Novel epitope evoking CD138 antigen-specific cytotoxic T lymphocytes targeting multiple myeloma and other plasma cell disorders

Abstract

The development of an immunotherapeutic strategy targeting CD138 antigen could potentially represent a new treatment option for multiple myeloma (MM). This study evaluated the immune function of CD138 peptide-specific cytotoxic T lymphocytes (CTL), generated ex vivo using an HLA-A2-specific CD138 epitope against MM cells. A novel immunogenic HLA-A2-specific CD138(260-268) (GLVGLIFAV) peptide was identified from the full-length protein sequence of the CD138 antigen, which induced CTL specific to primary CD138(+) MM cells. The peptide-induced CD138-CTL contained a high percentage of CD8(+) activated/memory T cells with a low percentage of CD4(+) T cell and naive CD8(+) T cell subsets. The CTL displayed HLA-A2-restricted and CD138 antigen-specific cytotoxicity against MM cell lines. In addition, CD138-CTL demonstrated increased degranulation, proliferation and γ-interferon secretion to HLA-A2(+) /CD138(+) myeloma cells, but not HLA-A2(-) /CD138(+) or HLA-A2(+) /CD138(-) cells. The immune functional properties of the CD138-CTL were also demonstrated using primary HLA-A2(+) /CD138(+) cells isolated from myeloma patients. In conclusion, a novel immunogenic CD138(260-268) (GLVGLIFAV) peptide can induce antigen-specific CTL, which might be useful for the treatment of MM patients with peptide-based vaccine or cellular immunotherapy strategies.

Figure 1. Affinity and stability of CD138_260-268 (GLVGLIFAV) peptide binding to HLA-A2 molecules

(a) The HLA-A2 binding affinity of CD138_260-268 (GLVGLIFAV) peptide on T2 cells was analysed by flow cytometry. T2 cells were pulsed with CD138 peptide (50 μg/ml) or influenza virus matrix protein_58-66 (30 μg/ml) in AIM-V serum-free media. After overnight incubation, the cells were washed to remove unbound peptides and stained with HLA-A2-FITC mAb. The binding affinity of peptides was determined as the Fluorescence Index (FI), which was calculated as [mean channel fluorescence of T2 cells pulsed with the peptide plus β2-microglobulin ÷ mean channel fluorescence of T2 cells pulsed with β2-microglobulin]. Among the peptides tested, the CD138_260-268 (GLVGLIFAV) peptide displayed the highest HLA-A2 binding affinity. The values represent the mean ± SE of three separate experiments. (b) The HLA-A2 binding stability of CD138_260-268 (GLVGLIFAV) peptide on T2 cells was measured at different time points. After overnight peptide pulsing, the T2 cells were incubated with Brefeldin A, followed by staining with HLA-A2 FITC mAb at 0, 2, 4, 6 or 14 h incubation. The peptide binding stability was measured as HLA-A2 MFI. The stability of CD138_260-268 (GLVGLIFAV) peptide was comparable to influenza virus matrix protein_58-66 (GILGFVFTL) peptide, which was used as an HLA-A2-specific positive control. The values represent the mean ± SE of three separate experiments.

Figure 1. Affinity and stability of CD138_260-268 (GLVGLIFAV) peptide binding to HLA-A2 molecules

(a) The HLA-A2 binding affinity of CD138_260-268 (GLVGLIFAV) peptide on T2 cells was analysed by flow cytometry. T2 cells were pulsed with CD138 peptide (50 μg/ml) or influenza virus matrix protein_58-66 (30 μg/ml) in AIM-V serum-free media. After overnight incubation, the cells were washed to remove unbound peptides and stained with HLA-A2-FITC mAb. The binding affinity of peptides was determined as the Fluorescence Index (FI), which was calculated as [mean channel fluorescence of T2 cells pulsed with the peptide plus β2-microglobulin ÷ mean channel fluorescence of T2 cells pulsed with β2-microglobulin]. Among the peptides tested, the CD138_260-268 (GLVGLIFAV) peptide displayed the highest HLA-A2 binding affinity. The values represent the mean ± SE of three separate experiments. (b) The HLA-A2 binding stability of CD138_260-268 (GLVGLIFAV) peptide on T2 cells was measured at different time points. After overnight peptide pulsing, the T2 cells were incubated with Brefeldin A, followed by staining with HLA-A2 FITC mAb at 0, 2, 4, 6 or 14 h incubation. The peptide binding stability was measured as HLA-A2 MFI. The stability of CD138_260-268 (GLVGLIFAV) peptide was comparable to influenza virus matrix protein_58-66 (GILGFVFTL) peptide, which was used as an HLA-A2-specific positive control. The values represent the mean ± SE of three separate experiments.

