Cell-associated Molecule Research Articles

The constant domain of CRTAM is essential for high-affinity interaction with Nectin-like 2

CRTAM (Class-I MHC restricted T cell-associated molecule) is a member of the Nectin-like family, composed of two extracellular domains, one constant domain (IgC) and another variable domain (IgV), expressed in activated CD8 T cells, epithelial cells, natural killer (NK) cells, and in a subpopulation of CD4 T cells. CRTAM recognizes the ligand Nectin-like 2 (Necl2) through the IgV domain. However, the role of the IgC domain during this ligand recognition has yet to be understood. In this study, we show the purification of soluble-folded Ig domains of CRTAM, and we demonstrate that the IgC domain forms a homodimer in solution via hydrophobic interactions. By surface plasmon resonance (SPR) analysis, we also demonstrate that CRTAM binds to Necl2 with an affinity of 2.16 nM. In conclusion, CRTAM's IgC is essential for a high-affinity interaction with Necl-2.

Rab39 and its effector UACA regulate basolateral exosome release from polarized epithelial cells.

Exosomes are small extracellular vesicles that originate from the intraluminal vesicles of multivesicular bodies (MVBs). We previously reported that polarized Madin-Darby canine kidney (MDCK) epithelial cells secrete two types of exosomes, apical and basolateral exosomes, from different MVBs. However, how these MVBs are selectively targeted to the apical or basolateral membrane remained unknown. Here, we analyze members of the Rab family small GTPases and show that different sets of Rabs mediate asymmetrical exosome release. Rab27, the best-known regulator of MVB transport for exosome release, is specifically but partially involved in apical exosome release, and Rab37, a close homolog of Rab27, is an additional apical exosome regulator. By contrast, Rab39 functions as a specific regulator of basolateral exosome release. Mechanistically, Rab39 interacts with its effector UACA, and UACA then recruits Lyspersin, a component of BLOC-1-related complex (BORC). Our findings suggest that the Rab39-UACA-BORC complex specifically mediates basolateral exosome release.

Modulation of CD8+ T cell antitumor function by an immune inhibitory molecule sushi domain containing 2

Abstract A central mechanism by which the immunosuppressive tumor microenvironment (TME) facilitates tumor growth is the inhibition of CD8+ effector T cell function. Indeed, restoration of CD8+ T cell antitumor functions by immune checkpoint blockade (ICB) treatment represents a significant breakthrough in cancer therapy. However, a major challenge remaining in ICB immunotherapy is the existence of multiple immune checkpoint molecules. As a result, targeting of a single molecule cannot override compensatory signals from other immune inhibitory molecules. Therefore, there is an urgent need to identify new therapeutic target(s) to rejuvenate CD8+ T cell antitumor immunity for cancer therapy. Through unbiased gene profiling assay, our recent study has identified SUSD2 (sushi domain containing 2) as a highly expressed immune cell-associated cell surface molecule in an experimental cancer model. Using a newly developed Susd2 gene-deletion (Susd2−/−) mouse model, we found that genetic deletion of Susd2 led to an improved control of tumor growth in syngeneic tumor models including colorectal carcinoma MC38 and thymoma EG7. This improved tumor control phenotype of Susd2−/− mice required the presence of CD8+ T cells. Single-cell RNA sequencing assay revealed increased production of effector molecules such as granzyme B (Gzmb) and interferon (IFN)-γ in tumor-infiltrating CD8+ T cells from Susd2−/− mice compared to those from wild-type (WT) mice. Mechanistically, we found that SUSD2 negatively regulated IL-2 receptor (IL-2R)-mediated generation of Gzmb and IFN-g in CD8+ T cells. In sum, our results identify SUSD2 as a new immune inhibitory molecule which can be targeted to augment CD8+ T cell antitumor response.

Identification of TENM4 as a Novel Cancer Stem Cell-Associated Molecule and Potential Target in Triple Negative Breast Cancer.

