Single Positive Cells Research Articles

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.

CCR2 Signal Facilitates Thymic Egress by Priming Thymocyte Responses to Sphingosine-1-Phosphate.

The signal mediated by sphingosine-1-phosphate receptor 1 (S1P1) is essential but seemingly insufficient for thymic export of newly generated T cells. Here, we reported the identification of CCR2 as an additional regulator of this process. CCR2 showed a markedly increased expression in the most mature subset of single-positive (SP) thymocytes. Its deficiency led to a reduction of recent thymic emigrants in the periphery and a simultaneous accumulation of mature SP cells in the thymus. The CCR2 signaling promoted thymic emigration primarily through modulating the chemotactic responses to S1P1 engagement. On the one hand, the chemokinesis induced by CCR2 activation endowed thymocytes with enhanced capacity to respond to S1P-induced migration. On the other hand, CCR2 signaling through Stat3 augmented forkhead box O1 activity, leading to increased expression of S1P1. Taken together, the present study highlights a unique and novel function of CCR2 signaling in the regulation of thymic egress.

Genetic Ablation of the ABC Transporter Abcc4 Impairs Lymphoid Leukemogenesis

ABCC4 is an ABC transporter of the C sub‐family that is ubiquitously expressed and exports key endogenous compounds including cAMP and prostaglandins, and cancer chemotherapeutic drugs. cAMP and prostaglandins are signaling molecules that, among other functions, regulate thymocyte development and function. Thymocyte progenitors, IL7 receptor positive cells, migrate from the bone marrow to the thymus and give rise to mature T‐cells. The maturation of thymocytes can be followed by monitoring for the presence of surface markers CD4 and CD8. Briefly, thymocytes are initially double negative (DN) cells, with neither CD4 nor CD8; after T‐cell receptor rearrangement, CD4 and CD8 are expressed on double positive cells (DP), which following positive and negative selection, become mature, single positive (SP) cells expressing either CD4 or CD8. Abcc4 is highly expressed in DP and CD4‐positve thymocytes, while being undetectable in DN thymocytes from wild‐type (WT) mice, suggesting a developmental regulation of Abcc4. Therefore, we hypothesized that ablation of Abcc4 from mice would lead to aberrant T‐cell development and function.ABCC4 absence had no measureable impact on the number of hematopoietic progenitors. We hypothesized that lymphoid progenitors in Abcc4‐knockout(KO) mice may have an impaired leukemogenesis potential. To test this hypothesis, hematopoietic progenitors from Abcc4‐KO or WT mice were transduced with a retrovirus harboring a MYCN cDNA, a known driver of lymphoid leukemia, and subsequently transplanted into lethally irradiated congenic WT recipient mice. Mice transplanted with either WT or Abcc4‐KO MYCN‐transduced hematopoietic progenitors developed both B‐ and T‐cell leukemia, with tumors expressing Abcc4 on the plasma membrane. Kaplan‐Meier survival analysis revealed that mice transduced with Abcc4‐KO progenitors had a dramatic increase in median survival from 81.5 days in mice who received WT progenitors to 112 days in mice who received Abcc4‐KO progenitors. These studies suggested the lymphoid progenitors from Abcc4‐KO mice harbored an intrinsic defect manifested by reduced lymphoid leukemia. This was further investigated by performing a competitive bone‐marrow transplantation experiment. Briefly, hematopoietic progenitors from Abcc4‐KO and wildtype mice were isolated and mixed in equal numbers (each harboring a different surface marker to enable identification), then transplanted into congenic wildtype mice following lethal irradiation; subsequently the differentiated lymphoid progeny were identified in the peripheral blood. These studies revealed that the number of CD4+ cells from Abcc4‐KO donors was reduced, thus confirming a defect in Abcc4‐KO lymphoid progenitors. A defect in peripheral thymocyte function is also a potential mechanism, as genes in the arachidonic acid pathway regulate further differentiation and function of mature thymocytes. We discovered altered expression of genes in the arachidonic acid pathway such as Cox‐1 and Ptger4, when compared to age‐matched WT mice. These data suggest that genetic ablation of Abcc4 has an effect on arachidonic acid signaling, which may in turn affect thymocyte function.Overall, the data presented herein suggest that ABCC4 impacts lymphoid progenitors and differentiated thymocytes. One implication of these findings is that deficiency or insufficiency of ABCC4 may reduce the possibility of developing lymphoid malignancies.Support or Funding InformationThis work was funded by NIH and ALSACThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Metabolomic profiling identifies distinct phenotypes for ASS1 positive and negative GBM

