Kiyotaka Kuzushima

Differences between T cell-type and natural killer cell-type chronic active epstein-barr virus infection

Infections of T cells and natural killer (NK) cells play a central role in the pathogenesis of chronic active Epstein-Barr virus (CAEBV) infection. To characterize the virologic and cytokine profiles of T cell-type and NK cell-type infection, 39 patients with CAEBV infection were analyzed. Patients with T cell-type infection had higher titers of immunoglobulin G against early and late EBV antigens, suggesting lytic cycle infection. However, the pattern of EBV gene expression was latency type II; BZLF1, which is a hallmark of lytic cycle infection, could not be detected in any patients, regardless of infection type. Patients with CAEBV infection had high concentrations of proinflammatory, T helper cell type 1, and anti-inflammatory cytokines. The cytokine profile in patients with NK cell-type infection was similar to that in patients with T cell-type infection, but the concentration of IL-13 was high in patients with NK cell-type infection. These findings should help to clarify the pathogenesis of CAEBV infection and facilitate the development of more-effective treatments.

Myeloma Cells Are Highly Sensitive to the Granule Exocytosis Pathway Mediated by WT1-Specific Cytotoxic T Lymphocytes

Purpose: Because WT1 is a universal tumor antigen, we examined the sensitivity of myeloma cells to WT1-specific cytotoxic T lymphocyte (CTL)-mediated cytotoxicity. Experimental Design: WT1 expression in hematologic malignant cells was examined by quantitative reverse transcription-polymerase chain reaction. The cytotoxicity of a WT1-specific CTL clone against hematologic malignant cells, including myeloma cells, was examined by standard chromium-51 release assays. The extent of membrane damage induced by purified perforin was examined. Induction of WT1-specific CTLs from the patients with multiple myeloma (MM) was attempted, and we examined their function against myeloma cells. Results: The expression levels of WT1 mRNA in myeloma and lymphoma cells were significantly lower than that in acute leukemia cells. Although the WT1 expression levels in myeloma and lymphoma cells were almost same, only myeloma cells were lysed efficiently by WT1-specific CTLs in a HLA-restricted manner. The amounts of interferon-γ produced by WT1-specific CTLs in response to stimulation with myeloma cells and with lymphoma cells were almost the same, suggesting that WT1 protein is processed and expressed in the context of HLA class I molecules similarly on both myeloma and lymphoma cells. The extent of membrane damage induced by purified perforin appeared to be significantly higher in myeloma cells than in lymphoma cells. WT1-specific CTLs appeared to be present in patients with MM. Conclusions: The present study has shown that susceptibility of membranes to perforin is an important factor determining the sensitivity of target cells to CTL-mediated cytotoxicity and that WT1 is an ideal target antigen for cellular immunotherapy of MM.

The human Cathepsin H gene encodes two novel minor histocompatibility antigen epitopes restricted by HLA-A*3101 and -A*3303

Minor histocompatibility antigens (mHags) play crucial roles in the induction of graft versus host disease (GVHD) and/or graft versus leukaemia (GVL) effects following human leucocyte antigen (HLA)‐identical haematopoietic stem cell transplantation (HSCT). Using HLA‐A*3101‐ and ‐A*3303‐restricted cytotoxic T lymphocyte (CTL) clones generated from different post‐HSCT recipients, we identified two novel mHag epitopes encoded by the leader sequence of cathepsin H (CTSH) isoform a. The nonameric sequence ATLPLLCAR was defined as an HLA‐A*3101‐restricted epitope (CTSHR/A31), while a decameric peptide featuring a one N‐terminal amino acid extension, WATLPLLCAR, was presented by HLA‐A*3303 (CTSHR/A33). The immunogenicity of both epitopes was totally dependent on the polymorphic C‐terminal arginine residue and substitution with glycine completely abolished binding to the corresponding HLA molecules. Thus, the immunogenicity of this mHag is exerted by differential HLA binding capacity. CTSH is relatively ubiquitously expressed at protein levels, thus it may be involved in GVHD and anti‐leukaemic/tumour responses. Interestingly, however, CTL clones predominantly lysed targets of haematopoietic cell origin, which could not be explained in terms of the immunoproteasome system. Although the mechanisms involved in the differential susceptibility remain to be determined, these data suggest that CTSH‐encoded mHags could be targets for GVL effects.

