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Human thymic stromal lymphopoietin (TSLP) promotes CD4+ T-cell proliferation both directly and indirectly through dendritic cell (DC) activation. Although human TSLP-activated DCs induce CD8+ T-cell proliferation, it is not clear whether TSLP acts directly on CD8+ T cells. In this study, we show that human CD8+ T cells activated by T-cell receptor stimulation expressed TSLP receptor (TSLPR), and that TSLP directly enhanced proliferation of activated CD8+ T cells. Although non-stimulated human CD8+ T cells from peripheral blood did not express TSLPR, CD8+ T cells activated by anti-CD3 plus anti-CD28 did express TSLPR. After T-cell receptor stimulation, TSLP directly enhanced the expansion of activated CD8+ T cells. Interestingly, using monocyte-derived DCs pulsed with a cytomegalovirus (CMV)-specific pp65 peptide, we found that although interleukin-2 allowed expansion of both CMV-specific and non-specific CD8+ T cells, TSLP induced expansion of only CMV-specific CD8+ T cells. These results suggest that human TSLP directly enhances expansion of CD8+ T cells and that the direct and indirect action of TSLP on expansion of target antigen-specific CD8+ T cells may be beneficial to adoptive cell transfer immunotherapy. [ABSTRACT FROM AUTHOR]

HA-1H is one of the most attractive minor histocompatibility antigens (mHA) as a target for immunotherapy of hematopoietic malignancies, but HLA-A*0201 and HLA-B60 molecules capable of presenting HA-1H-derived peptides are less common in eastern Asian populations when compared with Caucasian populations. Therefore, an attempt was made to search for novel epitopes presented by HLA alleles other than those previously reported by generating CTL lines from patients undergoing HLA-identical, HA-1 disparate hematopoietic stem cell transplantation (hematopoietic SCT) by stimulation with a 29-mer HA-1H peptide spanning a central polymorphic histidine (His). Two CTL clones established were found to be restricted by HLA-A*0206, which is the second or third most common HLA-A2 subtype worldwide. Epitope mapping revealed that the clones recognized the same nonameric peptide as A*0201-restricted HA-1H, VLHDDLLEA. This epitope was unexpected, since it does not contain any preferred anchor motifs for HLA-A*0206. However, an HLA peptide binding assay revealed stronger binding of this peptide to A*0206 than to A*0201. Interestingly, HLA-A*0206-restricted CTL clones could lyse both HLA-A*0206+ and HLA-A*0201+ targets (including leukemic blasts) that express HA-1H peptide endogenously, whereas an HLA-A*0201-restricted, HA-1H-specific CTL clone failed to lyse HLA-A*0206+ targets. This finding will expand the patient population who can benefit from HA-1H-based immunotherapy.Bone Marrow Transplantation (2007) 40, 165–174; doi:10.1038/sj.bmt.1705689; published online 28 May 2007 [ABSTRACT FROM AUTHOR]

Breast cancer, the most common malignancy among women, is closely associated with mutations in the tumor suppressor gene BRCA. DSS1, a component of the TRanscription-EXport-2 (TREX-2) complex involved in transcription and mRNA nuclear export, stabilizes BRCA2 expression. DSS1 is also related to poor prognosis in patients with breast cancer owing to the induction of chemoresistance. Recently, BRCA2 was shown to be associated with the TREX-2 component PCID2, which prevents DNA:RNA hybrid R-loop formation and transcription-coupled DNA damage. This study aimed to elucidate the involvement of these TREX-2 components and BRCA2 in the chemosensitivity of breast carcinomas. Our results showed that compared with that in normal breast tissues, DSS1 expression was upregulated in human breast carcinoma, whereas PCID2 expression was comparable between normal and malignant tissues. We then compared patient survival time among groups divided by high or low expressions of DSS1, BRCA2, and PCID2. Increased DSS1 expression was significantly correlated with poor prognosis in recurrence-free survival time, whereas no differences were detected in the high and low BRCA2 and PCID2 expression groups. We performed in vitro analyses, including propidium iodide nuclear staining, single-cell gel electrophoresis, and clonogenic survival assays, using breast carcinoma cell lines. The results confirmed that DSS1 depletion significantly increased chemosensitivity, whereas overexpression conferred chemoresistance to breast cancer cell lines; however, BRCA2 expression did not affect chemosensitivity. Similar to DSS1, PCID2 expression was also inversely correlated with chemosensitivity. These results strongly suggest that DSS1 and PCID2 depletion is closely associated with increased chemosensitivity via BRCA2-independent DNA damage. Together with the finding that DSS1 is not highly expressed in normal breast tissues, these results demonstrate that DSS1 depletion confers a druggable trait and may contribute to the development of novel chemotherapeutic strategies to treat DSS1-depleted breast carcinomas independent of BRCA2 mutations., (© 2021. The Author(s), under exclusive licence to United States and Canadian Academy of Pathology.)

