Glycolysis Changes in Alloreactive Memory B Cells in Highly Sensitized Kidney Transplant Recipients Undergoing Desensitization Therapy

Abstract

Despite the growing use of desensitization strategies, hyperimmune patients remain at high risk of antibody-mediated rejection suggesting that, even when donor-specific antibodies (DSA) are effectively depleted, anti-donor specific B cells persist. We included 10 highly sensitized recipients that underwent desensitization with plasmapheresis and B cell depletion prior to kidney transplantation. We quantified changes in DSA (luminex), total B-cell subsets (flow cytometry), anti-donor HLA B cells (fluorospot), and single-cell metabolism in serially collected samples before desensitization, at the time of transplant, and at 6 and 12 months thereafter. Desensitization was associated with a decrease in DSA and total memory B cell and naive B cell percentage, while plasma cells and memory anti-donor HLA circulating B cells persisted up to 12 months after transplant. At 12-month post-transplantation, memory B cells increased their glycolytic capacity, while proliferative KI67+ plasma cells modified their metabolism by increasing fatty acid and amino acid oxidation capacity and decreasing their glucose dependence. Despite effective DSA depletion, anti-donor B cells persist in kidney transplant recipients. Due to the reliance of these cells on glycolysis, glycolysis-targeting therapies might represent a valuable treatment strategy.

Keywords: desensitization; donor-specific antibody; glycolysis; kidney transplantation; memory B cells; metabolism.

FIGURE 1

Donor-specific antibodies change post desensitization and transplantation. Trends in the number of class I DSA (A), class II DSA (B), in all class I and II DSA MFI (C), in immunodominant class I and class II DSA (D), in class I sum of MFI (E) and in class II sum of MFI (F). Blue is for class I DSAs and Red is for class II DSAs. Pre-D: pre-desensitization.

FIGURE 2

B cell composition and change post desensitization and transplantation. Trends in B cells and plasma cells before and after desensitization and transplantation: total B cells (CD19⁺CD3⁻CD14⁻) (A), memory B cells (CD19⁺CD38⁻ CD24⁺ CD27⁺) (B), plasma cells (CD19⁺CD27^hi CD38^hi) (C), naïve B cells (CD19⁺CD27^– IgD⁺) (D). Transitional B cells (CD19⁺ CD27^– CD24^hi) (E). *p < .05 by Anova test. PBMC were also available for all 10 patients at the 4 timepoints.

FIGURE 3

Proliferative B cell composition and change post desensitization and transplantation. Trends in KI67 + B cells and plasma cells before and after desensitization and transplantation: total B cells (CD19⁺CD3⁻CD14⁻) (A), memory B cells (CD19⁺CD38⁻ CD24⁺ CD27⁺) (B), plasma cells (CD19⁺CD27^hi CD38^hi) (C), naïve B cells (CD19⁺CD27^– IgD⁺) (D). Transitional B cells (CD19⁺ CD27^– CD24^hi) (E). *p < .05 by Anova test. PBMC were also available for all 10 patients at the 4 timepoints.

FIGURE 4

Desensitization does reduce donor-specific HLA memory B cells. Donor-specific memory B cell differentiation into plasma cells assessed by FluoroSpot measuring donor-specific antibodies (spots) and expressed as mean number of spots per million of PBMC (A) and ratio between HLA-specific mBCs over the total polyclonal IgG mBCs in each patient (B). Red dots correspond to patients with rejection. *p < .05 by Anova test. PBMC were also available for all 10 patients at the 4 timepoints.

FIGURE 5

Desensitization and transplantation impact on B cell metabolism. Trends in B cell subsets metabolism profile evolution before desensitization (Pre-D), pre-transplantation (M0), at month-6 (M6) and at month-12 (M12) post-transplantation; total B cells (CD19⁺CD3⁻CD14⁻) (A), memory B cells (CD19⁺CD38⁻ CD24⁺ CD27⁺) (B), plasma cells (CD19⁺CD27hi CD38hi) (C), naïve B cells (CD19⁺CD27⁻ IgD⁺) (D). Transitional B cells (CD19⁺ CD27⁻ CD24hi) (E). The p-value is indicated the evolution of metabolism percentage between Pre-D and M12. *p < .05 by Wilcoxon test. FAO: fatty acids oxidation; AAO: amino acids oxidation. 4 patients had 1 timepoint missing.

FIGURE 6

Glycolysis inhibition results in a significant decrease in IgG secretion by activated memory B cell. IgG secretion assessed by ELISpot in 5 kidney transplant recipients and expressed as raw number of spots. OXPHOS: oxidative phosphorylation.