CD8 Single-positive Cells Research Articles

Thymosin β4 Regulates the Differentiation of Thymocytes by Controlling the Cytoskeletal Rearrangement and Mitochondrial Transfer of Thymus Epithelial Cells.

The thymus is one of the most crucial immunological organs, undergoing visible age-related shrinkage. Thymic epithelial cells (TECs) play a vital role in maintaining the normal function of the thymus, and their degeneration is the primary cause of age-induced thymic devolution. Thymosin β4 (Tβ4) serves as a significant important G-actin sequestering peptide. The objective of this study was to explore whether Tβ4 influences thymocyte differentiation by regulating the cytoskeletal rearrangement and mitochondrial transfer of TECs. A combination of H&E staining, immunofluorescence, transmission electron microscopy, RT-qPCR, flow cytometry, cytoskeletal immunolabeling, and mitochondrial immunolabeling were employed to observe the effects of Tβ4 on TECs' skeleton rearrangement, mitochondrial transfer, and thymocyte differentiation. The study revealed that the Tβ4 primarily regulates the formation of microfilaments and the mitochondrial transfer of TECs, along with the formation and maturation of double-negative cells (CD4-CD8-) and CD4 single-positive cells (CD3+TCRβ+CD4+CD8-) thymocytes. This study suggests that Tβ4 plays a crucial role in thymocyte differentiation by influencing the cytoskeletal rearrangement and mitochondrial transfer of TECs. These effects may be associated with Tβ4's impact on the aggregation of F-actin. This finding opens up new avenues for research in the field of immune aging.

The Wiskott-Aldrich syndrome protein is required for positive selection during T-cell lineage differentiation.

The Wiskott-Aldrich syndrome (WAS) is an X-linked primary immune deficiency caused by a mutation in the WAS gene. This leads to altered or absent WAS protein (WASp) expression and function resulting in thrombocytopenia, eczema, recurrent infections, and autoimmunity. In T cells, WASp is required for immune synapse formation. Patients with WAS show reduced numbers of peripheral blood T lymphocytes and an altered T-cell receptor repertoire. In vitro, their peripheral T cells show decreased proliferation and cytokine production upon aCD3/aCD28 stimulation. It is unclear whether these T-cell defects are acquired during peripheral activation or are, in part, generated during thymic development. Here, we assessed the role of WASp during T-cell differentiation using artificial thymic organoid cultures and in the thymus of humanized mice. Although CRISPR/Cas9 WAS knockout hematopoietic stem and progenitor cells (HSPCs) rearranged the T-cell receptor and differentiated to T-cell receptor (TCR)+ CD4+ CD8+ double-positive (DP) cells similar to wild-type HSPCs, a partial defect in the generation of CD8 single-positive (SP) cells was observed, suggesting that WASp is involved in their positive selection. TCR repertoire analysis of the DP and CD8+ SP population, however, showed a polyclonal repertoire with no bias toward autoreactivity. To our knowledge, this is the first study of the role of WASp in human T-cell differentiation and on TCR repertoire generation.

Severe CD8+ T Lymphopenia in WHIM Syndrome Caused by Selective Sequestration in Primary Immune Organs.

Warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome is an ultra-rare combined primary immunodeficiency disease caused by heterozygous gain-of-function mutations in the chemokine receptor CXCR4. WHIM patients typically present with recurrent acute infections associated with myelokathexis (severe neutropenia due to bone marrow retention of mature neutrophils). Severe lymphopenia is also common, but the only associated chronic opportunistic pathogen is human papillomavirus and mechanisms are not clearly defined. In this study, we show that WHIM mutations cause more severe CD8 than CD4 lymphopenia in WHIM patients and WHIM model mice. Mechanistic studies in mice revealed selective and WHIM allele dose-dependent accumulation of mature CD8 single-positive cells in thymus in a cell-intrinsic manner due to prolonged intrathymic residence, associated with increased CD8 single-positive thymocyte chemotactic responses invitro toward the CXCR4 ligand CXCL12. In addition, mature WHIM CD8+ T cells preferentially home to and are retained in the bone marrow in mice in a cell-intrinsic manner. Administration of the specific CXCR4 antagonist AMD3100 (plerixafor) in mice rapidly and transiently corrected T cell lymphopenia and the CD4/CD8 ratio. After lymphocytic choriomeningitis virus infection, we found no difference in memory CD8+ T cell differentiation or viral load between wild-type and WHIM model mice. Thus, lymphopenia in WHIM syndrome may involve severe CXCR4-dependent CD8+ T cell deficiency resulting in part from sequestration in the primary lymphoid organs, thymus, and bone marrow.

