Alloreactive CD154-expressing T-cell subsets with differential sensitivity to the immunosuppressant, belatacept: potential targets of novel belatacept-based regimens

Abstract

Belatacept blocks CD28-mediated T-cell costimulation and prevents renal transplant rejection. Understanding T-cell subset sensitivity to belatacept may identify cellular markers for immunosuppression failure to better guide treatment selection. Here, we evaluate the belatacept sensitivity of allo-antigen-specific CD154-expressing-T-cells, whose T-cytotoxic memory (TcM) subset predicts rejection with high sensitivity after non-renal transplantation. The belatacept concentration associated with half-maximal reduction (EC50) of CD154 expression was calculated for 36 T-cell subsets defined by combinations of T-helper (Th), Tc, T-memory and CD28 receptors, following allostimulation of peripheral blood leukocytes from 20 normal healthy subjects. Subsets were ranked by median EC50, and by whether subset EC50 was correlated with and therefore could be represented by the frequency of other subsets. No single subset frequency emerged as the significant correlate of EC50 for a given subset. Most (n = 25) T-cell subsets were sensitive to belatacept. Less sensitive subsets demonstrated a memory phenotype and absence of CD28 receptor. Potential drug-resistance markers for future validation include the low frequency highly differentiated, Th-memory-CD28-negative T-cells with the highest median EC50, and the least differentiated, high-frequency Tc subset, with the most CD28-negative T-cells, the third highest median EC50, and significant correlations with frequencies of the highest number of CD28-negative and memory subsets.

Figure 1

(A) Histogram showing the effect of belatacept on CD154+T-cells. Figure 1B. The effect of belatacept concentrations on nine CD154+T-cell subsets is modeled with best-fit four-parameter log-logistic function. Insets show calculated EC₅₀. Similar analyses have been performed for nine subsets of each of the Th, Tc and double negative (Dn, CD3+CD4−CD8−) T-cells.

Figure 2. T-cell subsets ranked by increasing (median±SEM) EC₅₀.

Red and green boxes identify subsets with the highest (>20 μg/ml) and lowest (<10 μg/ml) EC₅₀ values, respectively. T = T-cell, Tc = T-cytotoxic cell, Th = T-helper cell, M = memory, N = naïve, Dn=double negative T-cell (CD3 + CD4-CD8-).

Figure 3. Heatmap from hierarchical clustering analysis shows Spearman correlations between EC₅₀ values for each CD154+T-cell subset with frequencies of each of 36 T-cell subsets for 20 responders.

Spearman rho values range from −0.6 (green) to 0.6 (red). Tc EC₅₀ is correlated with frequencies of several T-cell subsets. ThM28- frequencies are correlated with EC₅₀ of several T-cell subsets. T = T-cell, Tc = T-cytotoxic cell, Th = T-helper cell, M = memory, N = naïve, Dn = double negative T-cell (CD3 + CD4-CD8-).

Figure 4. Plots showing mean Immunoreactivity Index (IR) values for T-cytotoxic Memory (TcM), T- cytotoxic (Tc) and T-helper CD28 negative (Th + 28−) cells which express CD154 in blood samples from 11 children who have received liver or intestine transplantation.

For each subset, mean values are shown for rejectors (n = 4) and non-rejectors (n = 7). Significantly greater IR values are seen in rejectors compared with non rejectors for the TcM and Tc subsets but not the ThM + 28− subset. Subjects received tacrolimus based immunosuppression and not belatacept.