Targeting human B-cell malignancies through Ig light chain-specific cytotoxic T lymphocytes

Abstract

Purpose: The variable regions of Ig (idiotype, Id) expressed by malignant B cells can be used as tumor-specific antigens that induce humoral and cellular immunity. However, epitopes derived from Id that stimulate human CD8(+) T-cell immunity are incompletely characterized.

Experimental design: The clonal Ig V(L) of human myeloma cell line U266 and five primary B-cell tumors were sequenced, and peptides corresponding to the Ig V(L) region were tested for their ability to stimulate CTLs from 10 HLA-A*0201-positive normal donors. The CTLs thus generated were tested against peptide-pulsed T2 cells and autologous tumor cells.

Results: Fourteen peptides derived from Ig light chain (V(L)) of U266 and primary B-cell tumors were used to generate 68 CTLs lines that specifically produced IFN-γ when cocultured with peptide-pulsed T2 cells. These CTLs lysed peptide-pulsed T2 cell as well as U266 or autologous tumor targets in an HLA class I-dependent manner. Sequence analysis revealed shared V(L) T-cell epitopes in U266 and primary B-cell tumors, not previously reported within Ig heavy chain (V(H)) sequences.

Conclusion: This study thus identifies novel immunogenic CTLs epitopes from Id V(L), suggests that they are naturally presented on the surface of B-cell malignancies, and supports their inclusion in next-generation Id vaccines. The ability to prime T cells derived from normal HLA-matched donors, rather than patients, may also have direct application to current strategies, designed to generate allogeneic tumor-specific T cells for adoptive transfer.

Figure 1

Generation of Id V_L peptide-specific T cells from 10 normal donors (ND1–10). (A) IFN-γ secretion by U266 myeloma cell line V_L peptide-specific CTLs after stimulation with peptide-pulsed T2 cells or HIV-Gag77 peptide-pulsed T2 cells (negative control). Influenza A virus M1(58–66) peptide was used as a positive control. Id V_L peptide sequences and characteristics are shown in Table.1 (B) IFN-γ secretion by primary B-cell tumor-derived V_L peptide specific CTLs against peptide pulsed or HIV-Gag77 peptide-pulsed T2 cells. (C) Intracellular cytokine staining assay of peptide-specific CTLs in response to V_L peptide or HIV-Gag77 peptide-pulsed T2 cells. * indicates P<0.05.

Figure 2

The cytotoxic function of V_L peptide-specific CTLs against Id peptide or control HIV peptide pulsed T2 cells. (A)The cytotoxic function of U266 derived V_L peptide-specific CTLs was tested against T2 cells pulsed with U266 or control HIV peptide. (B) The cytotoxic function of donor CTL raised against primary B-cell tumor-derived V_L peptides was tested against T2 cells pulsed with the respective human tumor-derived or control HIV peptide. Results are representative of three independent experiments.

Figure 3

Cytotoxic function of V_L peptide-specific normal donor CTLs against U266 and primary human B-cell tumor targets. (A) U266 V_L peptide-specific (P19, P20, P21, P23, P25, P26, P27, P28, P29) CTLs generated from a normal donor (ND1) lysed U266 myeloma cells but not ARP-1 (HLA A2−, irrelevant Id) or XG-1 (HLA A2+, irrelevant Id) myeloma cell lines. (B) Human tumor-derived V_L peptide-specific (L50 and K18) CTLs lysed the respective primary tumor cells: Tumor (CLL1) and Tumor (PL1), but not tumors expressing irrelevant Id: Tumor (control), or autologous patient’s tumor-free PBMC: PBMC(CLL1) and PBMC(PL1).

Figure 4

HLA restriction of of V_L peptide specific CTLs. (A) HLA class I but not HLA class II specific antibody or isotype control antibody (10 µg/ml) blocked the cytolysis of U266 tumor cells by U266 V_L peptide-specific CTLs. (B) HLA class I but not HLA class II antibody or isotype control antibody (10 µg/ml) blocked the cytolysis of primary human CLL and PL tumor cells, respectively by V_L peptide-specific CTLs. (C) U266 P21 peptide-specific CTLs specifically lysed HLA-A*0201+ primary FL cells that shared the P21 epitope: Tumor (FL4, P21+), but not the same patients’ PBMC: PBMC(FL4) or HLA-A*0201 tumor cells that did not express P21(FL5,P21−).