NPAT Supports CD8+ Immature Single-Positive Thymocyte Proliferation and Thymic Development.

Thymocytes need to proliferate into a significant cell mass to allow a subsequent selection process during the double-positive (DP) stage. However, it is not clear at what stage this massive cell proliferation occurs. Immature CD8 single-positive (ISP) cells are a well-defined thymocyte subpopulation. However, the function of this cell subset has not yet been characterized. In this study, we analyzed the transcription pattern of mouse ISP cells and observed higher expression levels of cell cycling genes. We also found out that ISP cells exhibited the highest cell proliferative capacity among thymocytes in different developmental stages. Nuclear protein ataxia-telangiectasia (NPAT/p220) is one of the highly expressed cell cycling genes in ISP cells, which is known to play a critical role in coordinating histone gene expression necessary for rapid cell proliferation. Selective deletion of NPAT at the ISP stage led to reduced thymus size and significant loss of DP cells, secondary to reduced histone gene expression and impaired ISP cell proliferation capacity. A block of thymocyte development at the ISP stage was also observed, which was due to increased IL-7R expression. Continuous IL-7R signal served as a compensating mechanism for cell proliferation upon NPAT deletion, but in turn inhibited the expression of transcription factors TCF-1 and LEF-1, which is essential for the transition of ISP to DP cells. In summary, our study revealed the proliferation capacity of the ISP subpopulation during thymocyte differentiation as well as a vital role of NPAT in this developmental stage.

TET proteins regulate T cell and iNKT cell lineage specification in a TET2 catalytic dependent manner.

TET proteins mediate DNA demethylation by oxidizing 5-methylcytosine to 5-hydroxymethylcytosine (5hmC) and other oxidative derivatives. We have previously demonstrated a dynamic enrichment of 5hmC during T and invariant natural killer T cell lineage specification. Here, we investigate shared signatures in gene expression of Tet2/3 DKO CD4 single positive (SP) and iNKT cells in the thymus. We discover that TET proteins exert a fundamental role in regulating the expression of the lineage specifying factor Th-POK, which is encoded by Zbtb7b. We demonstrate that TET proteins mediate DNA demethylation - surrounding a proximal enhancer, critical for the intensity of Th-POK expression. In addition, TET proteins drive the DNA demethylation of site A at the Zbtb7b locus to facilitate GATA3 binding. GATA3 induces Th-POK expression in CD4 SP cells. Finally, by introducing a novel mouse model that lacks TET3 and expresses full length, catalytically inactive TET2, we establish a causal link between TET2 catalytic activity and lineage specification of both conventional and unconventional T cells.

Salt inducible kinases 2 and 3 are required for thymic T cell development

Salt Inducible Kinases (SIKs), of which there are 3 isoforms, are established to play roles in innate immunity, metabolic control and neuronal function, but their role in adaptive immunity is unknown. To address this gap, we used a combination of SIK knockout and kinase-inactive knock-in mice. The combined loss of SIK1 and SIK2 activity did not block T cell development. Conditional knockout of SIK3 in haemopoietic cells, driven by a Vav-iCre transgene, resulted in a moderate reduction in the numbers of peripheral T cells, but normal B cell numbers. Constitutive knockout of SIK2 combined with conditional knockout of SIK3 in the haemopoietic cells resulted in a severe reduction in peripheral T cells without reducing B cell number. A similar effect was seen when SIK3 deletion was driven via CD4-Cre transgene to delete at the DP stage of T cell development. Analysis of the SIK2/3 Vav-iCre mice showed that thymocyte number was greatly reduced, but development was not blocked completely as indicated by the presence of low numbers CD4 and CD8 single positive cells. SIK2 and SIK3 were not required for rearrangement of the TCRβ locus, or for low level cell surface expression of the TCR complex on the surface of CD4/CD8 double positive thymocytes. In the absence of both SIK2 and SIK3, progression to mature single positive cells was greatly reduced, suggesting a defect in negative and/or positive selection in the thymus. In agreement with an effect on negative selection, increased apoptosis was seen in thymic TCRbeta high/CD5 positive cells from SIK2/3 knockout mice. Together, these results show an important role for SIK2 and SIK3 in thymic T cell development.

