Human heat shock protein-specific cytotoxic T lymphocytes display potent antitumour immunity in multiple myeloma

Abstract

Tumour cell-derived heat shock proteins (HSPs) are used as vaccines for immunotherapy of cancer patients. However, it is proposed that the peptide chaperoned on HSPs, not HSPs themselves, elicited a potent immune response. Given that HSPs are highly expressed by most myeloma cells and vital to myeloma cell survival, we reasoned that HSPs themselves might be an ideal myeloma antigen. In the present study, we explored the feasibility of targeting HSPs themselves for treating multiple myeloma. We identified and chose HLA-A*0201-binding peptides from human HSPB1 (HSP27) and HSP90AA1 (HSP90), and confirmed their immunogenicity in HLA-A*0201 transgenic mice. Dendritic cells pulsed with HSPB1 and HSP90AA1 peptides were used to stimulate peripheral blood mononuclear cells from healthy volunteers and myeloma patients to generate HSP peptide-specific cytotoxic T lymphocytes (CTLs). HSP peptide-specific CTLs efficiently lysed HLA-A*0201(+) myeloma cells (established cell lines and primary plasma cells) but not HLA-A*0201(-) myeloma cells in vitro, indicating that myeloma cells naturally express HSP peptides in the context of major histocompatibility complex class I molecules. More importantly, HSP peptide-specific CTLs effectively reduced tumour burden in the xenograft mouse model of myeloma. Our study clearly demonstrated that HSPs might be novel tumour antigens for immunotherapy of myeloma.

Keywords: heat shock proteins; immunotherapy; multiple myeloma; vaccine.

Figure 1. Expression of HSP genes and proteins in normal and myeloma plasma cells

(A) HSP expression is elevated in most myeloma tumour cells. Levels of HSP90AA1, HSPA13, and HSPB1 expression were assessed by gene array in bone marrow CD138⁺ plasma cells from 24 healthy controls and 350 newly diagnosed myeloma patients. Each vertical bar represents an individual sample. Immunohistochemical staining of MM bone marrow biopsies for HSP90AA1 (B), HSPA13 (C) and HSPB1 (D) expression in bone marrow CD138⁺ plasma cells, which show that only myeloma plasma cells but no normal haematopoietic cells were positively stained (original magnification × 400).

Figure 2. Binding affinity and in vivo immunogenicity of HSP peptides

Peptide binding assay showing (A) affinity and (B) stability (fluorescence index) of HLA-A*0201-binding peptides from human HSP. HIV-pol and Flu-matrix peptides were used as positive controls. In these studies, T2 cells were incubated with100 µg/ml peptides overnight, or with 100 µg/ml peptides for different time points, and analysed as detailed in Materials and methods. We identified and selected four peptides that could potentially bind to HLA-A*0201 molecules: two peptides (aa362 and aa670) derived from HSP90AA1α, one (hp27) from HSPB1, and one (hp70) from HSPA13. We chose the HSP90AA1α-derived (aa670) and HSPB1-derived (hp27) peptides for the following experiments on the basis of their high binding affinity (Figure 2A). Also shown are (C) T cell proliferation detected by CFSE-labelled assay, (D) Intracellular staining for CD8⁺ IFN-γ–expressing T cells, (E) HLA-A*0201-peptide-tetramer staining showing HSP peptide-specific CD8⁺ T cells, and (F) Cytotoxicity of splenocytes of mice immunized with Flu-matrix peptide (Flu), hp27, or aa670 peptides. Splenocytes were re-stimulated with (+Peptide) or without (-Peptide) the immunizing peptides for 5 days before analysis. Representative results of 3 independent experiments are shown. *P < 0.05; **P < 0.01.

Figure 3. Circulating and stimulated HSP-specific T cells in healthy volunteers and patients with MM

(A) HLA-A*0201-peptide-tetramer staining showing the presence and percentages of HSP peptide-specific CD8⁺ T cells in the blood of 3 HLA-A*0201⁺ patients (pt1-pt3) with multiple myeloma (MM) and a healthy volunteer (HV). (B) HLA-A*0201 peptide-tetramer staining showing the representative frequency of HSP peptide-specific CD8⁺ T cells in cultures during in vitro stimulations. Similar results were obtained with T-cell lines from other MM patients or healthy volunteers. Also shown are proliferative responses measured by (C) CFSE dilution assay or (D) MTT assay of T cell lines specific for hp27 or aa670 generated from a HLA-A*0201⁺ healthy volunteers in response to unpulsed dendritic cells (DCs) or DCs pulsed with hp27 or aa670 peptides. In D, different ratios of T cell:DC were used. *P < 0.05; **P < 0.01.