Background: Germinal center-associated nuclear protein (GANP) is a phosphoprotein involved in mRNA export and regulation of DNA recombination. Although GANP expression in human breast cancer tissue is associated with breast cancer prognosis, the association between the genetic background of GANP and susceptibility and prognosis of breast cancer is unclear., Methods: We selected 694 breast cancer cases and 1376 age- and menopausal status-matched non-cancer controls from the Hospitable-based Epidemiologic Research Program, conducted at Aichi Cancer Center between 2001 and 2005. We evaluated the impact of two polymorphisms at the GANP locus (rs2839178 and rs11702450) on the susceptibility and prognosis of breast cancer. Reference alleles were defined as the A allele for rs2839178 and G allele for rs11702450., Results: The GG genotype of rs2839178 was statistically significantly associated with breast cancer risk (odds ratio [OR] 0.48, 95% confidence interval [CI] 0.30-0.76, P = 0.002). In prognostic analysis, compared to those with AA genotype of rs2839178, patients with AG or GG genotypes had longer disease-free survival (DFS) (hazard ratio [HR] 0.71, 95% CI 0.49-1.04 and HR 0.42, 95% CI 013-1.42, respectively, P for trend = 0.04). eQTL analysis indicated that association with rs2839178 can be explained by the effect of rs2839173 on expression of GANP/MCM3AP., Conclusions: The G allele of rs2839178 at the GANP locus was significantly associated with reduced breast cancer risk and longer DFS in breast cancer patients, showing a consistent direction in the association between susceptibility and clinical outcome. GANP is, therefore, important for the occurrence and progression of sporadic breast cancer.

This study presents an efficient method to improve TCR affinity, comprising 1) CDR-directed saturation mutation of TCR cDNA, 2) transient TCR display on CD3-expressing HEK293T (CD3-293T) cells by simple plasmid transfection, 3) staining with HLA-tetramers, and 4) multi-round sorting of cells with CD8-independent tetramer binding on a flow cytometer. Using these procedures, we successfully identified mutant TCRs with enhanced binding from an HLA-A*24:02-restricted, human telomerase reverse transcriptase (hTERT)-specific TCR. Two such clones, 2A7A and 2D162, harboring mutations in CDR1 and CDR2 of TCRβ, respectively, were isolated with both showing sequential four amino acid substitutions. When expressed on CD3-293T cells along with wild-type TCRα, the TCR molecules of these mutants as well as their combinatory mutation, bound to HLA-A24/hTERT-tetramers more strongly than the wild-type TCRs, without binding to control tetramers. Besides, in order to facilitate a functional study of TCR, we established an artificial T cell line, designated as CD8I-J2, which expresses a human CD8 and IFN-γ producing cassette by modifying Jurkat-derived J.RT3-T3.5 cells. CD8I-J2 cells expressing wild-type or affinity-enhanced hTERT-specific TCRs were analyzed for their recognition of serially diluted cognate peptide on HLA-A*24:02-transduced T2 cells. CD8I-J2 cells expressing each mutant TCR recognized the hTERT peptide at lower concentrations than wild-type TCR. The hierarchy of peptide recognition is concordant with tetramer binding on CD3-293T cells and none of these mutant TCRs were cross-reactive with irrelevant peptides reported to be present on HLA-A*24:02 molecules as far as tested. These methods might thus be useful for obtaining high affinity mutants from other TCRs of interest., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

