Virus and autoantigen-specific CD4+ T cells are key effectors in a SCID mouse model of EBV-associated post-transplant lymphoproliferative disorders.

Polyclonal Epstein-Barr virus (EBV)-infected B cell line (lymphoblastoid cell lines; LCL)-stimulated T-cell preparations have been successfully used to treat EBV-positive post-transplant lymphoproliferative disorders (PTLD) in transplant recipients, but function and specificity of the CD4+ component are still poorly defined. Here, we assessed the tumor-protective potential of different CD4+ T-cell specificities in a PTLD-SCID mouse model. Injection of different virus-specific CD4+ T-cell clones showed that single specificities were capable of prolonging mouse survival and that the degree of tumor protection directly correlated with recognition of target cells in vitro. Surprisingly, some CD4+ T-cell clones promoted tumor development, suggesting that besides antigen recognition, still elusive functional differences exist among virus-specific T cells. Of several EBV-specific CD4+ T-cell clones tested, those directed against virion antigens proved most tumor-protective. However, enriching these specificities in LCL-stimulated preparations conferred no additional survival benefit. Instead, CD4+ T cells specific for unknown, probably self-antigens were identified as principal antitumoral effectors in LCL-stimulated T-cell lines. These results indicate that virion and still unidentified cellular antigens are crucial targets of the CD4+ T-cell response in this preclinical PTLD-model and that enriching the corresponding T-cell specificities in therapeutic preparations may enhance their clinical efficacy. Moreover, the expression in several EBV-negative B-cell lymphoma cell lines implies that these putative autoantigen(s) might also qualify as targets for T-cell-based immunotherapy of virus-negative B cell malignancies

EBV-specific CD4+ T cells differ in their tumor-protective potential.

<p>(A) Survival of mice after adoptive transfer of different EBV-specific CD4+ T-cell clones. 1×10⁷ LCL and 1×10⁷ T cells were consecutively i.p. injected and mouse survival analyzed. As exemplified by the EBNA1-specific T-cell clone 1C3 and the EBNA3B-specific clone B9, injection of latent antigen-specific T cells had no effect on mouse survival, except for EBNA3C-specific CD4+ T cells that showed a trend towards delaying tumor growth (group sizes: EBNA1-1C3: LCL n = 20, LCL + T cells n = 11; EBNA3B-B9: LCL n = 20, LCL + T cells n = 7; EBNA3C-3H10: LCL n = 11, LCL + T cells n = 4). Adoptive transfer of the BLLF1-specific CD4+ T-cell clone 1D6 prolonged mouse survival (group sizes: LCL n = 10; LCL + T cells n = 10; summarized results of 2 independently performed experiments). (B) CFSE-labeled BLLF1- and EBNA1-specific T cells were i.p. injected into mice that had received autologous LCL 25 days before. Single cell suspensions of tumors were analyzed 24, 48, or 72 hours post injection by FACS for the presence of CFSE-labeled T cells as well as human CD20-expressing tumor cells. BLLF1- but not EBNA1-specific T cells infiltrated tumors and led to a decrease in the percentage of CD20+ cells. (C) Immunostaining of tumor sections from mice described in (B). Cryo-embedded tumor sections were double-stained with FITC- and BLLF1-specific antibodies to detect tumor infiltrated CFSE-labeled T cells (brown) and BLLF1-expressing tumor cells (blue). BLLF1-specific T cells infiltrated tumors and were found in proximity to antigen expressing cells while no EBNA1-specific T cells were found to infiltrate the tumors. Two immunostainings of two separate tumor sections are shown in each case. (D) Injection of the EBNA1-specific T-cell clone 3E10 and the BNRF1-specific T-cell clone 1H7 led to faster tumor development and shortened mouse survival (group sizes: EBNA1-3E10: LCL n = 20, LCL + T cells n = 10; BNRF1-1H7: LCL n = 20, LCL + T cells n = 4).</p

Induction of human PTLD-like tumors in immunodeficient mice.

<p>(A) Intraperitoneal injection of SCID mice with 1×10⁷ LCL of four different donors led to tumor development with an incidence of 75 – 100% and a latency of 20 and 46 days (group sizes: LCL FL and LCL MF n = 4; LCL GB n = 11; LCL JM n = 20; days p.i.: days post injection). (B) Injection of 1×10⁷ LCL, 1×10⁷ LCL Z(-), or 5×10⁷ PBMC from the same donor led to tumor development in all animals but with different latency (group sizes: LCL JM n = 20; PBMC JM n = 6; LCL Z(-) JM n = 6). All survival curves and donor dependent incidences and latencies were reproduced in several independent experiments. (C) Different numbers of LCL from the same donor were injected in mice and the survival determined. Results are depicted in a Kaplan-Meier curve (group sizes: 1×10⁷ n = 20; 5×10⁶ n = 4; 1×10⁶ n = 6; median survival 28, 30, and 38 days). (D) Developing tumors were confirmed as PTLD-like lymphomas. Formalin-fixed, paraffin-embedded tumor slides (3–8 µm) were stained with H&E (first row) and with antibodies against the human B cell marker CD20 (second row), as well as antibodies against the EBV latent proteins EBNA1 (third row) and EBNA2 (fourth row), whose co-expression is characteristic of PTLD.</p

Mouse survival is mediated by non-virus-specific T cells.