BackgroundTumour cells have a high demand for arginine. However, a subset of glioblastomas has a defect in the arginine biosynthetic pathway due to epigenetic silencing of the rate limiting enzyme argininosuccinate synthetase (ASS1). These tumours are auxotrophic for arginine and susceptible to the arginine degrading enzyme, pegylated arginine deiminase (ADI-PEG20). Moreover, ASS1 deficient GBM have a worse prognosis compared to ASS1 positive tumours. Since altered tumour metabolism is one of the hallmarks of cancer we were interested to determine if these two subtypes exhibited different metabolic profiles that could allow for their non-invasive detection as well as unveil additional novel therapeutic opportunities.MethodsWe looked for basal metabolic differences using one and two-dimensional gas chromatography-time-of-flight mass spectrometry (1D/2D GC-TOFMS) followed by targeted analysis of 29 amino acids using liquid chromatography-time-of-flight mass spectrometry (LC-TOFMS). We also looked for differences upon arginine deprivation in a single ASS1 negative and positive cell line (SNB19 and U87 respectively). The acquired data was evaluated by chemometric based bioinformatic methods.ResultsOrthogonal partial least squares-discriminant analysis (OPLS-DA) of both the 1D and 2D GC-TOFMS data revealed significant systematic difference in metabolites between the two subgroups with ASS1 positive cells generally exhibiting an overall elevation of identified metabolites, including those involved in the arginine biosynthetic pathway. Pathway and network analysis of the metabolite profile show that ASS1 negative cells have altered arginine and citrulline metabolism as well as altered amino acid metabolism. As expected, we observed significant metabolite perturbations in ASS negative cells in response to ADI-PEG20 treatment.ConclusionsThis study has highlighted significant differences in the metabolome of ASS1 negative and positive GBM which warrants further study to determine their diagnostic and therapeutic potential for the treatment of this devastating disease.

P38γ and p38δ Are Involved in T Lymphocyte Development.

p38 mitogen-activated protein kinase (MAPK) signal transduction pathways are essential regulators of the immune response. Particularly, p38γ and p38δ regulate many immune cell functions such as cytokine production, migration, or T cell activation; however, their involvement in immune cell development is largely unknown. Here, we analysed the role of p38 MAPK isoforms p38γ and p38δ in T cell differentiation in the thymus and in lymph nodes, using mice deficient in p38γ, p38δ, or in both. We found that the T cell differentiation program in the thymus was affected at different stages in p38γ-, p38δ-, and p38γ/δ-deficient mice, and also peripheral T cell homaeostasis was compromised. Particularly, p38δ deletion affects different stages of early CD4−CD8− double-negative thymocyte development, whereas lack of p38γ favours thymocyte positive selection from CD4+CD8+ double-positive to CD4+ or CD8+ single-positive cells. Our results identify unreported functions for p38γ and p38δ in T cells.

Gli3 in fetal thymic epithelial cells promotes thymocyte positive selection and differentiation by repression of Shh.

ABSTRACTGli3 is a Hedgehog (Hh)-responsive transcription factor that can function as a transcriptional repressor or activator. We show that Gli3 activity in mouse thymic epithelial cells (TECs) promotes positive selection and differentiation from CD4+ CD8+ to CD4+ CD8− single-positive (SP4) cells in the fetal thymus and that Gli3 represses Shh. Constitutive deletion of Gli3, and conditional deletion of Gli3 from TECs, reduced differentiation to SP4, whereas conditional deletion of Gli3 from thymocytes did not. Conditional deletion of Shh from TECs increased differentiation to SP4, and expression of Shh was upregulated in the Gli3-deficient thymus. Use of a transgenic Hh reporter showed that the Hh pathway was active in thymocytes, and increased in the Gli3-deficient fetal thymus. Neutralisation of endogenous Hh proteins in the Gli3−/− thymus restored SP4 differentiation, indicating that Gli3 in TECs promotes SP4 differentiation by repression of Shh. Transcriptome analysis showed that Hh-mediated transcription was increased whereas TCR-mediated transcription was decreased in Gli3−/− thymocytes compared with wild type.