Simple SummaryPatients with triple negative breast cancer (TNBC) experience shorter overall survival compared to non-TNBC patients because of the high incidence of recurrences and metastases. This is due to the capacity of aggressive cancer cell subpopulations named cancer stem cells (CSC) to resist current therapies. To design more effective therapeutic strategies for TNBC patients, in this study we sought to identify functional targets expressed on CSC. Our analyses led us to propose teneurin 4 (TENM4) as a promising candidate for drug- and immune-based therapies due to its role in CSC self-renewal and migratory capacity and the inverse correlation between its expression and survival of TNBC patients. In addition, TENM4 detection in the plasma of tumor-bearing patients endorses its potentiality as a disease detection marker.Triple-negative breast cancer (TNBC) is insensitive to endocrine and Her2-directed therapies, making the development of TNBC-targeted therapies an unmet medical need. Since patients with TNBC frequently show a quicker relapse and metastatic progression compared to other breast cancer subtypes, we hypothesized that cancer stem cells (CSC) could have a role in TNBC. To identify putative TNBC CSC-associated targets, we compared the gene expression profiles of CSC-enriched tumorspheres and their parental cells grown as monolayer. Among the up-regulated genes coding for cell membrane-associated proteins, we selected Teneurin 4 (TENM4), involved in cell differentiation and deregulated in tumors of different histotypes, as the object for this study. Meta-analysis of breast cancer datasets shows that TENM4 mRNA is up-regulated in invasive carcinoma specimens compared to normal breast and that high expression of TENM4 correlates with a shorter relapse-free survival in TNBC patients. TENM4 silencing in mammary cancer cells significantly impaired tumorsphere-forming ability, migratory capacity and Focal Adhesion Kinase (FAK) phosphorylation. Moreover, we found higher levels of TENM4 in plasma from tumor-bearing mice and TNBC patients compared to the healthy controls. Overall, our results indicate that TENM4 may act as a novel biomarker and target for the treatment of TNBC.

Short Disordered Epitope of CRTAM Ig-Like V Domain as a Potential Target for Blocking Antibodies.

Class-I Restricted T Cell-Associated Molecule (CRTAM) is a protein that is expressed after T cell activation. The interaction of CRTAM with its ligand, nectin-like 2 (Necl2), is required for the efficient production of IL-17, IL-22, and IFNγ by murine CD4 T cells, and it plays a role in optimal CD8 T and NK cell cytotoxicity. CRTAM promotes the pro-inflammatory cytokine profile; therefore, it may take part in the immunopathology of autoimmune diseases such as diabetes type 1 or colitis. Thus, antibodies that block the interaction between CRTAM and Necl2 would be useful for controlling the production of these inflammatory cytokines. In this work, using bioinformatics predictions, we identified three short disordered epitopes (sDE1-3) that are located in the Ig-like domains of murine CRTAM and are conserved in mammalian species. We performed a structural analysis by molecular dynamics simulations of sDE1 (QHPALKSSKY, Ig-like V), sDE2 (QRNGEKSVVK, Ig-like C1), and sDE3 (CSTERSKKPPPQI, Ig-like C1). sDE1, which is located within a loop of the contact interface of the heterotypic interaction with Nectl2, undergoes an order–disorder transition. On the contrary, even though sDE2 and sDE3 are flexible and also located within loops, they do not undergo order–disorder transitions. We evaluated the immunogenicity of sDE1 and sDE3 through the expression of these epitopes in chimeric L1 virus-like particles. We confirmed that sDE1 induces polyclonal antibodies that recognize the native folding of CRTAM expressed in activated murine CD4 T cells. In contrast, sDE3 induces polyclonal antibodies that recognize the recombinant protein hCRTAM-Fc, but not the native CRTAM. Thus, in this study, an exposed disordered epitope in the Ig-like V domain of CRTAM was identified as a potential site for therapeutic antibodies.

