High frequencies of less differentiated and more proliferative WT1-specific CD8+ T cells in bone marrow in tumor-bearing patients: an important role of bone marrow as a secondary lymphoid organ

Abstract

In tumor-bearing patients, tumor-associated antigen (TAA)-specific CTLs are spontaneously induced as a result of immune response to TAAs and play an important role in anti-tumor immunity. Wilms' tumor gene 1 (WT1) is overexpressed in various types of tumor and WT1 protein is a promising pan-TAA because of its high immunogenicity. In this study, to clarify the immune response to the WT1 antigen, WT1-specific CD8(+) T cells that were spontaneously induced in patients with solid tumor were comparatively analyzed in both bone marrow (BM) and peripheral blood (PB). WT1-specific CD8(+) T cells more frequently existed in BM than in PB, whereas frequencies of naïve (CCR7(+) CD45RA(+)), central memory (CCR7(+) CD45RA-), effector-memory (CCR7- CD45RA(-)), and effector (CCR7- CD45RA(+)) subsets were not significantly different between BM and PB. However, analysis of these subsets for the expression of CD57 and CD28, which were associated with differentiation, revealed that effector-memory and effector subsets of the WT1-specific CD8(+) T cells in BM had less differentiated phenotypes and more proliferative potential than those in PB. Furthermore, CD107a/b functional assay for WT1 peptide-specific cytotoxic potential and carboxyfluorescein diacetate succinimidyl ester dilution assay for WT1 peptide-specific proliferation also showed that WT1-specific CD8(+) T cells in BM were less cytotoxic and more proliferative in response to WT1 peptide than those in PB. These results implied that BM played an important role as a secondary lymphoid organ in tumor-bearing patients. Preferential residence of WT1-specific CD8(+) T cells in BM could be, at least in part, explained by higher expression of chemokine receptor CCR5, whose ligand was expressed on BM fibroblasts on the WT1-specific CD8(+) T cells in BM, compared to those in PB. These results should provide us with an insight into WT1-specific immune response in tumor-bearing patients and give us an idea of enhancement of clinical response in WT1 protein-targeted immunotherapy.

Figure 1

Frequencies of WT1 tetramer⁺ CD8⁺ T cells in bone marrow (BM) and peripheral blood (PB) in patients with solid tumors. (A) Representative flow cytometric analysis of WT1 tetramer⁺ CD8⁺ T cells. Mononuclear cells from BM and PB were gated on CD8⁺, CD4⁻, CD14⁻, CD16⁻, CD19⁻, CD33⁻, CD34⁻, and CD56⁻ cells, and WT1 tetramer⁺ CD8⁺ T cells were defined as WT1‐specific CD8⁺ T cells. (B) Frequencies of WT1 tetramer⁺ CD8⁺ T cells in CD8⁺ T cells in BM (closed circles) and PB (open circles) from patients (Pt), and PB from healthy donors (HD; open triangles). The horizontal bars indicate median values of the frequencies.

Figure 2

Subset composition of WT1 tetramer⁺ CD8⁺ T cells in bone marrow (BM) and peripheral blood (PB). (A) WT1 tetramer⁺ CD8⁺ T cells are divided into four subsets according to the expression of CCR7 and CD45RA and differentiate as follows: naïve (N), CCR7⁺ CD45RA⁺ → central memory (CM), CCR7⁺ CD45RA⁻ → effector‐ memory (EM), CCR7⁻ CD45RA⁻ → effector (E), CCR7⁻ CD45RA⁺. (B) Representative subset analysis of WT1 tetramer⁺ CD8⁺ T cells in BM and PB. Frequencies of N, CM, EM, and E subsets are shown. (C), Frequencies of each subset of WT1 tetramer⁺ CD8⁺ T cells in BM (closed circles) and PB (open circles) are shown. The horizontal bars indicate median values of the frequencies.

Figure 3

Effector‐memory (EM) and effector (E) subsets of WT1 tetramer⁺ CD8⁺ T cells in bone marrow (BM) had less differentiated and more proliferative phenotypes than those in peripheral blood (PB). (A) Frequencies of CD57⁺ cells in EM and E subsets of WT1 tetramer⁺ CD8⁺ T cells in BM (closed circles) and PB (open circles). (B) Representative dot‐plots of FACS analysis of CD28 and CD57 expression in the EM subset. Numbers represent frequencies (%) of cells in each fraction. (C) Frequencies of CD28⁺ T cells in the CD57⁺ or CD57⁻ EM subset in WT1 tetramer⁺ CD8⁺ T cells in BM (closed circles) and PB (open circles). (D) Frequencies of CD57⁺ T cells in the CD28⁺ or CD28⁻ EM subset in WT1 tetramer⁺ CD8⁺ T cells in BM (closed circles) and PB (open circles). Two patients were not evaluated because of the small number of cells. Horizontal bars indicate median values of the frequencies. n.s., not significant.

Figure 4

Proliferative potential of WT1‐reactive CD8⁺ T cells. Carboxyfluorescein diacetate succinimidyl ester (CFSE)‐labeled bone marrow (BM) and peripheral blood (PB) mononuclear cells were stimulated with WT1 peptides, and CFSE dilution was analyzed by FACS. (A) Representative flow cytometric analysis of CFSE dilution in interferon (IFN)‐γ⁺ CD8⁺ CD3⁺ T cells. (B) CFSE mean fluorescence intensity (MFI) of IFN‐γ⁺ CD8⁺ CD3⁺ T cells in BM (closed circles) and PB (open circles). Horizontal bars indicate median values of MFI.

Figure 5

Cytotoxic potential of WT1 tetramer⁺ CD8⁺ T cells. Mononuclear cells from bone marrow (BM) and peripheral blood (PB) were stimulated with WT1 peptide, then CD107a/b cell surface expression was examined. (A) Frequencies of CD107a/b⁺ cells in WT1 tetramer⁺ CD8⁺ T cells in BM (closed circles) and PB (open circles). (B) Frequencies of CD107a/b⁺ cells in effector‐memory (EM) and effector (E) subsets in WT1 tetramer⁺ CD8⁺ T cells in BM (closed circles) and PB (open circles). Horizontal bars indicate median values of the frequencies.

Figure 6

Expression of chemokine receptor CCR5 on WT1 tetramer⁺ CD8⁺ T cells. Frequencies of CCR5⁺ cells in WT1 tetramer⁺ CD8⁺ T cells in bone marrow (BM; closed circles) and peripheral blood (PB; open circles). Horizontal bars indicate median values of the frequencies.