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. 2016 Oct;186(1):96-105.
doi: 10.1111/cei.12842. Epub 2016 Aug 16.

Analysis of Nucleophosmin-Anaplastic Lymphoma Kinase (NPM-ALK)-reactive CD8(+) T Cell Responses in Children With NPM-ALK(+) Anaplastic Large Cell Lymphoma

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Analysis of Nucleophosmin-Anaplastic Lymphoma Kinase (NPM-ALK)-reactive CD8(+) T Cell Responses in Children With NPM-ALK(+) Anaplastic Large Cell Lymphoma

V K Singh et al. Clin Exp Immunol. .
Free PMC article

Abstract

Cellular immune responses against the oncoantigen anaplastic lymphoma kinase (ALK) in patients with ALK-positive anaplastic large cell lymphoma (ALCL) have been detected using peptide-based approaches in individuals preselected for human leucocyte antigen (HLA)-A*02:01. In this study, we aimed to evaluate nucleophosmin (NPM)-ALK-specific CD8(+) T cell responses in ALCL patients ensuring endogenous peptide processing of ALK antigens and avoiding HLA preselection. We also examined the HLA class I restriction of ALK-specific CD8(+) T cells. Autologous dendritic cells (DCs) transfected with in-vitro-transcribed RNA (IVT-RNA) encoding NPM-ALK were used as antigen-presenting cells for T cell stimulation. Responder T lymphocytes were tested in interferon-gamma enzyme-linked immunospot (ELISPOT) assays with NPM-ALK-transfected autologous DCs as well as CV-1 in Origin with SV40 genes (COS-7) cells co-transfected with genes encoding the patients' HLA class I alleles and with NPM-ALK encoding cDNA to verify responses and define the HLA restrictions of specific T cell responses. NPM-ALK-specific CD8(+) T cell responses were detected in three of five ALK-positive ALCL patients tested between 1 and 13 years after diagnosis. The three patients had also maintained anti-ALK antibody responses. No reactivity was detected in samples from five healthy donors. The NPM-ALK-specific CD8(+) T cell responses were restricted by HLA-C-alleles (C*06:02 and C*12:02) in all three cases. This approach allowed for the detection of NPM-ALK-reactive T cells, irrespective of the individual HLA status, up to 9 years after ALCL diagnosis.

Keywords: ALCL; CD8+ T cell; IFN-γ ELISPOT; NPM-ALK; immune response.

Figures

Figure 1
Figure 1
Ex‐vivo CD8+ T cell response to autologous nucleophosmin–anaplastic lymphoma kinase (NPM–ALK)‐transfected dendritic cells (FastDCs) and human leucocyte antigen (HLA) restriction. Unstimulated CD8+ T cells of patient NHL‐4JS2 were tested directly in an interferon (IFN)‐γ enzyme‐linked immunospot (ELISPOT) assay. (a) Recognition of autologous FastDCs (1·0 ×104/well) transfected with NPM–ALK in‐vitro‐transcribed RNA. Shown are ELISPOT filter scans and spot numbers determined via semi‐automated computer‐assisted video image analysis in a bar diagram. (b) Recognition of CV‐1 in Origin with SV40 genes (COS‐7) cells (2·0 × 104/well) co‐transfected with the patient's HLA class I alleles (except B*52:01) and NPM–ALK or control antigens (pp65) cDNA. The bars represent means of duplicates ± standard deviation.
Figure 2
Figure 2
Anti‐cytomegalovirus (CMV)/pp65 T cell responses after short‐term pp65‐RNA stimulation in blood samples from anaplastic large cell lymphoma (ALCL) patients and healthy donors. CD8+ T cells (1·0 × 105) were stimulated with irradiated autologous dendritic cells (FastDCs) transfected with pp65 in‐vitro‐transcribed RNA. Microcultures were restimulated weekly for 2 weeks with the pp65‐transfected FastDCs. On day 19, pp65 responder T cells were tested in a 20‐h interferon (IFN)‐γ enzyme‐linked immunospot (ELISPOT) assay for the recognition of autologous FastDCs expressing pp65 (3–10 × 103/well). Shown are representative microcultures against autologous pp65‐transfected FastDCs from (a) four ALCL patients (NHL‐P1SU could not be tested; only patient NHL‐FVUD was CMV‐seronegative) and (b) healthy donors (only NHL‐96YC was CMV‐seronegative). Bars represent the means of duplicates ± standard deviation.
Figure 3
Figure 3
Anti‐nucleophosmin–anaplastic lymphoma kinase (NPM–ALK) T cell responses after short‐term NPM–ALK in‐vitro‐transcribed RNA stimulation in anaplastic large cell lymphoma (ALCL) patients. CD8+ T cells were stimulated with irradiated autologous dendritic cells (FastDCs) transfected with NPM–ALK IVT‐RNA, comparable to the pp65 stimulation. After two weekly restimulations, day 19 responder T cells were tested in a 20‐h interferon (IFN)‐γ enzyme‐linked immunospot (ELISPOT) assay for the recognition of FastDCs expressing NPM–ALK fusion protein. Representative microcultures against autologous NPM–ALK RNA transfected FastDCs from four ALCL patients are shown (patient NHL‐P1SU could not be tested). Bars represent the means of duplicates ± standard deviation.
Figure 4
Figure 4
Identification of human leucocyte antigen (HLA) class I‐restriction alleles of CD8+ T cells reactive against nucleophosmin–anaplastic lymphoma kinase (NPM–ALK) in NPM–ALK+ anaplastic large cell lymphoma (ALCL) patients. To assess the restricting HLA class I allele, day 19 responder T cells were tested in a parallel IFN‐γ enzyme‐linked immunospot (ELISPOT) assay against CV‐1 in origin with SV40 genes (COS‐7) cells (2·0 × 104/well) co‐transfected with plasmids encoding the patients’ HLA class I alleles and NPM–ALK or cytomegalovirus (CMV)/pp65 antigen. Shown are representative microcultures reactive against COS‐7 cells co‐transfected with NPM–ALK and the individual HLA class I alleles from patients NHL‐P1SU, NHL‐4JS2 and NHL‐7AKR, after short‐term stimulation with autologous NPM–ALK transfected dendritic cells (FastDCs). Bars represent the means of duplicates ± standard deviation.

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