<p>T-cell lines were generated by four rounds of stimulation <i>in vitro</i> with LCL cultivated in media containing human serum (HS), fetal calf serum (FCS), or fetal calf serum plus acyclovir (FCS-ACV) to prevent the expression and presentation of virion antigens. (A) Specificity analysis of the generated T-cell lines. Autologous PBMC were pulsed with recombinant EBV latent proteins <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004068#ppat.1004068-Adhikary3" target="_blank">[20]</a> for 24 h and then probed with the T cells. Whereas all T-cell lines recognized LCL, none specifically responded against PBMC pulsed with any of the latent proteins of EBV. (B) Following i.p. injection with autologous LCL as tumor inducing cells, all three T-cell lines prolonged mouse survival significantly (*** p<0.0001). Group sizes: LCL n = 20; LCL + T-cell line HS n = 9; LCL + T-cell line FCS n = 12; LCL + T-cell line FCS-ACV n = 10 (summarized results of two independent experiments). (C) Injection of T-cell line HS together with LCL Z(-) significantly prolonged mouse survival, demonstrating that virion antigen-specific T cells are not required for the tumor protective effect. Group sizes: LCL Z(-) n = 7; LCL Z(-) + T-cell line HS n = 8 (*** p<0.0001).</p

Later passage T-cell preparations show increased virion antigen specificity but are less tumor-protective.

<p>(A) Reactivity of the T-cell lines against virion antigens. With increasing numbers of stimulation, the T-cell lines progressively responded against virion antigens transferred by viral particles. T-cell specificity was tested by cytokine secretion upon stimulation with autologous LCL Z(-). The target cells were either left untreated or loaded with virus particles for presentation of structural antigens (mainly late lytic antigens). (B) Tumor protection by early and late passage T-cell lines. 1×10⁷ LCL and 1×10⁷ T cells stimulated with autologous LCL four (p4) or ten (p10) times <i>in vitro</i> were simultaneously injected into SCID mice (group sizes: LCL n = 20; LCL + T cells p4 n = 13; LCL + T cells p10 n = 13; depicted results are combined from two independently performed experiments). Later passage T-cell lines prolonged mouse survival less efficiently.</p

LCL Z(-)- as well as miniLCL-stimulated T-cell lines recognize autoantigens and prolong mouse survival.

<p>(A) Recognition of autoantigens by LCL Z(-) or miniLCL-stimulated T cells. Specificity analysis of the T-cell line JM-W3 was performed using autologous LCL and LCL Z(-) as well as HLA-matched EBV-negative and EBV-positive BL30 cell lines. T-cell recognition of the EBV-negative BL30 cell line, but barely of BL30 cells that had been infected with the B95.8 or the P3HR1 EBV strains, demonstrated that these T cells recognized a non-viral antigen(s). Recognition of viral antigens by the GB-W3 T cells was excluded by probing the cells with the HLA-matched, EBV-negative Hodgkin lymphoma cell line L428 pulsed with recombinant latent proteins of EBV. (B) Analysis of the tumor-protective potential of these autoreactive T-cell lines <i>in vivo</i>. 1×10⁷ LCL Z(-) or miniLCL were i.p. injected in combination with 1×10⁷ autologous T cells and tumor development assessed (LCL Z(-) n = 6; LCL Z(-) + T cells JM-W3 n = 4; miniLCL n = 4; miniLCL + T cells GB-W3 n = 10; * p<0.05; *** p<0.001) (C) Phenotypic characterization of the autoreactive T cells GB-W3 by FACS. Autoreactive T cells displayed a CD3+CD4+ effector T-cell phenotype (CD62L-CCR7-) of differentiated T cells (CD27-CD28+CXCR3+CCR4-CCR6+/-), and produced granzyme A (black line) and B (grey line) (D).</p

Human IgG1 antibodies antagonizing activating receptor NKG2D on natural killer cells

NKG2D is a surface receptor expressed on NK cells but also on CD8+ T cells, γδ T cells, and auto-reactive CD4+/CD28− T cells of patients with rheumatoid arthritis. Various studies suggested that NKG2D plays a critical role in autoimmune diseases, e.g., in diabetes, celiac disease and rheumatoid arthritis (RA), rendering the activating receptor a potential target for antibody-based therapies. Here, we describe the generation and characteristics of a panel of human, high-affinity anti-NKG2D IgG1 monoclonal antibodies (mAbs) derived by phage display. The lead molecule mAb E4 bound with an affinity (KD) of 2.7 ± 1.4 × 10−11 M to soluble and membrane-bound human NKG2D, and cross-reacted with NKG2D from cynomolgus macaque, indicating potential suitability for studies in a relevant primate model. MAb E4 potently antagonized the cytolytic activity of NKL cells against BaF/3-MICA cells expressing NKG2D ligand, and blocked the NKG2D ligand-induced secretion of TNFα, IFNγ and GM-CSF, as well as surface expression of CRTAM by NK cells cultured on immobilized MICA or ULBP-1 ligands. The antibody did not show a detectable loss of binding to NKG2D after seven days in human serum at 37°C, and resisted thermal inactivation up to 70°C. Based on these results, anti-human NKG2D mAb E4 provides an ideal candidate for development of a novel therapeutic agent antagonizing a key receptor of NK and cytotoxic T cells with implications in autoimmune diseases