Megakaryocytic TGFβ1 Partitions Hematopoiesis into Amplifying Stem and Progenitor Cells and Maturing Effector Cells

Background: Chronic anemia is a significant problem affecting over 3 million Americans annually. Therapies are restricted to transfusion and Erythropoietin Stimulating Agents (ESA). There is a need for new approaches to treat chronic anemia. Immature erythroid progenitors are thought to be continuously produced and then permitted to survive and mature if there is sufficient erythropoietin (Epo) available. This model is elegant in that oxygen sensing within the kidney triggers Epo production so anemia can increase Epo and promote erythroid output. However, during homeostasis this model suggests that considerable energy is used to produce unneeded erythroid progenitors. We searched for independent control and compartmentalization of erythropoiesis that could couple early hematopoiesis to terminal erythroid commitment and maturation.Methods: We previously found the proportion of bone marrow megakaryocytes (MKs) staining for active, signaling-competent TGFβ transiently increases during bone marrow regeneration after chemotherapy. To assess the functional role of Mk-TGFβ, we crossed murine strains harboring a floxed allele of TGFβ1 (TGFβ1Flox/Flox) littermate with a Mk-specific Cre deleter to generate mice with Mk-specific deletion of TGFβ1 (TGFβ1ΔMk/ΔMk). We analyzed hematopoiesis of these mice using high-dimensional flow cytometry, confocal immunofluorescent microscopy and in vitro and in vivo assays of hematopoietic function (Colony forming assays, and in vivo transplantation).Results: Using validated, 9-color flow cytometry panels capable of quantifying hematopoietic stem cells (HSCs) and six other hematopoietic progenitor populations, we found that Mk-specific deletion of TGFβ1 leads to expansion of immature hematopoietic stem and progenitor cells (HSPCs) (Fig1A&B). Functional assays confirmed a more than three-fold increase in hematopoietic stem cells (HSCs) capable of serially-transplanting syngeneic recipients in the bone marrow (BM) of TGFβ1ΔMk/ΔMk mice compared to their TGFβ1Flox/Flox littermates. Expansion was associated with less quiescent (Go) HSCs implicating Mk-TGFβ in the control of HSC cell cycle entry. Similarly, in vitro colony forming cell assays and in vivo spleen colony forming assays confirmed expansion of functional progenitor cells in TGFβ1ΔMk/ΔMk mice. These results place Mk-TGFβ as a critical regulator of the size of the pool of immature HSPCs.We found that the blood counts and total BM cellularity of TGFβ1ΔMk/ΔMk mice was normal despite the dramatic expansion of immature HSPCs. Using a combination of confocal immunofluorescence microscopy (cleaved caspase 3) (Fig1C) and flow cytometry (Annexin V and cleaved caspase 3) (Fig1D), we found ~10-fold greater apoptosis of mature precursor cells in TGFβ1ΔMk/ΔMk BM and spleens. Coincident with this, we found the number of Epo receptor (EpoR) expressing erythroid precursors to be dramatically increased. Indeed, apoptosis of erythroid precursors peaked as they transitioned from dual positive Kit+EpoR+ precursors to single positive cells expressing EpoR alone. Epo levels were normal in the serum of these mice. We reasoned that the excess, unneeded EpoR+ cells were not supported physiologic Epo levels but might respond to even small doses of exogenous Epo. Indeed, we found that the excess erythroid apoptosis could be rescued by administration of very low doses of Epo (Fig1E). Whereas TGFβ1Flox/Flox mice showed minimal reticulocytosis and no change in blood counts, TGFβ1ΔMk/ΔMk mice responded with exuberant reticulocytosis and raised RBC counts almost 10% within 6 days (Fig. 1F). Low dose Epo also rescued survival of Epo receptor positive erythroid precursors in the bone marrow, spleen and blood of TGFβ1ΔMk/ΔMk mice. TGFβ1ΔMk/ΔMk mice showed a similarly brisk and robust erythropoietic response during recovery from phenylhydrazine-induced hemolysis (Fig.1G). Exogenous TGFβ worsened BM apoptosis and caused anemia in treated mice. Pre-treatment of wild-type mice with a TGFβ signaling inhibitor sensitized mice to low dose Epo.Conclusion: These results place megakaryocytic TGFβ1 as a gate-keeper that restricts the pool of immature HSPCs and couples immature hematopoiesis to the production of mature effector cells. This work promises new therapies for chronic anemias by combining TGFβ inhibitors to increase the outflow of immature progenitors with ESAs to support erythroid maturation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Significant expression of Foxp3 in murine extrathymic CD4+CD8+ double positive T cells.