Identification of an HLA-A24-restricted cytotoxic T lymphocyte epitope from human papillomavirus type-16 E6: The combined effects of bortezomib and interferon-γ on the presentation of a cryptic epitope

About 50% of cervical cancers are associated with human papillomavirus type 16 (HPV‐16), and since the HPV‐16 E6 and E7 oncoproteins are constitutively expressed in the tumor cells, they are attractive targets for cytotoxic T lymphocyte (CTL)‐mediated immunotherapy. Nevertheless, only a limited number of HPV‐16 E6 epitopes have been identified to date. Using reverse immunological methods, we have generated a CTL clone against the HPV‐16 E649–57 epitope restricted by HLA‐A*2402, which is the most common allele in Japan and relatively frequent worldwide, capable of lysing 293T cells transduced with HLA‐A*2402 and HPV‐16 E6. Although it was unable to recognize the SiHa cervical cancer cell line positive for HPV‐16 and HLA‐A*2402, the cells became susceptible to lysis when transduced with E6‐E7 genes, which was unexpectedly offset by pretreatment with interferon (IFN)‐γ alone. Interestingly, however, combined pretreatment with a proteasome inhibitor, bortezomib and IFN‐γ fully restored CTL‐mediated lysis of the original SiHa cells. Furthermore, such intervention of 2 of 4 other cervical cancer cell lines expressing HPV‐16 E6 and HLA‐A*2402 was found to induce IFN‐γ production by specific CTLs. Tetramer analysis further revealed that induction of E649–57‐specific T cells was possible in 5 of 7 patients with HPV‐16‐positive high grade cervical intraepithelial neoplasia or cervical cancer by in vitro stimulation with E649–57 peptide. Thus, these findings together indicate that E649–57 is a candidate epitope for immunotherapy and immunological monitoring of such patients.

CD137-guided isolation and expansion of antigen-specific CD8 cells for potential use in adoptive immunotherapy

The efficient isolation and ex vivo expansion of antigen-specific T cells are crucial for successful adoptive immunotherapy against uncontrollable infections and cancers. Several methods have been reported for this purpose, for example, employing MHC-multimeric complexes, interferon-gamma secretion, and antibodies specific for molecules expressed on T-cell surfaces, including CD25, CD69, CD107a, CD137, and CD154. Of the latter, CD137 has been shown to be one of the most promising targets since it is only expressed on CD8+ T cells early after encountering antigen, while being almost undetectable on resting cells. However, detailed comparisons between CD137-based and other methods have not yet been conducted. In this study, we therefore compared three approaches (with CD137, CD107a, and tetramers) using HLA-A24-restricted CMV pp65 and EBV BRLF1 epitopes as model antigens. We found that the CD137-based isolation of antigen-stimulated CD8+ T cells was comparable to tetramer-based sorting in terms of purity and superior to the other two methods in terms of subsequent cell expansion. The method was less applicable to CD4+ T cells since their CD137 upregulation is not sufficiently high. Collectively, this approach is most likely to be optimal among the methods tested for the isolation and expansion of antigen-specific CD8+ cells.

Epstein-Barr virus (EBV) latent membrane protein-1-specific cytotoxic T lymphocytes targeting EBV-carrying natural killer cell malignancies

Epstein‐Barr virus (EBV)‐encoded latent membrane protein (LMP) 1 is a potential target for immunotherapy of some proportion of Hodgkins disease cases, nasopharyngeal carcinomas, EBV‐associated natural killer (NK)/T lymphomas, and chronic active EBV infection (CAEBV). Since it is unknown whether EBV‐infected NK/T cells are susceptible to lysis by LMP1‐specific cytotoxic T lymphohcytes (CTL), we here tested the ability of mRNA‐transduced antigen‐presenting cells (APC) to stimulate rare LMP1‐specific CTL. A 43‐amino acid N‐terminal deletion mutant LMP1 (ΔLMP1) could be efficiently expressed in dendritic cells and CD40‐activated B cells upon mRNA electroporation. ΔLMP1‐expressing APC were found to stimulate LMP1‐specific CTL from a healthy donor and a CTL clone recognized a peptide, IIIILIIFI, presented by HLA‐A*0206 molecules. Processing and presentation of the antigenic peptide proved dependent on expression of an immunoproteasome subunit, low‐molecular‐weight protein‐7, as confirmed by RNA interference gene silencing. Furthermore, an EBV‐infected NK cell line derived from a patient with CAEBV, and another from an NK lymphoma with enforced HLA‐A*0206 expression, were specifically lysed by the CTL. Overall, these data suggest that immunotherapy targeting LMP1 in EBV‐associated NK lymphomas and CAEBV might serve as an alternative treatment modality.