Results of a Phase II Study of PD-1 Inhibition in Advanced Myeloproliferative Neoplasms

Background: Myelofibrosis (MF) is a hematopoietic stem cell neoplasm characterized by abnormal JAK-STAT signaling, increased inflammation, and evolution to acute myeloid leukemia. Most patients harbor a phenotypic driver mutation- JAK2, CALR or MPL. In the last decade, two JAK2 inhibitors have been approved, however, outside of hematopoietic stem cell transplantation (HSCT), there are no medications that meaningfully modify MF disease biology. Thus, additional treatments are needed. We previously demonstrated the presence of multiple immunosuppressive mechanisms in MPN patients, including expanded myeloid derived suppressor cell (MDSC) populations and elevated expression of immune checkpoint receptors, particularly PD1, on T cells from MPN patients compared to healthy donors (Cimen Bozkus, Cancer Discovery 2019). Therefore, we conducted a multi-center, open-label, phase 2, single-arm study of pembrolizumab in patients with primary, post-essential thrombocythemia or post-polycythemia vera myelofibrosis (MF) (NCT03065400). Methods: Patients with intermediate-2/high DIPSS MF, ineligible for or previously failed ruxolitinib were eligible. Pembrolizumab was administered at the FDA-approved dose of 200 mg every 3 weeks for 6 cycles. The study was a Simon two-stage design, 9 patients enrolled in the first arm. The study was terminated after completion of this arm due to the lack of responses. Response assessment was conducted after 6 cycles utilizing the International Working Group (IWG-MRT) criteria. In addition, patients with accelerated or blast phase (MPN-AP/BP) could enroll as a separate, ten patient, exploratory cohort. Results: 9 patients enrolled in the MF cohort and 1 in the BP cohort between 6/2017 and 3/2020. Baseline characteristics are summarized in Table 1. Median weeks on treatment were 14.7 (range 4-20). All 9 MF patients were evaluable for response as they received at least 1 dose of pembrolizumab. Of the 9 patients who were evaluable for response, all had SD, 4 (44%) patients discontinued therapy prior to the end of cycle 6. Reasons for discontinuation were adverse events (n=1), patient decision (n=1), physician decision (n=2). Grade 3/4 AEs included anemia (n=3), thrombocytopenia (n=2), leukopenia (n=1), hyperglycemia (n=2), hyperuricemia (n=1), dyspnea (n=1), headache (n=1). No grade 3/4 immune related AEs occurred (Table 2). The effects of pembrolizumab on the immune suppressive milieu observed in MPN were analyzed. PD-1 was detected on peripheral blood T cells by flow cytometry at baseline, but not post-treatment, likely due to receptor occupancy by pembrolizumab. In all patients analyzed, the levels of soluble PD1 in the plasma by Olink assay were significantly higher post-treatment. Other soluble factors associated with T cell activation such as class I-restricted T cell-associated molecule (CRTAM) and CD27 were also elevated after pembrolizumab administration. Furthermore, ARG1, a molecule that mediates T-cell suppression by MDSC, was significantly reduced in the plasma of treated patients. An increase in peripheral blood T cell frequencies was observed in a subset of patients after two cycles. Discussion: Pembrolizumab did not demonstrate clinical activity in this phase 2 trial. The relevance of the preliminary correlative findings will be further confirmed by in situ gene profiling of immune cells and their microenvironment. The complete results will be available at the meeting. These results suggest that pembrolizumab may promote a phenotypical and soluble signature suggestive of a restored immune response. The fact that these molecular changes were not associated with clinical responses indicate that pembrolizumab alone may not be sufficient to reverse the multifactorial causes of immune tolerance in MPN. Disclosures Hobbs: Constellation: Honoraria, Research Funding; Bayer: Research Funding; Jazz: Honoraria; Incyte: Research Funding; Novartis: Honoraria; Merck: Research Funding; Celgene/BMS: Honoraria. Stone:Syndax: Consultancy, Research Funding; Daiichi-Sankyo: Consultancy; Astellas: Consultancy; Takeda: Other: DSMB; Syntrix: Other: DSMB; Arog: Consultancy, Research Funding; Jazz: Consultancy; Trovagene: Consultancy; Syros: Consultancy; Abbvie: Consultancy, Research Funding; Biolinerx: Consultancy; Argenix: Other; Novartis: Consultancy, Research Funding; Celgene: Consultancy, Other; Agios: Consultancy, Research Funding; Gemoab: Consultancy; Janssen: Consultancy; Stemline: Consultancy; Pfizer: Consultancy; Aztra-Zeneca: Consultancy; Macrogenics: Consultancy; Actinium: Consultancy, Membership on an entity's Board of Directors or advisory committees. Mascarenhas:Incyte, Kartos, Roche, Promedior, Merck, Merus, Arog, CTI Biopharma, Janssen, and PharmaEssentia: Other: Research funding (institution); Celgene, Prelude, Galecto, Promedior, Geron, Constellation, and Incyte: Consultancy.