Forkhead box protein 3 (Foxp3) is a specific marker and the key factor in the development and function of regulatory T (Treg) cells. The present study investigates Foxp3 expression in murine head and neck lymph node (HNLN) and peripheral blood (PB) CD4+CD8+ double positive (DP) T cells and compares it with that in CD4+ or CD8+ single positive (SP) T cells. Here we provide evidence that murine extrathymic DP cells express Foxp3. We determined that the mean percentage of Foxp3-expressing cells within HNLN and PB DP cells was, respectively, 22.4 and 16.2. The obtained results clearly indicate that DP cells are very similar to CD4+ SP cells in respect of Foxp3 expression, and thus considerably deviate from CD8+ SP cells. It was found that Foxp3 expression in DP cells is positively correleted with CD25 expression. These results suggest that murine extrathymic Foxp3+ DP T cells may represent a unique regulatory T cell subset.

The corepressor NCOR1 regulates the survival of single-positive thymocytes

Nuclear receptor corepressor 1 (NCOR1) is a transcriptional regulator bridging repressive chromatin modifying enzymes with transcription factors. NCOR1 regulates many biological processes, however its role in T cells is not known. Here we show that Cd4-Cre-mediated deletion of NCOR1 (NCOR1 cKOCd4) resulted in a reduction of peripheral T cell numbers due to a decrease in single-positive (SP) thymocytes. In contrast, double-positive (DP) thymocyte numbers were not affected in the absence of NCOR1. The reduction in SP cells was due to diminished survival of NCOR1-null postselection TCRβhiCD69+ and mature TCRβhiCD69− thymocytes. NCOR1-null thymocytes expressed elevated levels of the pro-apoptotic factor BIM and showed a higher fraction of cleaved caspase 3-positive cells upon TCR stimulation ex vivo. However, staphylococcal enterotoxin B (SEB)-mediated deletion of Vβ8+ CD4SP thymocytes was normal, suggesting that negative selection is not altered in the absence of NCOR1. Finally, transgenic expression of the pro-survival protein BCL2 restored the population of CD69+ thymocytes in NCOR1 cKOCd4 mice to a similar percentage as observed in WT mice. Together, these data identify NCOR1 as a crucial regulator of the survival of SP thymocytes and revealed that NCOR1 is essential for the proper generation of the peripheral T cell pool.

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.

Neonatal immune response to rhinovirus A16 has diminished dendritic cell function and increased B cell activation.

BackgroundRhinovirus infections during infancy account for the majority of respiratory illness health care utilization and are an associated risk factor for subsequent development of allergic asthma. Neonatal type I interferon production is diminished compared to adults after stimulation with TLR agonists. However, broad profiling of immune cell responses to infectious rhinovirus has not been undertaken and we hypothesized that additional immune differences can be identified in neonates. In this study, we undertook a comparative analysis of neonatal and adult blood immune cell responses after in vitro incubation with infectious RV-A16 for 6 and 24 hours.MethodsIntracellular proinflammatory and type I interferon cytokines along with expression of surface co-stimulatory and maturation markers were measured using multi-parameter flow cytometry.ResultsBoth circulating myeloid dendritic cell (mDC) and plasmacytoid dendritic cell (pDC) frequency were lower in cord blood. Qualitative and quantitative plasmacytoid dendritic cell IFN-alpha + TNF- alpha responses to rhinovirus were significantly lower in cord pDCs. In cord blood samples, the majority of responsive pDCs were single-positive TNF-alpha producing cells, whereas in adult samples rhinovirus increased double-positive TNF-alpha+IFN-alpha+ pDCs. Rhinovirus upregulated activation and maturation markers on monocytes, mDCs, pDCs, and B cells, but CD40+CD86+ monocytes, mDCs, and pDCs cells were significantly higher in adult samples compared to cord samples. Surprisingly, rhinovirus increased CD40+CD86+ B cells to a significantly greater extent in cord samples compared to adults.ConclusionsThese findings define a number of cell-specific differences in neonatal responses to rhinovirus. This differential age-related immune response to RV may have implications for the immune correlates of protection to viral respiratory illness burden and determination of potential biomarkers for asthma risk.

