Selective targeting of human alloresponsive CD8+ effector memory T cells based on CD2 expression

Abstract

Costimulation blockade (CoB), specifically CD28/B7 inhibition with belatacept, is an emerging clinical replacement for calcineurin inhibitor-based immunosuppression in allotransplantation. However, there is accumulating evidence that belatacept incompletely controls alloreactive T cells that lose CD28 expression during terminal differentiation. We have recently shown that the CD2-specific fusion protein alefacept controls costimulation blockade-resistant allograft rejection in nonhuman primates. Here, we have investigated the relationship between human alloreactive T cells, costimulation blockade sensitivity and CD2 expression to determine whether these findings warrant potential clinical translation. Using polychromatic flow cytometry, we found that CD8(+) effector memory T cells are distinctly high CD2 and low CD28 expressors. Alloresponsive CD8(+) CD2(hi) CD28(-) T cells contained the highest proportion of cells with polyfunctional cytokine (IFNγ, TNF and IL-2) and cytotoxic effector molecule (CD107a and granzyme B) expression capability. Treatment with belatacept in vitro incompletely attenuated allospecific proliferation, but alefacept inhibited belatacept-resistant proliferation. These results suggest that highly alloreactive effector T cells exert their late stage functions without reliance on ongoing CD28/B7 costimulation. Their high CD2 expression increases their susceptibility to alefacept. These studies combined with in vivo nonhuman primate data provide a rationale for translation of an immunosuppression regimen pairing alefacept and belatacept to human renal transplantation.

Figure 1

Characterization of memory T cell subsets by polychromatic flow cytometry. (A) PBMCs were isolated from healthy volunteers and surface staining was performed. Lymphocytes were identified using forward and side scatter. CD3⁺ T cells were then subdivided based on CD4 and CD8 expression. Cells were segregated into memory subsets (T_N, T_CM, T_EM, T_EMRA) according to CCR7 and CD45RA expression. (B) CD2 mean fluorescence intensity (MFI) of T cell subsets as measured by polychromatic flow cytometry was performed in triplicate, and a representative sample is shown. (C) CD8⁺ and (D) CD4⁺ T cells were divided into CD2^hi and CD2^lo populations and then evaluated for memory phenotypes based on CCR7 and CD45RA expression.

Figure 2

Cytokine and cytotoxic effector molecule expression in alloreactive CD8⁺ cells. Responder PBMCs were incubated with irradiated, CD3⁺-depleted stimulator PBMCs for 6 hours directly ex vivo. Gating was performed by taking the nonaggregate population and selecting CD3⁺ cells. CD8⁺CD2⁺ T cells were subdivided into CD2^hi and CD2^lo subsets and analyzed for expression of (A) IFNγ, TNF and IL-2 or (B) IFNγ, CD107a and granzyme B, via intracellular cytokine staining.

Figure 3

Characterization of memory T cell subsets based on CD2 and CD28 expression. (A) CD8⁺ and (B) CD4⁺ T cells were gated based on CD2 and CD28 expression and divided into 3 subsets: CD2^loCD28⁺, CD2^hiCD28⁺ and CD2^hiCD28⁻. All three subsets were subsequently evaluated for memory T cell phenotypes by CCR7 and CD45RA expression. Representative results from one individual are shown.

Figure 4

Expression of intracellular cytokines and cytotoxic effector molecules by CD8⁺ T cells after alloantigen stimulation. CD8⁺ T cells were divided into subsets based on CD2 and CD28 expression. (A) Expression of IFNγ, TNF and IL-2 or (B) IFNγ, CD107a and granzyme B by CD8⁺ T cell subsets after 6 hour allostimulation.

Figure 5

Percentage of CD8⁺ T cells expressing multiple cytokine or cytotoxic effector molecules after alloantigen stimulation. (A) Percentage of dual (IFNγ and TNF) and (B) triple (IFNγ, TNF and IL-2) cytokine producers, and (C) percentage of dual cytotoxic effector molecule expressors (CD107a and granzyme B) among CD8⁺ CD2/CD28 T cell subsetsd.

Figure 6

Proliferation of alloreactive CD8⁺ T cells in one-way mixed lymphocyte reactions (MLRs). (A and B) CFSE-labeled responder PBMCs were allostimulated for 5 days in one-way MLRs. CD8⁺ T cells were divided based on proliferation and analyzed for memory subsets (CCR7 and CD45RA, left) or CD2 and CD28 expression (right). Percentage of divided CD8⁺ T cells is shown. PBMCs were treated with (A) PBS or (B) belatacept (100 µg/ml) at the start of incubation. CD2 MFI of nondividing and dividing populations in both (C) untreated and (D) belatacept treated samples was measured. Results of 7 experiments are shown.

Figure 7

Inhibition of proliferation of alloreactive CD8⁺ T cells in one-way MLRs. (A) CFSE-labeled PBMCs were allostimulated in one-way MLRs and treated with increasing doses of alefacept (0 to 10.0 µg/ml) alone (top row) or increasing doses of alefacept in combination with belatacept (100 µg/ml, bottom row). Percentage of divided CD8⁺ T cells is shown. (B) Percent of divided CD8⁺ T cells in untreated, belatacept, alefacept, and combined belatacept and alefacept treated samples from 7 unique responders is shown. Compared to untreated samples, * p<0.05, ** p<0.001.