Early cytotoxic lymphocyte localization to the brain following resuscitation in a porcine model of asphyxial cardiac arrest: A pilot study

IntroductionOut-of-hospital cardiac arrest (OHCA) is a major cause of morbidity and mortality in the US. Of major concern is a lack of therapies to mitigate associated brain injury. Immune cell infiltration (ICI) into the brain, which may exacerbate injury post-resuscitation, is one possible therapeutic target, although the post-OHCA immune response has not been fully characterized. ObjectiveIn this pilot study, we aimed to detect early post-resuscitation cytotoxic lymphocyte ICI in porcine brain using a model of opioid-mediated asphyxial OHCA. MethodsTen young, healthy swine (26.7+/-3.4 kg) were sedated, anaesthetized and paralyzed. In eight of the animals, this was followed by induction of asphyxial OHCA via fentanyl bolus and concurrent airway occlusion. The remaining two ‘sham’ animals were instrumented but did not undergo asphyxia. After nine minutes of asphyxia, mechanical CPR and manual ventilations were started, in an initial BLS followed by ALS configuration. At termination of resuscitation or euthanasia, the whole brain was removed. Immune cells were extracted and analyzed via flow cytometry. Results304 +/− 62.2 cells/g were discovered to be CD8 single positive cells in animals that achieved ROSC, 481 +/− 274.4 cells/g in animals that did not achieve ROSC, and 40 +/− 11.31 cells/g in sham animals. CD8 single positive cells made up 0.473 +/− 0.24% of detected cells in animals that achieved ROSC, 0.395 +/− 0.062% in animals that did not achieve ROSC, and 0.19 +/− 0.014% in sham animals (No ROSC vs Sham, p = 0.012). ConclusionsThese data suggest that cytotoxic lymphocytes may be localizing to the brain during cardiac arrest resuscitation.

Cofilin1-driven actin dynamics controls migration of thymocytes and is essential for positive selection in the thymus.

Actin dynamics is essential for T-cell development. We show here that cofilin1 is the key molecule for controlling actin filament turnover in this process. Mice with specific depletion of cofilin1 in thymocytes showed increased steady-state levels of actin filaments, and associated alterations in the pattern of thymocyte migration and adhesion. Our data suggest that cofilin1 is controlling oscillatory F-actin changes, a parameter that influences the migration pattern in a 3-D environment. In a collagen matrix, cofilin1 controls the speed and resting intervals of migrating thymocytes. Cofilin1 was not involved in thymocyte proliferation, cell survival, apoptosis or surface receptor trafficking. However, in cofilin1 mutant mice, impaired adhesion and migration resulted in a specific block of thymocyte differentiation from CD4/CD8 double-positive thymocytes towards CD4 and CD8 single-positive cells. Our data suggest that tuning of the dwelling time of thymocytes in the thymic niches is tightly controlled by cofilin1 and essential for positive selection during T-cell differentiation. We describe a novel role of cofilin1 in the physiological context of migration-dependent cell differentiation.

Adhesion between medullary thymic epithelial cells and thymocytes is regulated by miR-181b-5p and miR-30b*

In this work, we demonstrate that adhesion between medullary thymic epithelial cells (mTECs) and thymocytes is controlled by miRNAs. Adhesion between mTECs and developing thymocytes is essential for triggering negative selection (NS) of autoreactive thymocytes that occurs in the thymus. Immune recognition is mediated by the MHC / TCR receptor, whereas adhesion molecules hold cell-cell interaction stability. Indeed, these processes must be finely controlled, if it is not, it may lead to aggressive autoimmunity. Conversely, the precise molecular genetic control of mTEC-thymocyte adhesion is largely unclear. Here, we asked whether miRNAs would be controlling this process through the posttranscriptional regulation of mRNAs that encode adhesion molecules. For this, we used small interfering RNA to knockdown (KD) Dicer mRNA in vitro in a murine mTEC line. A functional assay with fresh murine thymocytes co-cultured with mTECs showed that single-positive (SP) CD4 and CD8 thymocyte adhesion was increased after Dicer KD and most adherent subtype was CD8 SP cells. Analysis of broad mTEC transcriptional expression showed that Dicer KD led to the modulation of 114 miRNAs and 422 mRNAs, including those encoding cell adhesion or extracellular matrix proteins, such as Lgals9, Lgals3pb, Tnc and Cd47. Analysis of miRNA-mRNA networks followed by miRNA mimic transfection showed that these mRNAs are under the control of miR-181b-5p and miR-30b*, which may ultimately control mTEC-thymocyte adhesion. The expression of CD80 surface marker in mTECs was increased after Dicer KD following thymocyte adhesion. This indicates the existence of new mechanisms in mTECs that involve the synergistic action of thymocyte adhesion and regulatory miRNAs.

