*Purpose: Background: Kidney recipients treated with the T-cell costimulation blocker, belatacept, experience a higher incidence of acute cellular rejection (ACR) and lower incidence of donor-specific anti-HLA antibodies. In our previous in vitro studies, the alloresponse of most T-cell subsets was inhibited half-maximally (EC50) at belatacept concentrations 10-20 mcg/ml. These belatacept EC50s were well below the belatacept trough concentration of 35 mcg/ml seen early during pivotal kidney transplant trials but exceeded belatacept trough levels of 4 mcg/ml seen later during the first post-transplant year, when ACR is more likely. Our previous studies have shown that EC50 of common immunosuppressants determined with in vitro testing are encompassed by the range of therapeutic trough concentrations of these agents. Purpose: To determine whether belatacept inhibits B-cell alloantigen presentation and B-cell alloresponse within known therapeutic ranges.

*Methods: Peripheral blood leucocytes (PBL) from healthy adults were cultured with fluorochrome-labeled antigenic lysate, or with intact PBL from HLA-mismatched healthy adults in the presence of belatacept at 0, 0.05, 0.5, 5, 20, 50 and 100 mcg/ml. Frequencies of B-cell subsets that presented alloantigenic lysate or expressed CD154 upon allostimulation were measured with flow cytometry. Subsets were CD19+B-cells, and their IgD+CD27-naïve, IgD+CD27+unswitched memory, IgD+CD27-CD24highCD38high transitional, IgD-CD27highCD38high plamablasts, and CD27- and CD27+isotype switched IgD-memory B-cells. EC50 was calculated with best-fit four-parameter log-logistic function under a Poisson assumption: f (x) = c + \frac{d−c}{1 + \ exp(b(\ log(x) − \ log(e)))} where b = slope, c = lower limit, d = upper limit, and e = EC50

*Results: Increasing concentrations of belatacept a) did not inhibit alloantigen presentation by any B-cell subset (n=3), b) were associated with decreasing frequencies of alloreactive CD154+B-cells and their subsets after overnight allostimulation in 10 replicate co-cultures. The median (range) EC50s were 1.3 (0.01-37) mcg/ml for unfractionated B-cells, 2 (0.03-50) mcg/ml for naïve, 3.6 (0.2-92) mcg/ml for unswitched memory, 6.2 (0.05-32) mcg.ml for transitional, and 7.8 (0.01-101) mcg/ml for plasmablasts. Median EC50s were highest at 17.9 and 31 mcg/ml, respectively for CD27- and CD27+ isotype-switched memory B-cells.

*Conclusions: In the first post-transplant year, B-cell alloresponses are likely to be suppressed half-maximally in most belatacept-treated kidney recipients, explaining why belatacept limits the development of DSA. This beneficial effect may not extend to those recipients with larger fractions of isotype-switched memory B-cells, which are less sensitive to belatacept.

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