Abstract 3416: Androgen deprivation following JAK1/2 and PD-L1 inhibition improves antitumor efficacy in mouse models of Pten-deficient prostate cancer

Abstract Recently, the tumor suppressor PTEN has been shown to function as an immune modulator and its inactivation is associated with mediating tumor immune evasion. We previously examined the effects of programmed death-ligand 1 (PD-L1) immune checkpoint blockade (ICB) in transgenic mouse models of Pten-null prostate cancer. However, anti-PD-L1 monotherapy was largely ineffective in immunologically cold castration-naïve tumors from intact mice but showed some activity when administered to castrated mice. Our previous immune profiling studies also revealed that while androgen withdrawal increased tumor inflammation, it also mediated the recruitment and accumulation of immunosuppressive cells such as regulatory T cells, M2-polarized macrophages and myeloid-derived suppressor cells (MDSCs). Interleukin-6 (IL-6)/signal transducer and activator of transcription-3 (STAT3) signaling is a key immune modulating pathway that is upregulated in both intact and androgen deprived tumors. Here, we evaluate the effectiveness of targeting PD-L1 and JAK/STAT signaling in preclinical models of Pten-null prostate cancer. Intact tumor bearing conditional Pten-knockout were treated for four weeks with an a-PD-L1 blocking antibody (clone D265A, mouse/IgG1 kappa) and/or the JAK1/2 inhibitor, AZD1480. Pharmacological treatments targeting PD-L1 and JAK1/2 (PJ) combinations were also evaluated as concurrent therapy with androgen deprivation (AD) by orchidectomy (PJ+AD), and as sequential therapy; two weeks AD followed by PJ (AD>PJ), and one week of PT followed by AD (PJ>AD). Combined PD-L1/JAK followed by AD was the only treatment combination that improved antitumor immune responses over monotherapy. Notably, flow cytometry studies showed that combined PD-L1/JAK potently abrogated PD-L1 expression in circulating dendritic cells in all settings. Evidence for enhanced antitumor immune response in PJ>AD was supported by increased numbers circulating effector memory CD8 T cells and CD355+CD8+ T cells (CD355 Class I MHC-restricted T cell-associated molecule (CRTAM) a marker for activated CD8 T cell), and increased CD8 T cell infiltration in tumors and a reduction of CD25+CD4+ regulatory T cells. The efficacy of the PJ>AD treatment combination was further tested and confirmed in a subcutaneous syngeneic allograft model using mice grafted with tumor blocks from Pten-deficient castration-resistant prostate cancer tumors. Together these results indicate that pretreatment with combined PD-L1/JAK blockade can decrease the immunosuppressive effects of androgen withdrawal and have the potential to restore antitumor immune activity in Pten-deficient prostate cancer. Citation Format: Marco A. De Velasco, Yurie Kura, Naomi Ando, Noriko Sako, Kazuko Sakai, Kazuhiro Yoshikawa, Kazuto Nishio, Hirotsugu Uemura. Androgen deprivation following JAK1/2 and PD-L1 inhibition improves antitumor efficacy in mouse models of Pten-deficient prostate cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3416.

Prokaryotic Expression of the Immunoglobulin's Domains of CRTAM to Characterize a Monoclonal Antibody.