Abstract 5686: Utilization of fluorescent multiplex IHC and digital image analysis for studying LAG-3, CD3 and CD8 positive TIL subsets in NSCLC tissue

Abstract Targeting checkpoint molecules expressed on immune cells has shown promising results in the treatment of non-small cell lung cancer (NSCLC). One interesting molecule that is frequently expressed on exhausted tumor-infiltrating lymphocytes (TIL) is lymphocyte activation gene-3 (LAG-3). We recently implemented chromogenic anti-LAG-3/CD3 dual immunohistochemistry (IHC) and digital image analysis to quantify LAG-3 positive T cells in NSCLC tissue. Here, we extended this work by establishing an anti-LAG-3/CD3/CD8/pan-Cytokeratin (pan-CK) fluorescent multiplex IHC (mIHC) assay followed by digital image analysis to examine the composition of T cells infiltrating NSCLC tissue in more detail. Tyramide signal amplification (TSA) based fluorescent 5-color mIHC (LAG-3/CD3/CD8/pan-CK + DAPI) was developed by Indivumed on the Leica BOND RX automated staining platform and applied to formalin-fixed paraffin-embedded (FFPE) NSCLC tissue samples. Image analysis was performed by OracleBio. Tumor and stroma regions of interest (ROI) were classified according to the pan-CK and DAPI signals. LAG-3, CD3 and CD8 single positive cells, as well as dual and triple positive cells, were then quantified in the tumor and stroma ROIs. Fluorescent mIHC allowed for a specific quantification of LAG-3, CD3 and CD8 single, LAG-3/CD3, LAG-3/CD8, CD3/CD8 dual and LAG-3/CD3/CD8 triple labeled cells in the tumor and stroma ROIs of the analyzed NSCLC samples. As part of the assay validation, similar ratios of the differently labeled cells, in particular LAG-3 positive T cells (LAG-3/CD3 dual positive), were observed with fluorescent mIHC and chromogenic IHC, demonstrating a good concordance between the two approaches. Among the analyzed NSCLC samples, different ratios of LAG-3 positive TILs in adeno- and non-adenocarcinoma samples were detected. Determining the ratios of dual or triple positive TIL subsets by mIHC in combination with digital image analysis allows for a clearer understanding of the lymphocyte composition within NSCLC tissue than a simple quantification of LAG-3, CD3 and CD8 single positive cells. Furthermore, such information will contribute to a deeper understanding of the role of LAG-3 positive immune cell sub-populations in the progression and treatment of NSCLC. Citation Format: Malik Khenkhar, Daniel Biljes, Nickels Winkler, Philipp C. Uhlig, Hartmut Juhl, Alison L. Bigley, Lorcan Sherry. Utilization of fluorescent multiplex IHC and digital image analysis for studying LAG-3, CD3 and CD8 positive TIL subsets in NSCLC tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5686.

Murine CD8+ Invariant Natural Killer T Cells are Negatively Selected by CD1d Expressed on Thymic Epithelial Cells and Dendritic Cells

ABSTRACTBackground: CD1d-dependent invariant natural killer (iNKT) cells are found as either CD4 single positive (SP) or CD4/CD8 double negative (DN) cells in mice. The size of the CD8+ iNKT population is extremely small. It is known that CD1d expression on developing thymocytes is sufficient for iNKT development and co-receptor choice, which is driven by Th-POK expression. This study aimed to examine the factors involved in the CD4/CD8 co-receptor choice of iNKT cells in addition to Th-POK-driven silencing of CD8 expression. Methods: In this study, we compared iNKT cells of wild-type (WT) mice with those of transgenic mice in which CD1d expression is restricted to developing thymocytes by the proximal Lck (pLCK) promoter. CD8 positive iNKT cell population were analyzed by flow cytometry. Results: We found that there was a substantial population of CD8+ iNKT cells in the thymus and spleen of transgenic mice, and these cells are negatively selected in between Stage 2 and Stage 3 of their developmental program by the CD1d expressed on Thymic epithelial cell (TEC) and Dendritic cells in WT mice. Conclusion: We conclude that TEC expression of CD1d in the murine thymus contributed to co-receptor choice of iNKT cells, in addition to Th-POK-driven silencing of CD8. Therefore, mostly CD4 SP and DN iNKT cells are produced under normal physiological conditions in mice.