CD4 + T helper (Th) cell activation is essential for inducing cytotoxic T lymphocyte (CTL) responses against malignancy. We reprogrammed a Th clone specific for chronic myelogenous leukemia (CML)-derived b3a2 peptide to pluripotency and re-differentiated the cells into original TCR-expressing T-lineage cells (iPS-T cells) with gene expression patterns resembling those of group 1 innate lymphoid cells. CD4 gene transduction into iPS-T cells enhanced b3a2 peptide-specific responses via b3a2 peptide-specific TCR. iPS-T cells upregulated CD40 ligand (CD40L) expression in response to interleukin-2 and interleukin-15. In the presence of Wilms tumor 1 (WT1) peptide, antigen-specific dendritic cells (DCs) conditioned by CD4-modified CD40L high iPS-T cells stimulated WT1-specific CTL priming, which eliminated WT1 peptide-expressing CML cells in vitro and in vivo. Thus, CD4 modification of CD40L high iPS-T cells generates innate lymphoid helper-like cells inducing bcr-abl-specific TCR signaling that mediates effectiveanti-leukemic CTL responses via DC maturation, showing potential for adjuvant immunotherapy against leukemia., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

The advent of tyrosine kinase inhibitor (TKI) therapy markedly improved the outcome of patients with chronic-phase chronic myeloid leukemia (CML). However, the poor prognosis of patients with advanced-phase CML and the lifelong dependency on TKIs are remaining challenges; therefore, an effective therapeutic has been sought. The BCR-ABL p210 fusion protein's junction region represents a leukemia-specific neoantigen and is thus an attractive target for antigen-specific T-cell immunotherapy. BCR-ABL p210 fusion-region-specific CD4 + T-helper (Th) cells possess antileukemic potential, but their function remains unclear. In this study, we established a BCR-ABL p210 b3a2 fusion-region-specific CD4 + Th-cell clone (b3a2-specific Th clone) and examined its dendritic cell (DC)-mediated antileukemic potential. The b3a2-specific Th clone recognized the b3a2 peptide in the context of HLA-DRB1*09:01 and exhibited a Th1 profile. Activation of this clone through T-cell antigen receptor stimulation triggered DC maturation, as indicated by upregulated production of CD86 and IL-12p70 by DCs, which depended on CD40 ligation by CD40L expressed on b3a2-specific Th cells. Moreover, in the presence of HLA-A*24:02-restricted Wilms tumor 1 (WT1) 235-243 peptide, DCs conditioned by b3a2-specific Th cells efficiently stimulated the primary expansion of WTI-specific cytotoxic T lymphocytes (CTLs). The expanded CTLs were cytotoxic toward WT1 235-243 -peptide-loaded HLA-A*24:02-positive cell lines and exerted a potent antileukemic effect in vivo. However, the b3a2-specific Th-clone-mediated antileukemic CTL responses were strongly inhibited by both TKIs and interferon-α. Our findings indicate a crucial role of b3a2-specific Th cells in leukemia antigen-specific CTL-mediated immunity and provide an experimental basis for establishing novel CML immunotherapies.

To expand our knowledge of the ontogeny of the T-cell receptor (TCR) repertoire of antigen-specific T-cell subsets, we combined next-generation deep sequencing and single-cell multiplex clonotype analysis to evaluate the diversity and frequency of paired TCRs, their functions and whether clonotypic TCRs are shared among different individuals. Using an HLA-A*02-restricted cytomegalovirus (CMV) pp65-derived immunogenic peptide, we found that the more dominant pp65-specific TCR clonotypes in the blood of healthy donors have higher binding affinities for the CMV peptide and arise from clonotypes that are highly shared among individuals. Interestingly, these highly shared HLA-A*02-restricted CMV-specific TCRs were detected in a CMV-seronegative individual as well as in HLA-A*02-negative donors albeit at lower frequency. More intriguingly, these shared TCR clonotypes were abundant in the stem memory T-cell subset, and TCR diversity of the stem memory T-cell repertoire was significantly lower than in the central memory and effector memory T-cell repertoires. These results suggest that the stem memory T-cell subset may serve as a reservoir of highly shared and highly functional memory T-cells.

