, including CD27⁺IgD⁻ (switched), CD27⁺IgD⁺ (unswitched), and CD27⁻IgD⁻ (exhausted) memory subsets. Alemtuzumab induction and belatacept-based immunotherapy significantly inhibits the repopulation of memory B cells. (c) Analysis of naïve and memory B cells using Bm1-Bm5 classification based on CD38/IgD expression shows that repopulating B cells are arrested at the Bm2 stage, consisting largely of naïve B cells. The Bm2’ subset, considering mainly regulatory/transitional cells, increases significantly over time during B cell repopulation. The box borders indicate 75^th and 25^th percentile, and the line within box indicates median. Upper and lower whiskers represent 90^th and 10^th percentiles. The dots represent fifth and 95^th percentiles.

Figure 3.. Postdepletional reconstitution of naïve and memory B cell subsets after kidney transplantation (n=40).

(a) Absolute numbers and frequencies over time of CD27⁻IgD⁺ naïve B cells showing rapid repopulation post–alemtuzumab induction. (b) Absolute numbers and frequencies over time of memory B cells, including CD27⁺IgD⁻ (switched), CD27⁺IgD⁺ (unswitched), and CD27⁻IgD⁻ (exhausted) memory subsets. Alemtuzumab induction and belatacept-based immunotherapy significantly inhibits the repopulation of memory B cells. (c) Analysis of naïve and memory B cells using Bm1-Bm5 classification based on CD38/IgD expression shows that repopulating B cells are arrested at the Bm2 stage, consisting largely of naïve B cells. The Bm2’ subset, considering mainly regulatory/transitional cells, increases significantly over time during B cell repopulation. The box borders indicate 75^th and 25^th percentile, and the line within box indicates median. Upper and lower whiskers represent 90^th and 10^th percentiles. The dots represent fifth and 95^th percentiles.

Figure 4.. Alemtuzumab induction and belatacept/rapamycin-based immunotherapy promotes transitional/regulatory B cell repopulation after kidney transplantation (n=40).

(a) Transitional B cells characterized as CD27⁻IgD⁺CD28^hi subset demonstrate rapid expansion postdepletion induction. (b) CD24^hiCD38^hi regulatory B cells rapidly repopulate over baseline levels, posttransplantation, and maintain significant high levels through 36 months of study when compared with baseline. The box borders indicate 75^th and 25^th percentile, and the line within box indicates median. Upper and lower whiskers represent 90^th and 10^th percentiles. The dots represent fifth and 95^th percentiles.

Figure 5.. Measurement of serum total IgM and IgG concentrations before and after transplantation.

Serum samples longitudinally collected from patients (n=40) were tested for total IgM and IgG levels as measured by ELISA. (a) Patients undergoing depletion induction followed by belatacept-based immunotherapy demonstrate reduction of IgG but not IgM over 36 months of study. (b) Patients with detectable donor-specific alloantibody (DSA) demonstrated lower IgM levels after transplantation, and higher IgG levels before transplantation when compared with patients without DSA. The box borders indicate 75^th and 25^th percentile, and the line within box indicates median. Upper and lower whiskers represent 90^th and 10^th percentiles. The dots represent fifth and 95^th percentiles.