Figure 2. Generation of activated memory cells in CTL cultures stimulated with CD138_260-268 (GLVGLIFAV) peptide

HLA-A2⁺/CD3⁺ T cells were stimulated weekly with irradiated antigen-presenting cells pulsed with CD138_260-268 peptide (GLVGLIFAV). One week after their fourth stimulation, the phenotype of the T cell cultures was analysed by flow cytometry. (a) The percentage of CD8⁺ T cells was increased in the CD3⁺ T cell cultures stimulated with the CD138_260-268 peptide as compared to the control (non-peptide stimulated) cells, which contained a lower percentage of CD8⁺ T cells and higher percentage of CD4⁺ T cells. (b) The percentage of CD69⁺/CD45RO⁺ (activated memory) cells was increased with a corresponding decrease in the percentage of CCR7⁺/CD45RA⁺ (naive) cells in the cultures stimulated with the CD138_260-268 peptide compared the control unstimulated cells.

Figure 2. Generation of activated memory cells in CTL cultures stimulated with CD138_260-268 (GLVGLIFAV) peptide

HLA-A2⁺/CD3⁺ T cells were stimulated weekly with irradiated antigen-presenting cells pulsed with CD138_260-268 peptide (GLVGLIFAV). One week after their fourth stimulation, the phenotype of the T cell cultures was analysed by flow cytometry. (a) The percentage of CD8⁺ T cells was increased in the CD3⁺ T cell cultures stimulated with the CD138_260-268 peptide as compared to the control (non-peptide stimulated) cells, which contained a lower percentage of CD8⁺ T cells and higher percentage of CD4⁺ T cells. (b) The percentage of CD69⁺/CD45RO⁺ (activated memory) cells was increased with a corresponding decrease in the percentage of CCR7⁺/CD45RA⁺ (naive) cells in the cultures stimulated with the CD138_260-268 peptide compared the control unstimulated cells.

Figure 3. Functional activity of CD138-CTL in response to HLA-A2⁺/CD138⁺ MM cell lines

(a) The tumour-specific cytotoxic activity of the CD138-CTL was tested in calcein-release cytotoxicity assays one week after the fourth peptide stimulation. CD138-CTL demonstrated HLA-A2 restricted lysis of U266 (CD138⁺/HLA-A2⁺) and McCAR (CD138⁺/HLA-A2⁺) cells, but not MM1S (CD138⁺/HLA-A2⁻) cells. The CD138-CTL did not show any significant lysis of antigen mismatched ML-2 (CD138⁻/HLA-A2⁺) AML cells or NK-sensitive K562 cells. (b) Specific CD138-CTL proliferation was examined by flow cytometry after stimulating CFSE-labelled CTL with tumour cell lines for 5 days. The proliferating cell population was measured as the percent decrease in CFSE expression (M1-gated cells). The CTL proliferated in response to McCAR (CD138⁺/HLA-A2⁺) cells, but not in response to MHC-mismatched MM1S (CD138⁺/HLA-A2⁻) or antigen mismatched ML-2 (CD138⁻/HLA-A2⁺) cells. Background proliferation was determined using CFSE-labelled CD138-CTL cultured in media alone. (c) IFN-γ secretion by CD138-CTL was measured by ELISA in the culture supernatants collected 1 day following stimulation with tumour cell lines. CD138-CTL showed a significantly higher level of IFN-γ secretion (*p < 0.05) in response to McCAR cells (CD138⁺/HLA-A2⁺) as compared to control CD138-CTL cultured in media alone. CD138-CTL IFN-γ secretion in response to MHC-mismatched MM1S (CD138⁺/HLA-A2⁻) or antigen-mismatched ML-2 (CD138⁻/HLA-A2⁺) tumour cell lines was similar to background IFN-γ production.