Purpose: Mogamulizumab (Mog), a humanized anti-CC chemokine receptor 4 (CCR4) mAb that mediates antibody-dependent cellular cytotoxicity (ADCC) using FcγR IIIa (CD16)-expressing effector cells, has recently been approved for treatment of CCR4-positive adult T-cell leukemia (ATL) in Japan. However, Mog failure has sometimes been observed in patients who have accompanying chemotherapy-associated lymphocytopenia. In this study, we examined whether adoptive transfer of artificial ADCC effector cells combined with Mog would overcome this drawback., Experimental Design: We lentivirally gene-modified peripheral blood T cells from healthy volunteers and ATL patients expressing the affinity-increased chimeric CD16-CD3ζ receptor (cCD16ζ-T cells). Subsequently, we examined the ADCC effect mediated by those cCD16ζ-T cells in the presence of Mog against ATL tumor cells both in vitro and in vivo, Results: cCD16ζ-T cells derived from healthy donors killed in vitro Mog-opsonized ATL cell line cells (n = 7) and primary ATL cells (n = 4) depending on both the number of effector cells and the dose of the antibody. cCD16ζ-T cells generated from ATL patients (n = 3) also exerted cytocidal activity in vitro against Mog-opsonized autologous ATL cells. Using both intravenously disseminated model (n = 5) and subcutaneously inoculated model (n = 4), coadministration of Mog and human cCD16ζ-T cells successfully suppressed tumor growth in xenografted immunodeficient mice, and significantly prolonged their survival (P < 0.01 and P = 0.02, respectively)., Conclusions: These data strongly suggest clinical feasibility of the novel combined adoptive immunotherapy using cCD16ζ-T cells and Mog for treatment of aggressive ATL, particularly in patients who are ineligible for allogeneic hematopoietic stem cell transplantation. Clin Cancer Res; 22(17); 4405-16. ©2016 AACR., (©2016 American Association for Cancer Research.)

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., (©2016 American Association for Cancer Research.)

Leukemic Epstein-Barr virus (EBV)-associated post-transplant lymphoproliferative diseases (PTLD) following allogeneic hematopoietic stem cell transplantation are extremely rare. We can successfully treat an EBV-associated leukemic lymphoma patient with rituximab, cidofovir, and donor lymphocyte infusion (DLI). In the present case, EBV-specific T cells that were present in the peripheral blood before rituximab administration treatment rapidly increased after DLI in association with a decrease in the EBV-DNA load., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Aurora Kinase A is a cancer-associated protein normally involved in the regulation of mitosis. Being over-expressed in a range of cancers, it is a suitable target for cell-based immunotherapy. Gene transfer of T-cell receptor sequences cognisant of HLA-A*0201-restricted Aurora Kinase A antigen has previously been shown to transfer specific immunoreactivity against the target peptide in a Human Lymphocyte Antigen-restricted manner. While T cell receptor gene-transfer has great potential in overcoming the difficulties of isolating and expanding tumour-reactive lymphocytes from a patient's own cells, one hurdle is potential mispairing and competition between exogenous and endogenous T cell receptor chains. We have used a retroviral vector design bearing a short-interfering RNA that downregulates endogenous T cell receptor chains, without affecting expression of the transgenic T cell receptor sequences. The T cell receptor expression cassette also includes a 2A self-cleaving peptide, resulting in equimolar expression of the T cell receptor alpha and beta chains, further enhancing formation of the desired T cell receptor. Via a simple, modular cloning method, we have cloned the alpha and beta chains of the anti-Aurora Kinase A-reactive T cell receptor into this 'siTCR' vector. We then compared the activity of this vector against the original, 'conventional' vector across a panel of assays. T cell receptors expressed from the siTCR-vector retained the cytotoxic functionality of the original vector, with evidence of reduced off-target reactivity. The rate of expression of correctly-formed T cell receptors was superior using the siTCR design, and this was achieved at lower vector copy numbers. Maintaining T cell receptor efficacy with a reduced vector copy number reduces the risk of genotoxicity. The siTCR design also reduces the risk of mispairing and cross-reactivity, while increasing the functional titre. Such improvements in the safety of T cell receptor gene-transfer will be crucial for clinical applications of this technology.