Figure 4. Cytotoxicity of HSP peptide-specific CTL lines against myeloma cells

Shown are percentages of cytotoxicity detected by ⁵¹Cr-release (⁵¹Cr, upper panels) or lactate dehydrogenase (LDH) release (LDH; lower panels) assays of hp27- and aa670-specific T cell lines from HLA-A*0201⁺ myeloma patients and healthy volunteers. The primary myeloma cells isolated from the bone marrow of multiple myeloma (MM) patients could not survive more than 48 h in vitro, indicating that all of the primary myeloma cells examined in the cytotoxicity assay were allogeneic. The HSPB1- and HSP90AA1-specific T cell lines were derived from different patients and healthy volunteers, details can be found in Table III. The HSP-specific T cell lines from the same origin were used to test their cytotoxicity not only on primary myeloma cells but also the established cell lines. In brief, T cell lines examined in the LDH release of HSPB1 and HSP90AA1 on MM cell lines, and the ⁵¹Cr-release of HSPB1 and HSP90AA1 were all derived from 2 healthy volunteers and 1 patient. The allogeneic CTL cell lines examined in the LDH release of HSPB1 and HSP90AA1 on primary MM cells and the ⁵¹Cr-release of HSPB1 and HSP90AA1 were all derived from 2 patients and 1 healthy volunteer. Target cells include myeloma cell lines and allogeneic primary myeloma cells from patients. HLA-A*0201⁺ autologous peripheral blood mononuclear cells (PBMCs) were used as controls. MM patients 1, 2, 3, 4, 6 and 7 were HLA-A*0201⁺, and patients 5 and 8 were HLA-A*0201⁻. Different effector:target (E:T) cell ratios were used. *P < 0.05; **P < 0.01.

Figure 5. Phenotype and cytokine expression profiles of HSP peptide-specific CTL lines

Flow cytometry analysis showing the expression of (A) surface CD45RO and CD69; (B) intracellular staining of interleukin 4 (IL4) and γ-interferon (IFN-γ) in unstimulated T cells and HSP peptide-specific CTL lines one week after the fourth cycle of peptide stimulation. The results are representative of three independent experiments. (C) Inhibition of the T-cell lines–mediated cytotoxicity against peptide-pulsed T2 cells by mAbs against MHC class I (aMHC-I), MHC class II (aMHC-II), or HLA-A*0201 (aHLA-A2). Isotypic IgG was used as control. An effector-target (E/T) ratio of 10:1 was used in panels C. Results of 4 independent experiments are shown. Error bars show standard deviation. Similar results were obtained with other T-cell lines from blood donors and patients with myeloma. *P < 0.05. CTL, cytotoxic T lymphocytes

Figure 6. Cytolytic machinery of HSP peptide-specific CTL lines

Flow cytometry analysis showing the expression of (A) perforin, FasL, tumour necrosis factor (TNF)-β by the cytotoxic T lymphocytes (CTL) lines, (B) granzyme B by target cells labelled with fluorescent dye, TFL4, alone or after 4-h coculture with effector CTLs at two different E:T ratios, and (C) CD107a by the CTL lines and unstimulated T cells from the same donor, before and after restimulation with myeloma cell lines LP-1 (HLA-A*0201⁻) or U266 (HLA-A*0201⁺). Similar results were obtained with other T cell lines derived from other MM patients or healthy volunteers. **P < 0.01.

Figure 7. In vivo anti-myeloma effects of HSP peptide-specific CTL lines

Non-obese diabetic/severe combined immunodeficiency mice (5 per group) were injected subcutaneously with 2 × 10⁷ U266 cells on day 0. When tumour areas reached 4 mm², aa670-specific CTLs (1 × 10⁷) or unstimulated T cells from the same donors were injected intravenously into myeloma-bearing mice together with subcutaneous injection of interleukin 2 at 10,000 iu/mouse. Tumour area was measured twice every week. Shown are tumour sizes in mice receiving injection of (A) control T cells or (B) HSP peptide-specific CTLs and (C) mouse survival data.