Figure 3. Functional activity of CD138-CTL in response to HLA-A2⁺/CD138⁺ MM cell lines

(a) The tumour-specific cytotoxic activity of the CD138-CTL was tested in calcein-release cytotoxicity assays one week after the fourth peptide stimulation. CD138-CTL demonstrated HLA-A2 restricted lysis of U266 (CD138⁺/HLA-A2⁺) and McCAR (CD138⁺/HLA-A2⁺) cells, but not MM1S (CD138⁺/HLA-A2⁻) cells. The CD138-CTL did not show any significant lysis of antigen mismatched ML-2 (CD138⁻/HLA-A2⁺) AML cells or NK-sensitive K562 cells. (b) Specific CD138-CTL proliferation was examined by flow cytometry after stimulating CFSE-labelled CTL with tumour cell lines for 5 days. The proliferating cell population was measured as the percent decrease in CFSE expression (M1-gated cells). The CTL proliferated in response to McCAR (CD138⁺/HLA-A2⁺) cells, but not in response to MHC-mismatched MM1S (CD138⁺/HLA-A2⁻) or antigen mismatched ML-2 (CD138⁻/HLA-A2⁺) cells. Background proliferation was determined using CFSE-labelled CD138-CTL cultured in media alone. (c) IFN-γ secretion by CD138-CTL was measured by ELISA in the culture supernatants collected 1 day following stimulation with tumour cell lines. CD138-CTL showed a significantly higher level of IFN-γ secretion (*p < 0.05) in response to McCAR cells (CD138⁺/HLA-A2⁺) as compared to control CD138-CTL cultured in media alone. CD138-CTL IFN-γ secretion in response to MHC-mismatched MM1S (CD138⁺/HLA-A2⁻) or antigen-mismatched ML-2 (CD138⁻/HLA-A2⁺) tumour cell lines was similar to background IFN-γ production.

Figure 3. Functional activity of CD138-CTL in response to HLA-A2⁺/CD138⁺ MM cell lines

(a) The tumour-specific cytotoxic activity of the CD138-CTL was tested in calcein-release cytotoxicity assays one week after the fourth peptide stimulation. CD138-CTL demonstrated HLA-A2 restricted lysis of U266 (CD138⁺/HLA-A2⁺) and McCAR (CD138⁺/HLA-A2⁺) cells, but not MM1S (CD138⁺/HLA-A2⁻) cells. The CD138-CTL did not show any significant lysis of antigen mismatched ML-2 (CD138⁻/HLA-A2⁺) AML cells or NK-sensitive K562 cells. (b) Specific CD138-CTL proliferation was examined by flow cytometry after stimulating CFSE-labelled CTL with tumour cell lines for 5 days. The proliferating cell population was measured as the percent decrease in CFSE expression (M1-gated cells). The CTL proliferated in response to McCAR (CD138⁺/HLA-A2⁺) cells, but not in response to MHC-mismatched MM1S (CD138⁺/HLA-A2⁻) or antigen mismatched ML-2 (CD138⁻/HLA-A2⁺) cells. Background proliferation was determined using CFSE-labelled CD138-CTL cultured in media alone. (c) IFN-γ secretion by CD138-CTL was measured by ELISA in the culture supernatants collected 1 day following stimulation with tumour cell lines. CD138-CTL showed a significantly higher level of IFN-γ secretion (*p < 0.05) in response to McCAR cells (CD138⁺/HLA-A2⁺) as compared to control CD138-CTL cultured in media alone. CD138-CTL IFN-γ secretion in response to MHC-mismatched MM1S (CD138⁺/HLA-A2⁻) or antigen-mismatched ML-2 (CD138⁻/HLA-A2⁺) tumour cell lines was similar to background IFN-γ production.

Figure 4. CD138-CTL cytotoxic activity against primary CD138⁺ cells isolated from HLA-A2⁺ MM patients

(a) The cytotoxic activity of CD138-CTL was evaluated in calcein-release assay against primary cells isolated from HLA-A2⁺ myeloma patients. Compared to unstimulated T cells (▲), CD138-CTL (■) generated from Donor A or Donor B showed effective lysis of the primary CD138⁺ cells isolated from bone marrow mononuclear cells of two HLA-A2⁺ MM patients. (b) Degranulation of CD138-CTL was evaluated by measuring CD107ab up-regulation on CD8⁺ CTL after stimulation with HLA-A2⁺/CD138⁺ primary MM cells using flow cytometry. CD138-CTL stimulated with primary cells from three HLA-A2⁺ MM patients showed higher level of CD107ab⁺/CD8⁺ cells as compared to unstimulated control cells. (c) Specific cell proliferation of CD138-CTL was evaluated in response to primary HLA-A2⁺/CD138⁺ cells isolated from MM patients by CFSE analysis. CD138-CTL showed a higher level of cell proliferation (Q1 gated cells) in response to the primary cells from three HLA-A2⁺ MM patients, compared to CD138-CTL cultured in media alone.