Abstract 4622: Characterization of sulfatide reactive type II NKT cells from mouse lung

Abstract The immune system plays a major role in the elimination of tumors. CD8 T cell infiltration is known to be a good prognostic indicator. The development of therapies based on checkpoint inhibitor antibodies was an important breakthrough in increasing survival by limiting the exhaustion of cytotoxic cells and increasing tumor cell eradication. However, the immune system also contains regulatory cells that protect the organism from inappropriate activation of immune cells against self-antigens. Thus, the fact that tumor cells are autologous leads regulatory cells to inhibit the activation of anti-tumor cytotoxic cells and thus increase tumor escape. Therefore, understanding the function and the activation of regulatory cells might help to develop therapies to limit the activation of regulatory cells in order to increase tumor clearance. Natural killer T (NKT) cells are lymphocytes with features of natural killer (NK) and of T cells placing them at the interface of innate and adaptive immunity. Like NK cells, they rapidly produce cytokines after stimulation, orienting the immune response. As T cells, they express a T cell receptor (TCR) that allows the recognition of specific lipids presented by the non-classical MHC-I molecule CD1d. According to their TCR usage, two populations of NKT cells are described: type I and type II. All type I NKT cells express a semi-invariant TCR (Valpha24Jalpha18 in humans, Valpha14Jalpah18 in mice) that recognizes α-galactosylceramide (αGalCer). They can be identified with the αGalCer-loaded CD1d tetramer. In contrast, type II NKT cells express a more diverse TCR repertoire. There is no currently identified lipid antigen recognized by all type II NKT cells, making their identification more difficult. A fraction of them recognize sulfatide. By using sulfatide-loaded CD1d-tetramers, we observed for the first time that sulfatide-reactive type II NKT cells were enriched in the lung, a major site of tumor metastasis. Moreover, we previously showed that in vivo stimulation of type II NKT cells with sulfatide increased the number of tumor nodules in the lung. An in-depth phenotype analysis revealed that they were CD4 or CD8 single positive cells, like conventional T cells, whereas type I NKT cells are either CD4+CD8- or double negative. Type II NKT cells do not express PLZF, the master regulator of NKT cell development and exist in PLZF-/- mice contrary to type I NKT cells. We showed that type II NKT cells also expressed markers of myeloid cells, c-Kit, CD11b and Ly6C even though histological analysis revealed lymphocytic morphology. Interestingly, at steady state, type II NKT cells expressed granzyme A but not granzyme B or perforin. The in vivo injection of sulfatide increased the expression of the activation markers CD69 and CD44 as well as granzyme A. Since the regulatory functions of type II NKT cells have been shown to be critical in tumor immunity, the detailed characterization of these cells could help to develop a new immunotherapy for cancer. Citation Format: Lise Pasquet, Shingo Kato, Tony Adams, Susan Sharrow, Theresa Davies-Hill, Elaine Jaffe, Xia Zheng, Motoshi Suzuki, Damian Kovalovsky, Jay Berzofsky, Masaki Terabe. Characterization of sulfatide reactive type II NKT cells from mouse lung [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4622. doi:10.1158/1538-7445.AM2017-4622