Construction and molecular characterization of a T-cell receptor-like antibody and CAR-T cells specific for minor histocompatibility antigen HA-1H

The genetic transfer of T-cell receptors (TCRs) directed toward target antigens into T lymphocytes has been used to generate antitumor T cells efficiently without the need for the in vitro induction and expansion of T cells with cognate specificity. Alternatively, T cells have been gene-modified with a TCR-like antibody or chimeric antigen receptor (CAR). We show that immunization of HLA-A2 transgenic mice with tetramerized recombinant HLA-A2 incorporating HA-1 H minor histocompatibility antigen (mHag) peptides and β2-microglobulin (HA-1 H/HLA-A2) generate highly specific antibodies. One single-chain variable region moiety (scFv) antibody, #131, demonstrated high affinity (KD=14.9 nM) for the HA-1 H/HLA-A2 complex. Primary human T cells transduced with #131 scFV coupled to CD28 transmembrane and CD3ζ domains were stained with HA-1 H/HLA-A2 tetramers slightly more intensely than a cytotoxic T lymphocyte (CTL) clone specific for endogenously HLA-A2- and HA-1 H-positive cells. Although #131 scFv CAR-T cells required >100-fold higher antigen density to exert cytotoxicity compared with the cognate CTL clone, they could produce inflammatory cytokines against cells expressing HLA-A2 and HA-1 H transgenes. These data implicate that T cells with high-affinity antigen receptors reduce the ability to lyse targets with low-density peptide/MHC complexes (~100 per cell), while they could respond at cytokine production level.

Induction of HLA‐B*40:02‐restricted T cells possessing cytotoxic and suppressive functions against haematopoietic progenitor cells from a patient with severe aplastic anaemia

Acquired aplastic anaemia (AA) is a syndrome characterized by bone marrow (BM) hypoplasia and pancytopenia. Autoimmunity to haematopoietic stem/progenitor cells (HSPCs) has been considered as a possible cause (Young et al, 2006). We recently described AA patients possessing clonal/oligoclonal haematopoiesis who had specifically lost either human leucocyte antigen (HLA) haplotype containing the HLA-A*02:06 or -B*40:02 (Katagiri et al, 2011). We surmized that selective pressure against HSPCs by specific cytotoxic T lymphocytes (CTLs) via these HLA molecules led to an escape of HSPCs without the causative HLA haplotype. We sought to determine whether HLA-B*40:02-restricted CTLs specific for HSPCs were present in patient peripheral blood or BM, and if so, how the CTLs induced BM failure as well as clonal haematopoiesis in AA patients who improved with immunosuppressive therapy (IST). Method details are provided in Data S1. This case report describes a 14-year-old male diagnosed with severe AA. Because he had no suitable donor, he received antithymocyte globulin and ciclosporin (CsA) and subsequently achieved remission (Fig 1A). His AA recurred twice but was successfully treated with CsA, indicating his haematopoiesis was CsA-dependent. He possessed a risk allele of HLA-B*40:02. His blood cells lacked one HLA haplotype containing HLA-B*40:02 (Figure S1) due to acquired copy number-neutral loss of heterozygosity of the 6p arms (6pLOH) (data not shown). To isolate HLA-B*40:02-restricted CTLs that could have triggered his BM failure (time point 4 in Fig 1A), we chose K562/B*40:02 as alternative antigen-presenting cells in place of the patient’s rare BM HSPCs because K562 has characteristics of HSPCs (Lozzio et al, 1981). The T cell line generated lysed K562/B*40:02 more intensely than K562/A*02:01, but did not lyse the non-haematopoietic cell line 293T/B*40:02 (Fig 1B). Of 84 putative clones after CTL cloning, clone A6 (CTL-A6) was selected for further analyses as a representative clone (Fig 1C). CTL-A6 did not recognize immortalized B and T lymphocytes expressing HLA-B*40:02 (Fig 1D), suggesting that mature lymphocytes might not express the target antigen. We also examined whether CTL-A6 recognized HLA-B*40:02-transduced cell lines derived from various haematological malignancies, but none of them was lysed by CTL-A6 (Fig 1E), indicating that CTL-A6 recognized antigen expressed only in K562 among the cell lines tested and might have been involved in the development of AA in the patient. Finally, we questioned whether reactivity of CTL-A6 to K562/B*40:02 would be altered with differentiation of K562. Phorbol 12-myristate 13-acetate (PMA) treatment (Witt et al, 2000) completely abrogated reactivity of CTL-A6 against K562/B*40:02 (Fig 1F) while reactivity of CTL-EH6 (Torikai et al, 2007) against simultaneously treated K562/A*02:01 expressing HLA-A*02:01-restricted minor histocompatibility antigen (mHag) HA-1H (den Haan et al, 1998) was retained (Fig 1G). Butyrate treatment produced a similar trend, but to a lesser extent (Colamonici et al, 1986). Morphological changes were apparent but their viability remained unchanged (Figure S2A). Flow cytometry revealed slight upregulation of HLA class I expression and diminished glycophorin A expression by PMA (Figure S2B). These data suggest that CTL-A6 recognized an antigen expressed in HSPC with limited maturation. We then examined the effects of CTL-A6 on HSPC growth using CD34 cells from normal BM. We identified BM from a HLA-A*02:01, B*40:02 healthy donor that was positive for HLA-B*40:02-restricted mHag encoded by SLC1A5 (Kamei et al, 2009), but negative for HA-1H. CTL-3B6 (Kamei et al, 2009) and CTL-EH6, corresponding to these mHags, were used as positive and negative controls, respectively (Fig 2A). While CD34 cells cultured with CTL-EH6 allowed expansion of immature and differentiated cells, those with CTL-3B6 showed no growth (Fig 2B). When CD34 cells were cocultured with CTL-A6, either partial growth inhibition (Experiment 1 with erythropoietin, left) or no inhibition (Experiment 2 without erythropoietin, right) was observed. After 14 d coculture in Experiment 2, cells cocultured with CTL-EH6 contained mature myeloid cells and some less mature myeloid cells (Fig 2C, upper left); the majority of those with CTL-A6 were mature myeloid cells, including some with degraded morphology (upper right); cells in the CTL-EH6 coculture contained CD13-, CD14or CD33-antigen high fractions (Fig 2C, lower left), whereas cells in the CTL-A6 coculture contained weakly positive and negative cells for CD13, CD14 or CD33; one-third of the latter contained CD8 cells most probably corresponding to residual CTL-A6 (lower right). All types of colony-forming units (CFU) were diminished by two to threefold, with the exception of mixed lineage CFU (CFU-GEMM), which showed greater than fourfold decrease, in the coculture with CTL-A6 compared with those with CTL-EH6 (Fig 2D). Altogether, correspondence

