Human regulatory T cells kill tumor cells through granzyme-dependent cytotoxicity upon retargeting with a bispecific antibody

Abstract

A major mechanism by which human regulatory T cells (T(regs)) have been shown to suppress and kill autologous immune cells is through the granzyme-perforin pathway. However, it is unknown whether T(regs) also possess the capacity to kill tumor cells using similar mechanisms. Bispecific antibodies (bscAbs) have emerged as a promising class of therapeutics that activate T cells against tumor antigens without the need for classical MHC-restricted TCR recognition. Here, we show that a bscAb targeting the tumor-specific mutation of the epidermal growth factor receptor, EGFRvIII, redirects human CD4(+)CD25(+)FoxP3(+) T(regs) to kill glioblastoma (GBM) cells. This activity was significantly abrogated by inhibitors of the granzyme-perforin pathway. Notably, analyses of human primary GBM also displayed diffuse infiltration of granzyme-expressing FoxP3(+) T cells. Together, these data suggest that despite their known suppressive functions, tumor-infiltrating T(regs) possess potent cytotoxic mechanisms that can be co-opted for efficient tumor cell lysis.

Keywords: Bispecific Antibodies; Glioblastoma; Granzymes; Immunomodulation; Regulatory T Cells.

Figure 1

BscEGFRvIIIxCD3 activates highly-purified T_regs but does not reverse defects in cytokine secretion and proliferation in response to stimulation. (A) Representative flow cytometric analysis of purified CD4⁺CD25⁺CD127^dim/− T_regs demonstrates greater than 97% purity as determined by FoxP3 and CD25 phenotypic markers. (B) Purified T_regs express elevated levels of activation markers CD69 and CD25 in response to bscEGFRvIIIxCD3 specifically in the presence of EGFRvIII-expressing tumors when compared to cells incubated with non-specific, control BiTE. These activity was consistent and statistically significant among lymphocyte donors from three separate individuals (C). (D) Supernatants from wells containing U87MG.ΔEGFR, bscEGFRvIIIxCD3 and T_regs contained significantly lower levels inflammatory cytokines compared to wells in which responder cells consisted of purified CD4⁺CD25⁻ helper T cells (T_h). (E) Proliferation of T_regs and T_h in response to bscEGFRvIIIxCD3 and solid phase EGFRvIII as measured by ³H-thymidine incorporation demonstrates that proliferative defects in the T_reg compartment persist following activation with bscEGFRvIIIxCD3. Statistical analysis in (D) and (E) were performed in triplicate wells with lymphocytes from a single donor and all experiments were repeated twice. Horizontal bars represent a statistical significance of P < 0.05.

Figure 2

BscEGFRvIIIxCD3 activates T_regs to elevate expression of granzymes and perforin specifically in the presence of tumor cells expressing EGFRvIII. (A) Human PBMCs were incubated in the presence of U87MG.ΔEGFR and control BiTE (left) or EGFRvIII BiTE (right), harvested and stained for flow cytometric analysis. CD4⁺ cells were isolated and gated for high CD25 and FoxP3 expression and then analyzed for Perf, GrA and GrB expression with positivity determined by isotype control. Plots are representative of at least three repeated experiments. (B) Percent positive cells expressing Perf, GrA or GrB were determined to be significantly elevated in the presence of EGFRvIII BiTE over non-specific control BiTE across lymphocytes from three separate healthy donors. (C) Flow cytometric plot of backgated GrB-positive cells from Figure 2A corresponds to a discrete population (dots) with dual positivity for CD25 and FoxP3. All experiments were repeated twice. Horizontal bars represent a statistical significance of P < 0.05 between groups of three donors each defined by the presence of either EGFRvIII-specific or control BiTE.

Figure 3

T_regs expressing GrB are present in human glioma and possess cytotoxic activity against EGFRvIII-expressing tumor in the presence of EGFRvIII BiTE. (A) Upon redirection with bscEGFRvIIIxCD3, but not a non-specific control bscAb (Control), activated T_regs demonstrate enhanced lysis against EGFRvIII-expressing tumor (T_reg:target, 20:1; incubation time 18 hours; [BiTE] 10 μg/mL). (B) Specific lysis against target tumor cells expressing EGFRvIII is not significantly inhibited by blockade of Fas ligand- and TRAIL-mediated apoptosis but is significantly abrogated by partial inhibitors of the granzyme-perforin pathway, Z-AAD-CMK, EGTA and CMA. Pairwise comparisons with respect to T_reg, BiTE and inhibitors of the granzyme-perforin pathway were made. All tests were performed in triplicate wells and independently repeated. Horizontal bars represent a statistical significance between compared groups of P < 0.05. (C) IHC analysis of human GBM shows diffuse infiltration of FoxP3⁺ T_regs (AEC) expressing detectable levels of granzyme B (DAB).