Functionality of lung type II NKT cells developing in CD1d−/− animals

Abstract NKT cells are lymphocytes that recognize lipid antigens presented by CD1d. Type I NKT cells with semi-invariant TCR recognize α-galactosylceramide while many type II NKT cells with a more diverse TCR repertoire recognize sulfatide. We developed a stable sulfatide-loaded CD1d tetramer that allows the specific identification of type II NKT cells and found that they are enriched in the lung, a major site of tumor metastasis. An in-depth phenotypical analysis revealed that lung type II NKT cells were CD4 or CD8 single positive cells, like conventional T cells, whereas type I NKT cells were either CD4+CD8- or CD4-CD8-. We showed that type II NKT cells expressed markers of myeloid cells, c-Kit, CD11b and Ly6C, even though morphological analysis revealed lymphocytic morphology. In contrast to type I NKT cells, type II NKT existed in PLZF−/− and CD1d−/− mice. Thus, we asked the importance of CD1d in the in vivo and in vitro activation of type II NKT cells. In the steady state, lung type II NKT cells expressed granzyme A but not granzyme B or perforin. By injecting sulfatide in vivo, we observed that WT but not CD1d−/− lung type II NKT cells upregulated granzyme A and Ki67 expression. Using syngeneic WT bone marrow-derived dendritic cells (BMDC) as APCs, ex-vivo stimulation of WT but not CD1d−/− lung type II NKT cells with sulfatide induced the secretion of IFN-γ, IL-13 and IL-17. This was consistent with the expression of specific transcription factors, T-bet and RORγt, by lung type II NKT cells. In conclusion type II NKT cells still develop in CD1d−/− mice but are not functional. The ability to study responses of type II NKT cells in vitro can now help elucidate the mechanism by which these regulatory cells interact with other cells and modulate tumor immunity in the lung.

Ubc9 Is Required for Positive Selection and Late-Stage Maturation of Thymocytes.

SUMOylation is an important posttranslational modification that regulates protein function in diverse biological processes. However, its role in early T cell development has not been genetically studied. UBC9 is the only E2 enzyme for all SUMOylation. In this study, by selectively deleting Ubc9 gene in T cells, we have investigated the functional roles of SUMOylation in T cell development. Loss of Ubc9 results in a significant reduction of CD4 and CD8 single-positive lymphocytes in both thymus and periphery. Ubc9-deficient cells exhibit defective late-stage maturation post the initial positive selection with increased apoptosis and impaired proliferation, among which attenuated IL-7 signaling was correlated with the decreased survival of Ubc9-deficent CD8 single-positive cells. Furthermore, NFAT nuclear retention induced by TCR signals was regulated by SUMOylation during thymocytes development. Our study thus reveals a novel posttranslational mechanism underlying T cell development.

CD31, a Valuable Marker to Identify Early and Late Stages of T Cell Differentiation in the Human Thymus.

Although CD31 expression on human thymocytes has been reported, a detailed analysis of CD31 expression at various stages of T cell development in the human thymus is missing. In this study, we provide a global picture of the evolution of CD31 expression from the CD34+ hematopoietic precursor to the CD45RA+ mature CD4+ and CD8+ single-positive (SP) T cells. Using nine-color flow cytometry, we show that CD31 is highly expressed on CD34+ progenitors and stays high until the early double-positive stage (CD3-CD4+CD8α+β-). After β-selection, CD31 expression levels become low to undetectable. CD31 expression then increases and peaks on CD3highCD4+CD8+ double-positive thymocytes. However, following positive selection, CD31 expression differs dramatically between CD4+ and CD8+ lineages: homogeneously high on CD8 SP but lower or negative on CD4 SP cells, including a subset of CD45RA+CD31- mature CD4+ thymocytes. CD31 expression on TCRγδ thymocytes is very similar to that of CD4 SP cells. Remarkably, there is a substantial subset of semimature (CD45RA-) CD4 SP thymocytes that lack CD31 expression. Moreover, FOXP3+ and ICOS+ cells are overrepresented in this CD31- subpopulation. Despite this CD31-CD45RA- subpopulation, most egress-capable mature CD45RA+ CD4 SP thymocytes express CD31. The variations in CD31 expression appear to coincide with three major selection processes occurring during thymopoiesis: β-selection, positive selection, and negative selection. Considering the ability of CD31 to modulate the TCR's activation threshold via the recruitment of tyrosine phosphatases, our results suggest a significant role for CD31 during T cell development.

