Antigen-dependent CD28 signaling selectively enhances survival and proliferation in genetically modified activated human primary T lymphocytes

Abstract

Most tumor cells function poorly as antigen-presenting cells in part because they do not express costimulatory molecules. To provide costimulation to T lymphocytes that recognize tumor cells, we constructed a CD28-like receptor specific for GD2, a ganglioside overexpressed on the surface of neuroblastoma, small-cell lung carcinoma, melanoma, and other human tumors. Recognition of GD2 was provided by a single-chain antibody derived from the GD2-specific monoclonal antibody 3G6. We demonstrate that the chimeric receptor 3G6-CD28 provides CD28 signaling upon specific recognition of the GD2 antigen on tumor cells. Human primary T lymphocytes retrovirally transduced with 3G6-CD28 secrete interleukin 2, survive proapoptotic culture conditions, and selectively undergo clonal expansion in the presence of an antiidiotypic antibody specific for 3G6-CD28. Polyclonal CD8(+) lymphocytes expressing 3G6-CD28 are selectively expanded when cultured with cells expressing allogeneic major histocompatibility complex class I together with GD2. Primary T cells given such an antigen-dependent survival advantage should be very useful to augment immune responses against tumor cells.

Figure 1

G_D2 expression in Jurkat and EL4 cells. Cells were stained with mAb 3F8 (solid line) or isotype-matched mAb 20.4 (broken line), directed against G_D2 and human LNGFR, respectively. (a) Jurkat cells; (b) wild-type EL4; (c) EL4^GD2−. The three cell lines are, respectively, G_D2 ^low, G_D2 ⁺, and G_D2 ⁻. G_D2 expression by these cells is closely correlated with the costimulatory activity described in Table 1.

Figure 2

Retroviral gene transfer of 3G6-CD28, 3G6-CD28TR, and NTP in human primary lymphocytes. PBLs were stained 72 h after retroviral infection with antibodies to CD8 (x-axis) and either A1G4 mAb to stain 3G6-CD28 and 3G6-CD28TR or 20.4 mAb to stain NTP (y-axis). Staining with secondary antibody alone (GAR-PE; A) indicates the background staining. Staining with A1G4 (antiidiotype; B and C) and with 20.4 (anti– NTP; D), followed by the corresponding PE conjugates.

Figure 3

3G6-CD28–transduced primary T lymphocytes selectively survive CD3-dependent cell death in the presence of A1G4. Primary T lymphocytes transduced with NTP, 3G6-CD28, or 3G6-CD28TR were kept for 3 d under one of the following conditions: medium without antibody, with OKT3, with OKT3 plus 9.3, or with OKT3 plus A1G4. On day 3, the T cells were released from the cross-linking conditions, and cell viability was measured by trypan blue exclusion and FACS^® analysis. The data represent the mean ± SD of four experiments. Cell viability decreased in the presence of the plate-bound OKT3 from 77 ± 9% to 47 ± 7% in the NTP-transduced T cells, and from 77 ± 8% to 56 ± 4 and 54 ± 7% in the 3G6-CD28 and 3G6-CD28TR–transduced groups, respectively. Viability was unchanged by the addition of A1G4 to NTP- and 3G6-CD28TR– transduced T cell groups (51 ± 3 and 53 ± 7%, respectively), but increased to 77 ± 7% in the 3G6-CD28–transduced T cell populations (which were ∼30% A1G4⁺). Survival in all three groups was comparable in the presence of anti-CD3 and anti-CD28 antibodies (85 ± 8%).

Figure 4

Antiidiotypic mAb A1G4 and cell-bound G_D2 provide costimulatory signals to 3G6-CD28–transduced human primary T lymphocytes. (A and B) Primary T lymphocytes transduced with 3G6-CD28 or 3G6-CD28TR were cultured with soluble OKT3 (10 ng/ml) alone or with the addition of soluble A1G4 or 9.3 mAb as described in Materials and Methods. The percentage of transduced cells was measured by FACS^® analysis on days 6 and 12. When cultured in the presence of OKT3 and A1G4 antibodies, T lymphocytes transduced with 3G6-CD28 increased from 30% A1G4⁺ on day 0 to 60% on day 6 and to 88% on day 12, but remained at ∼30% when cultured otherwise (data represent one of three independent experiments). (C and D) HLA A2.1⁻ primary T lymphocytes transduced with 3G6-CD28 (C) or 3G6-CD28TR (D) were cocultured in triplicate wells with irradiated monolayers of fibroblasts as described in Materials and Methods. The fraction of A1G4⁺ cells was measured by FACS^® analysis in both CD4⁺ and CD8⁺ subsets on days 6 and 12. In cultures with 3T3-A2.1/G_D2 the percentage of CD8⁺A1G4⁺ T cells in the 3G6-CD28–transduced population increased from 10 ± 2% on day 0 to 15 ± 2% on day 6 and to 32 ± 4% on day 12 (C). The percentage of CD4⁺A1G4⁺ cells in the CD4⁺ T cell population remained unchanged under all of these coculture conditions (data not shown). Data represent one of three independent experiments.