Frontline Science: A hyporesponsive subset of rat NK cells negative for Ly49s3 and NKR-P1B are precursors to the functionally mature NKR-P1B+ subset.

Rat NK cells are divided into major subsets expressing either Ly49 receptors or the inhibitory NKR-P1B receptor in conjunction with NKG2A/C/E receptors. A minor subset of NKp46+ cells lacking expression of both Ly49 receptors and NKR-P1B is present in blood and spleen and is associated with decreased functional competence. We hypothesized that this subset may represent precursors to Ly49+ and/or NKR-P1B+ NK cells. When cultured in vitro in IL-2 and IL-15 or adoptively transferred to syngeneic hosts, a portion of NKR-P1B-Ly49s3- cells transformed to express NKR-P1B, but very little Ly49s3. Acquisition of NKR-P1B by NKR-P1B-Ly49s3- cells coincided with increased degranulation. In addition, although NKR-P1B-Ly49s3- cells highly proliferate, proliferative activity was reduced upon acquisition of NKR-P1B at comparable levels to bona fide NKR-P1B+ NK cells. A fraction of NKR-P1B-Ly49s3- cells remained negative for NKR-P1B, both in vitro and after adoptive transfer in vivo. Most NKR-P1B-Ly49s3- cells expressed the transcription factor Eomesodermin and NK cell markers, indicating that these cells represent conventional NK cells. Our findings suggest that the NKR-P1B-Ly49s3- NK cells are precursors to NKR-P1B single-positive cells and that functional competence is acquired upon expression of NKR-P1B.

Abstract 651: Evaluation of the expression and function of TIM-3 relative to PD-1 in human tumors

Abstract While immunotherapies directed against PD-1 and PD-L1 have proven effective across multiple indications, there is still a large unmet medical need for therapies for patients who do not respond or who develop acquired resistance during the course of treatment. There are several emerging hypotheses to explain the lack of response, including the overall presence and localization of immune cells, as well as the up-regulation of additional T-cell checkpoints, including TIM-3 in tumor infiltrating lymphocytes. To investigate the potential role of TIM-3, we set out to evaluate the expression and function of TIM-3 and the relationship to PD-1 in several systems. Firstly, we developed a flow cytometry methodology to enumerate T-cell, including CD4 and CD8 positive cells, as well as other immune cell populations in a panel of human tumor samples, including non-small cell lung cancer (NSCLC). In these samples, we investigated the expression profiles of TIM-3 and PD-1 in both T-cell and non-T-cell populations. In addition, we performed complementary genomic studies to explore gene expression profiles of not only the bulk tumor samples but also of isolated PD1 positive/TIM-3 negative versus PD1 and TIM-3 double positive cell populations. The profiling identified tumors with a range of tumor infiltrating lymphocyte content and also differences in PD1 and TIM-3 expression. It was found that in addition to T-cells, TIM-3 was expressed on myeloid cell populations and furthermore, that there were distinct differences in the gene expression profiles of PD1 single positive versus PD1 and TIM-3 double positive cells. Building on the expression studies, the functional role of Tim-3 in T-cells and also on myeloid-derived cells was explored. Taken together, these studies provide further evidence for TIM-3 as an important immunological checkpoint and as a relevant therapeutic target for the treatment of cancer with the potential for biological effects in both the T-cell and myeloid compartments of the tumor microenvironment. Citation Format: Kristen McEachern, Srimoyee Ghosh, Yonghong Zhao, Qiyao Zhang, Norman Zhang, David W. Jenkins. Evaluation of the expression and function of TIM-3 relative to PD-1 in human tumors [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 651. doi:10.1158/1538-7445.AM2017-651

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.