Regeneration of Tumor Antigen Specific CTLs Utilizing iPSC Technology for Off-the-Shelf Immunotherapy

In the tumor immunotherapy field tumor antigen specific T cell therapy is currently attracting attention. However, a critical issue still awaits resolution; it is difficult to efficiently expand tumor antigen specific T cells without exhaustion. To solve this problem, we are now utilizing induced pluripotent stem cell (iPSC) technology. When iPSCs are established from tumor antigen specific cytotoxic T cells (CTLs), T cells regenerated from these iPSCs (T-iPSCs) are expected to express the same T cell receptors (TCRs) as the original CTLs. Therefore, it will become possible to obtain de novo generated tumor antigen specific CTLs almost unlimitedly. In line with this concept, we have succeeded in establishing iPSCs from CTLs specific for the melanoma antigen MART-1 and regenerating MART-1 specific CTLs (Vizcardo et al, Cell Stem Cell, 2013).The strategy mentioned above was supposed to be applied as autologous transfusion. Autologous transfusion setting, however, has some problems; i) high cost to establish T-iPSCs from each patient, ii) heterogeneity of the quality as iPSCs, iii) difficulty in getting T-iPSCs with high affinity TCRs. To solve these problems, we are now planning to apply this method to the allogeneic transfusion setting. In allogeneic setting, we can select high quality T-iPSC clones with high affinity TCRs and such clones can be utilized to many patients with the same HLA type. In this context, it is preferable to establish T-iPSCs from HLA haplotype homozygous donors, since CTLs generated from these T-iPSCs can be administered to the patients carrying the same HLA haplotype at least on one allele. Thus by utilizing allogenic T-iPSCs the cost would be reduced and the quality of T-iPSCs can be ensured.Now we are trying to establish T-iPSCs from CTLs specific for various tumor antigen from healthy donors. We succeeded in establishing T-iPSCs from CTLs specific for EB virus related antigen LMP2 and cancer testis antigen WT1, both of which are restricted to HLA A2402, the commonest HLA-A in the Japanese. We improved in vitro culture condition and have succeeded in regenerating CD8 single positive cells expressing CD8 alpha-beta heterodimers. These regenerated CTLs exhibited very high antigen specific cytotoxicity comparable to the original CTLs. The CTLs derived from WT1 specific T-iPSCs were able to lyse some leukemia cell lines and primary leukemia cells which express endogenous WT1 protein. Administration of regenerated CTLs prolonged the survival of mice inoculated with leukemia cell line in in vivo xenograft model. In addition, we succeeded in transducing iPSCs derived from non-T cells with WT1-specific TCR genes (TCR-iPSCs) and generating CTLs from them. Generated CTLs from TCR-iPSCs exhibited comparable TCR specific cytotoxicity as regenerated CTLs from T-iPSCs. Using this method, it would be much easier to produce CTLs with defined high affinity TCRs specific for various tumor antigen.We are currently endeavoring to apply this method clinically. In the future, we intend to establish an allogeneic transfusion system, in which various tumor antigen specific T-iPSCs/TCR-iPSCs from a wide range of HLA haplotype homozygous donors will be lined up as a "T-iPS/TCR-iPSCs cell bank", with the aim of making off- the-shelf tumor immunotherapy a reality. DisclosuresKataoka:Boehringer Ingelheim: Honoraria; Kyowa Hakko Kirin: Honoraria; Yakult: Honoraria. Ogawa:Sumitomo Dainippon Pharma: Research Funding; Kan research institute: Consultancy, Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding. Kawamoto:Regcel: Research Funding.

A Histone Methyltransferase ESET Is Critical for T Cell Development.

ESET/SETDB1, one of the major histone methyltransferases, catalyzes histone 3 lysine 9 (H3K9) trimethylation. ESET is critical for suppressing expression of retroviral elements in embryonic stem cells; however, its role in the immune system is not known. We found that thymocyte-specific deletion of ESET caused impaired T cell development, with CD8 lineage cells being most severely affected. Increased apoptosis of CD8 single-positive cells was observed, and TCR-induced ERK activation was severely inhibited in ESET(-/-) thymocytes. Genome-wide comprehensive analysis of mRNA expression and H3K9 trimethylation revealed that ESET regulates expression of numerous genes in thymocytes. Among them, FcγRIIB, whose signaling can inhibit ERK activation, was strongly and ectopically expressed in ESET(-/-) thymocytes. Indeed, genetic depletion of FcγRIIB in ESET(-/-) thymocytes rescued impaired ERK activation and partially restored defective positive selection in ESET(-/-) mice. Therefore, impaired T cell development in ESET(-/-) mice is partly due to the aberrant expression of FcγRIIB. Collectively, to our knowledge, we identify ESET as the first trimethylated H3K9 histone methyltransferase playing a crucial role in T cell development.