Expansion of human CMV-specific cytotoxic T lymphocytes to a clinical scale: a simple culture system using tetrameric HLA–peptide complexes

BACKGROUND Recipients of allogeneic stem cell transplants (SCT) are at risk of human CMV infection during their immunocompromised period. The increasing number of reports of CMV isolates resistant to ganciclovir after transplantation has led us to attempt to develop alternative strategies for preventing or treating CMV infection. This study describes a system for generating sufficient numbers of CMV-specific cytotoxic T lymphocytes (CTL) for adoptive immunotherapy after SCT. METHODS CMV-specific CTL were isolated from a single blood draw of a CMV-seropositive donor using PE-labeled HLA-A*0201/pp65(495-503) tetramers and anti-PE magnetic beads. A mixture of a tetramer-positive population and CD4(+) T lymphocytes was expanded to sufficient numbers for clinical application with IL-2 and immobilized anti-CD3 stimulation. RESULT Starting from 50 mL of blood, we generated >10(7)/m(2) tetramer-positive CTL within 2 weeks. Flow cytometric analysis of expanded lymphocytes showed that purity of CMV peptide-specific CTL was >75%. Upon stimulation of HLA-A*0201-restricted CMV peptide, expanded CD8 T lymphocytes produced intracellular IFN-gamma. Purified CTL exhibited cytotoxic activity against CMV peptide-pulsed T2 cells and CMV-infected HLA-A*0201-positive fibroblasts, but not against HLA mismatched or uninfected target cells. Alloreactivity could be excluded in MLC. DISCUSSION This simple, rapid culture system can be useful for adoptive immunotherapy after allogeneic SCT. We are now trying to adapt our laboratory scale study to a clinical scale study under good manufacturing practices (GMP) conditions.

Systemic DC Activation Modulates the Tumor Microenvironment and Shapes the Long-Lived Tumor-Specific Memory Mediated by CD8+ T Cells.

Strategies to reprogram the tumor microenvironment are being explored to improve cancer immunotherapy. In one approach, we have targeted dendritic cells (DC) to improve their function with adjuvant vector cells (aAVC) that are engineered from NKT ligand-loaded CD1d(+) allogeneic cells transfected with tumor antigen mRNAs. Here, we report the finding that this approach also programs local immune responses by establishing tertiary lymphoid structures (TLS), which include expanded antigen-specific CD8(+) T-cell clones, mobilized DCs, and normalized tumor vasculature. aAVC therapy also expanded specific Vβ-expressing antitumor T-cell clones, leading to the formation of long-term memory T cells. When combined with PD-1 blockade, aAVC infusion triggered regression of poorly immunogenic tumor cells that did not respond to PD-1 blockade alone, as well as expansion of antigen-specific CD8(+) T-cell clones in the tumor. The findings of this study help to inform a next-generation platform for the generation of efficacious cancer vaccines. Cancer Res; 76(13); 3756-66. ©2016 AACR.