Alefacept promotes co-stimulation blockade based allograft survival in nonhuman primates

Abstract

Memory T cells promote allograft rejection particularly in co-stimulation blockade-based immunosuppressive regimens. Here we show that the CD2-specific fusion protein alefacept (lymphocyte function-associated antigen-3-Ig; LFA -3-Ig) selectively eliminates memory T cells and, when combined with a co-stimulation blockade-based regimen using cytotoxic T lymphocyte antigen-4 (CTLA-4)-Ig, a CD80- and CD86-specific fusion protein, prevents renal allograft rejection and alloantibody formation in nonhuman primates. These results support the immediate translation of a regimen for the prevention of allograft rejection without the use of calcineurin inhibitors, steroids or pan-T cell depletion.

Figure 1

(a.) Rejection-free survival defined as the interval between the time of transplantation and the first allograft rejection event shown in days by treatment group. The duration of therapy is shown in the shaded bars along the x-axis. P values were determined by Student’s t-test (two-tailed) comparing each individual group versus the treatment group receiving LFA3-Ig, CTLA4-Ig, sirolimus, DST. This group had significantly prolonged rejection-free survival compared to all groups except the group receiving the combined therapy without DST. Similarly, the two groups receiving both LFA3-Ig and CTLA4-Ig, when considered together, had significantly prolonged survival compared to all other groups combined (p=0.002, two tailed Student’s t-test). The groups did not differ by sirolimus level or donor-directed mixed lymphocyte reactivity (See Supplementary Table 1 online). Two animals in the combined therapy group were killed with normal graft function after developing alloantibody. (b.) Polychromatic flow cytometry (PFC) was used to analyze LFA3-Ig’s depletional influence on PBMC T-cell subsets: naïve (T_N; CD4⁺CD28⁺CD95^low/−, β7 integrin^int and CD8⁺CD28⁺CD95^low/−, CD11a^low), central memory (T_CM; CD28⁺CD95⁺ or CD45RA⁻CD62L⁺ in both CD4⁺ and CD8⁺ cells), and effector memory (T_EM; CD4⁺CD28⁻CD95⁺or CD45RA^high and CD8⁺CD28⁻CD95⁺ or CD11a^high). Shown is a representative gate defining the three subsets for T-cells previously gated for CD3. (c.) The influence of LFA3-Ig treatment of peripheral T_EM-cell count levels is shown for all animals pre-transplant, 3 weeks post-transplant, and at terminal end points. Animals receiving LFA3-Ig are shown to the left in blue, and animals that did not receive LFA3-Ig are shown to the right in green. The mean values for each group are shown as black horizontal bars. T_M-cells were significantly reduced after 3 weeks of LFA3-Ig therapy with the greatest depletion seen in the T_EM (CD28⁻CD95⁺) subset of CD4+ and CD8+ T-cells (p=0.007, two-tailed Student’s t-test comparing pre and post transplant cell counts) without significant alteration in the absolute numbers of naive T-cells. This was not seen in non-LFA3-Ig treated animals. No significant difference in naïve T-cells was observed between LFA3-Ig and non-LFA3-Ig treated animals at each time point. At the time of rejection or sacrifice, T_EM counts in LFA3-Ig treated animals had returned to normal. (d.) There is an inverse relationship between the surface expression of CD2 (the target of LFA3-Ig) and CD28 (the relevant receptor blocked with CTLA4-Ig). Shown are results from PFC experiments performed on freshly isolated peripheral blood lymphocytes obtained from untreated animals. T-cells prior to treatment have a bimodal expression of CD2 and CD3, with antigen experienced cells having lower CD3 expression and higher CD2 expression (center panel). Shown is this relationship for CD8+ T-cells based on PFC plots. The CD3^hiCD2^lo cells are predominently T_N (upper right) while the CD3^loCD2^hi cells are predominently T_EM (lower left). T_CM cells are less distinctly segregated by these markers. (e.) Summary data from five untreated animals relating the mean fluorescnce intensity (MFI) of CD2 to the surface phenotype for both CD4⁺ and CD8⁺ T-cells. (f.) Renal allograft histological examination of necropsy specimens from a healthy, rejection-free animal with immunohistochemical (IHC) staining for T-cells (blue – CD3⁺), B-cells (brown – CD79⁺), and macrophages (pink/red – Ham-56) at low (left images) and higher (right images) magnification. Despite normal renal allograft function and no clinical signs/symptoms of rejection, multiple focal and highly organized cellular infiltrates where observed (lower panels). Interestingly, the cellular infiltrates were predominately aggregates of T- and B-cells, with minimal or no macrophages. In comparison, examination of rejecting animals using identical IHC staining demonstrated diffuse infiltrates without focal organization displaying greater cellular heterogeneity. Importantly, there were greater numbers of macrophages observed per objective field compared to the infiltrates seen in non-rejecting allografts, which has been shown to be of functional significance. Note the Ham-56 macrophage marker is also present on mesangial cells. As a result, glomeruli will also stain positive (pink) for Ham-56 as seen throughout the histological specimens.

Figure 2

Shown are studies investigating the relationship between CD2 expression and alloresponsiveness in mixed lymphocyte culture (MLC) as measured by proliferation and cytokine production. (a.) CFSE-labeled T-cells responding in MLC progressively lose fluorescence intensity by a factor of two permitting alloresponsive cells to be distinguished from non-dividing cells and each generation of division (generation #) to be separately evaluated by PFC. (b.) PFC evaluation of each CFSE generation for accessory molecule expression (CD4 or CD8) and CD2, the target of LFA3-Ig demonstrates that each progressive cell division is associated with a concomitant increase in surface CD2 expression, suggesting that the most alloresponsive cells are the most susceptible to LFA3-Ig treatment. (c.) Intracellular cytokine staining relating CD2 expression and cytokine secretion (IFN-γ, TNF-α, and/or IL-2) produced following allostimulation shows that T-cells with the most developed cytokine production capabilities express the highest density of surface CD2, with poly-functional cells, those expressing IFN-γ, TNF-α, and IL-2 (red bars), having the greatest CD2 density. (d.) Proliferation of both CD4⁺ and (e.) CD8⁺ T-cells in CFSE MLC is inhibited with CTLA4-Ig and LFA3-Ig, and the combination of both agents is more effective than either agent alone across a 3-log concentration range. Results of MLCs are shown for untreated cells (top), and cells treated with both agents at 10ug/ml (middle) demonstrating that treatment markedly attenuated proliferations. The bottom panels display summary data expressed as percent cells divided during MLC across 4 different concentrations of CTLA4-Ig and/or LFA3-Ig, (0.0, 0.1, 1.0, and 10 ug/ml). A dose dependent decrease in proliferation is seen with the combination therapy having the lowest proliferative capacity at all concentrations tested.