Abstract B126: Regeneration of tumor antigen specific CTLs utilizing iPSC technology

Abstract Current cancer immunotherapies mainly aim to activate and expand tumor antigen specific cytotoxic T lymphocytes (CTLs) in vitro or in vivo, but these methods are not so successuful because of the difficulty in preparing sufficient number of CTLs. We are trying to overcome this problem by utilizing the induced pluripotent stem cell (iPSC) technology. Our strategy is based on the concept that i) when iPSCs are produced from antigen specific T cells, the rearranged configurations of T cell receptor (TCR) genes are inherited to the iPSCs (which we call T-iPSCs), and that ii) when T cells are regenerated from such T-iPSCs, all of them should come to express the same TCR as the original one. Therefore it will become possible to obtain de novo generated tumor specific CTLs almost unlimitedly, because it is possible to make a mass-culture of cells at the iPSC level. In line with this concept, we have succeeded in establishing T-iPSCs from CTLs specific for the melanoma antigen MART-1 derived from tumor infiltarating lymphocytes, and in regenerating MART-1 specific T cells from such T-iPSCs (Vizcardo et al, Cell Stem Cell, 2013). The strategy mentioned above has been conducted in the category of autologous transplantation. Autologous transplantation setting, however, has some problems; i) high cost to establish T-iPSCs from each patients, ii) heterogeneneity of the quality as iPSCs, iii) difficulty in getting high affinity TCR. To solve these problems we are now planing to apply this method to the allogeneic transplantation setting. In allogeneic setting, we can select high quality T-iPSC clone and such a clone can be used to many other patients. It is preferable to produce T-iPSCs from a HLA haplotype homo donor, since CTLs generated from these T-iPSCs can be administerd to the patients carrying the same HLA haplotype on one allele. Thus by utilizing allogenic T-iPSCs the cost would be reduced and the quality of T-iPSCs can be ensured. Now we are trying to establish T-iPSCs from CTLs specific for various cancer antigen from healthy donors. We succeded in establishing T-iPSCs from CTLs specific for EB virus related antigen LMP2 and cancer testis antigen WT1, both of which are restricted to HLA A2402, the commonest HLA-A in the Japanese. We improved in vitro culture condition and have succeded in regenerating CD8 single positive cells expressing CD8 alpha-beta heterodimers. These regenerated CTLs exhibited very high antigen specific killing activity comparable to the original CTLs. The CTLs derived from WT1 specific T-iPSCs were able to kill some leukemia cell lines which express endogenous WT1 protein. They can be expanded more than ten thousand fold by repeated TCR stimulation without reducing their killing activity. We are now examining in vivo killing activity of these CTLs using zenograft model. This method could bring about a breakthrough in cancer immunotherapy. Citation Format: Takuya Maeda, Seiji Nagano, Hiroshi Ichise, Kyoko Masuda, Toshio Kitawaki, Akifumi Kondo-Takaori, Hiroshi Kawamoto. Regeneration of tumor antigen specific CTLs utilizing iPSC technology. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B126.

Editorial: The Role of Aire, microRNAs and Cell-Cell Interactions on Thymic Architecture and Induction of Tolerance.

Editorial: The Role of Aire, microRNAs and Cell-Cell Interactions on Thymic Architecture and Induction of Tolerance.

Histone Deacetylase 3 Is Required for Efficient T Cell Development.

Hdac3 is a key target for Hdac inhibitors that are efficacious in cutaneous T cell lymphoma. Moreover, the regulation of chromatin structure is critical as thymocytes transition from an immature cell with open chromatin to a mature T cell with tightly condensed chromatin. To define the phenotypes controlled by Hdac3 during T cell development, we conditionally deleted Hdac3 using the Lck-Cre transgene. This strategy inactivated Hdac3 in the double-negative stages of thymocyte development and caused a significant impairment at the CD8 immature single-positive (ISP) stage and the CD4/CD8 double-positive stage, with few mature CD4(+) or CD8(+) single-positive cells being produced. When Hdac3(-/-) mice were crossed with Bcl-xL-, Bcl2-, or TCRβ-expressing transgenic mice, a modest level of complementation was found. However, when the null mice were crossed with mice expressing a fully rearranged T cell receptor αβ transgene, normal levels of CD4 single-positive cells were produced. Thus, Hdac3 is required for the efficient transit from double-negative stage 4 through positive selection.