CD117+CD44+ Stem T Cells Develop in the Thymus and Potently Suppress T-cell Proliferation by Modulating the CTLA-4 Pathway

BackgroundCD117 is expressed on double-negative (DN; CD4–CD8–) cells (Nat Rev Immunol 14:529–545; 2014), but whether it is expressed in other stages and its subsequent functions are unclear. We used an improved method of flow cytometry to analyze different populations of thymocytes (Sci Rep 4:5781; 2014). The expression of CD117 and CTLA-4 were directly assayed in the early stage of thymocytes.MethodsFlow cytometry was used to analyze different populations of thymocytes, and T-cell proliferation assays, RT-PCR, and real-time RT-PCR were used to characterize the stem cells and examine the function of CD44+CD117+ cells.ResultsIn DN cells, CD117 expression was greatest on CD44+CD25+ cells (DN2), followed by CD44+CD25– (DN1), CD44–CD25+ (DN3), and CD44–CD25– (DN4) cells. In thymocytes, CD117 expression was highest in DN cells, followed by single-positive (SP; CD4 or CD8) and double-positive (DP; CD4+CD8+) cells. Especially, CD117 expression was positively associated with CD44 and CTLA-4 expression. CTLA-4 expression was highest in DN cells, followed by SP and DP cells. CTLA-4 expression was positively associated with CD25, CD44, and Foxp3 expression. CD44+CD117+ T cells expressed more CTLA-4, which suppressed T-cell proliferation and blocked CTLA-4 to cause antibody-induced T-cell proliferation.ConclusionThese results suggest that CD44+CD117+ T cells are stem cells and a specific T-cell phenotype that initially develops in the thymus, but they do not progress through DN3 and DN4 stages, lack a DP stage, and potently suppress T-cell proliferation and modulate the CTLA-4 pathway.

Pim-3 Regulates Stemness of Pancreatic Cancer Cells via Activating STAT3 Signaling Pathway.

Due to its aggressiveness and unusual resistance to conventional therapies, pancreatic cancer is a highly lethal gastrointestinal malignancy with poor prognosis. According to the cancer stem cell hypothesis, there exists a fraction of cancer cells, that is, cancer stem cells, responsible for tumor maintenance and therapeutic failure. Herein we investigated the involvement of proto-oncogene Pim-3 in driving the stemness properties in pancreatic cancer. Expression levels of several stemness-associated markers were examined in several pancreatic cancer cell lines. The double positive (CD24+ESA+) and double negative (CD24-ESA-) pancreatic cancer cells were isolated from PANC-1 and L3.6pl, and their self-renewal ability, tumorigenicity as well as sensitivity to gemcitabine were then evaluated. Results showed that there existed heterogeneity in expression levels of stemness-associated surface markers among pancreatic cancer cell lines. CD24+ESA+ pancreatic cancer cells exhibited increased tumorigenicity and decreased chemosensitivity to gemcitabine as compared to CD24-ESA- cells. Besides, the double positive (CD24+ESA+) subpopulation also exhibited greater expression level of Pim-3 when compared with the double negative (CD24-ESA-) ones. Furthermore, silencing of Pim-3 in pancreatic cancer cells leads to decreased proportions of both single positive (CD24+ and ESA+) and double positive (CD24+ESA+) pancreatic cancer cells. Overexpression of Pim-3 was associated with increased levels of some stemness-associated transcription factors (STAT3, etc.). Moreover, the phosphorylation level and transcriptional activity of STAT3 were decreased in Pim-3 silenced pancreatic cancer cells and restoration of its activity results in restitution of stem cell-like phenotypes. Therefore, Pim-3 maintains stemness of pancreatic cancer cells via activating STAT3 signaling pathway and might be used as a novel therapeutic target in pancreatic cancer.

Gfi1-Foxo1 axis controls the fidelity of effector gene expression and developmental maturation of thymocytes

Double-positive (DP) thymocytes respond to intrathymic T-cell receptor (TCR) signals by undergoing positive selection and lineage differentiation into single-positive (SP) mature cells. Concomitant with these well-characterized events is the acquisition of a mature T-cell gene expression program characterized by the induction of the effector molecules IL-7Rα, S1P1, and CCR7, but the underlying mechanism remains elusive. We report here that transcription repressor Growth factor independent 1 (Gfi1) orchestrates the fidelity of the DP gene expression program and developmental maturation into SP cells. Loss of Gfi1 resulted in premature induction of effector genes and the transcription factors forkhead box protein O1 (Foxo1) and Klf2 in DP thymocytes and the accumulation of postselection intermediate populations and accelerated transition into SP cells. Strikingly, partial loss of Foxo1 function, but not restored survival fitness, rectified the dysregulated gene expression and thymocyte maturation in Gfi1-deficient mice. Our results establish the Gfi1-Foxo1 axis and the transcriptional circuitry that actively maintain DP identity and shape the proper generation of mature T cells.