Figure 4. CD138-CTL cytotoxic activity against primary CD138⁺ cells isolated from HLA-A2⁺ MM patients

(a) The cytotoxic activity of CD138-CTL was evaluated in calcein-release assay against primary cells isolated from HLA-A2⁺ myeloma patients. Compared to unstimulated T cells (▲), CD138-CTL (■) generated from Donor A or Donor B showed effective lysis of the primary CD138⁺ cells isolated from bone marrow mononuclear cells of two HLA-A2⁺ MM patients. (b) Degranulation of CD138-CTL was evaluated by measuring CD107ab up-regulation on CD8⁺ CTL after stimulation with HLA-A2⁺/CD138⁺ primary MM cells using flow cytometry. CD138-CTL stimulated with primary cells from three HLA-A2⁺ MM patients showed higher level of CD107ab⁺/CD8⁺ cells as compared to unstimulated control cells. (c) Specific cell proliferation of CD138-CTL was evaluated in response to primary HLA-A2⁺/CD138⁺ cells isolated from MM patients by CFSE analysis. CD138-CTL showed a higher level of cell proliferation (Q1 gated cells) in response to the primary cells from three HLA-A2⁺ MM patients, compared to CD138-CTL cultured in media alone.

Figure 4. CD138-CTL cytotoxic activity against primary CD138⁺ cells isolated from HLA-A2⁺ MM patients

(a) The cytotoxic activity of CD138-CTL was evaluated in calcein-release assay against primary cells isolated from HLA-A2⁺ myeloma patients. Compared to unstimulated T cells (▲), CD138-CTL (■) generated from Donor A or Donor B showed effective lysis of the primary CD138⁺ cells isolated from bone marrow mononuclear cells of two HLA-A2⁺ MM patients. (b) Degranulation of CD138-CTL was evaluated by measuring CD107ab up-regulation on CD8⁺ CTL after stimulation with HLA-A2⁺/CD138⁺ primary MM cells using flow cytometry. CD138-CTL stimulated with primary cells from three HLA-A2⁺ MM patients showed higher level of CD107ab⁺/CD8⁺ cells as compared to unstimulated control cells. (c) Specific cell proliferation of CD138-CTL was evaluated in response to primary HLA-A2⁺/CD138⁺ cells isolated from MM patients by CFSE analysis. CD138-CTL showed a higher level of cell proliferation (Q1 gated cells) in response to the primary cells from three HLA-A2⁺ MM patients, compared to CD138-CTL cultured in media alone.

Figure 5. Intracellular IFN-γ production and proliferation by CD138 or XBP1-peptide specific CTL in response to myeloma cell lines

(a) Intracellular IFN-γ production was measured by flow cytometry after stimulation of the respective CD138-CTL, XBP1 US-CTL or XBP1-SP-CTL with HLA-A2⁺ McCAR or U266 myeloma cell line. Intracellular IFN-γ production was detected in the CTL effector memory (CD3⁺/CD8⁺/CD45RO⁺/CCR7⁻) subset from each of the respective CTL generated from two individual donors (Donor A, Donor B). A higher level of IFN-γ production was detected in response to McCAR or U266 cells in the CD138-CTL than the XBP1-CTLs. Control T cells did not produce IFN-γ in response to the myeloma cell lines. (b) Specific cell proliferation of each peptide-specific CTL was evaluated following stimulation with the HLA-A2⁺ myeloma cell lines on day 5 using a CFSE assay. The percentage of proliferating CTL is shown in the P3 gate (CFSE-low). A higher level of cell proliferation was detected in the CD138-CTL than the XBP1-CTLs in response to McCAR or U266 cells.

Figure 5. Intracellular IFN-γ production and proliferation by CD138 or XBP1-peptide specific CTL in response to myeloma cell lines

(a) Intracellular IFN-γ production was measured by flow cytometry after stimulation of the respective CD138-CTL, XBP1 US-CTL or XBP1-SP-CTL with HLA-A2⁺ McCAR or U266 myeloma cell line. Intracellular IFN-γ production was detected in the CTL effector memory (CD3⁺/CD8⁺/CD45RO⁺/CCR7⁻) subset from each of the respective CTL generated from two individual donors (Donor A, Donor B). A higher level of IFN-γ production was detected in response to McCAR or U266 cells in the CD138-CTL than the XBP1-CTLs. Control T cells did not produce IFN-γ in response to the myeloma cell lines. (b) Specific cell proliferation of each peptide-specific CTL was evaluated following stimulation with the HLA-A2⁺ myeloma cell lines on day 5 using a CFSE assay. The percentage of proliferating CTL is shown in the P3 gate (CFSE-low). A higher level of cell proliferation was detected in the CD138-CTL than the XBP1-CTLs in response to McCAR or U266 cells.