Class-I restricted T cell-associated molecule (CRTAM) is a member of the immunoglobulin superfamily, and it is closely related to nectin-like protein. CRTAM is expressed in activated CD8 T cells, NKT cells, NK cells and in a subpopulation CD4 T cells. In this study, we produce as recombinant proteins, the Ig-domains of CRTAM (IgV-IgC), the IgV, and the IgC. These proteins were successfully purified in the soluble fraction only if the stalk region was included. The recombinant CRTAM recognizes its ligand nectin-like 2 in a cell-free system. We also demonstrate that the IgC domain of CRTAM is recognized by the anti-hCRTAM monoclonal antibody C8 with a 0.62nM affinity. In conclusion, the stalk region of CRTAM provides solubility for the expression of its Ig-domains as recombinant proteins.

CRTAM Protects Against Intestinal Dysbiosis During Pathogenic Parasitic Infection by Enabling Th17 Maturation

The gastrointestinal tract hosts the largest collection of commensal microbes in the body. Infections at this site can cause significant perturbations in the microbiota, known as dysbiosis, that facilitate the expansion of pathobionts, and can elicit inappropriate immune responses that impair the intestinal barrier function. Dysbiosis typically occurs during intestinal infection with Toxoplasma gondii. Host resistance to T. gondii depends on a potent Th1 response. In addition, a Th17 response is also elicited. How Th17 cells contribute to the host response to T. gondii remains unclear. Here we show that class I-restricted T cell-associated molecule (CRTAM) expression on T cells is required for an optimal IL-17 production during T. gondii infection. Moreover, that the lack of IL-17, results in increased immunopathology caused by an impaired antimicrobial peptide production and bacterial translocation from the intestinal lumen to the mesenteric lymph nodes and spleen.

CRTAM shapes the microbiota and enhances the severity of gut infection.

Abstract Gut lymphocytes and the microbiota establish a reciprocal relationship that tunes the host immune response. For example, the gut microbiota is required for T helper (Th)-17 cell differentiation; Th17 cytokines, in turn, stimulate the intestinal epithelium to produce antimicrobial proteins that control the gut microbiota. Residency and maintenance of T cells in the gut is controlled by Class I-restricted T cell-associated molecule (CRTAM). We thus evaluated whether CRTAM influences the gut microbiota composition. We found that CRTAM expression caused a shift in several members of the gut microbiota under homeostatic conditions. As the gut microbiota and T cells contribute to host defense against intestinal pathogens, we tested whether CRTAM plays a role during infection with the enteric pathogen Salmonella. We found lower levels of Salmonella colonization in the gut of Crtam−/− mice, along with lower expression of the Th17 cytokines Il17 and Il22 in the cecum. Salmonella-infected Crtam−/− mice exhibited fewer gut T cells and IL-17-producing cells. To investigate whether the altered gut microbiota composition of Crtam−/− mice influenced the host response to infection, we transplanted the fecal microbiota from WT or Crtam−/− littermate mice into germ-free mice, and then infected the mice with Salmonella. Relative to mice transplanted with “WT” fecal microbiota, mice transplanted with “Crtam−/−” microbiota exhibited lower numbers of T cells and IL-17-producing cells in the gut upon infection. Our results thus suggest an interplay between CRTAM expression and the gut microbiota, which ultimately impacts the mucosal immune system and the host response to enteric pathogens.

CD4 CTL, a Cytotoxic Subset of CD4+ T Cells, Their Differentiation and Function.

CD4+ T cells with cytotoxic activity (CD4 CTL) have been observed in various immune responses. These cells are characterized by their ability to secrete granzyme B and perforin and to kill the target cells in an MHC class II-restricted fashion. Although CD4 CTLs were once thought to be an in vitro artifact associated with long-term culturing, they have since been identified in vivo and shown to play important roles in antiviral and antitumor immunity, as well as in inflammation. Functional characterization of CD4 CTL suggests their potential significance for therapeutic purposes. However, in order to develop effective CD4 CTL therapy it is necessary to understand the differentiation and generation of these cells. Although the mechanisms regulating development of various CD4+ Th subsets have been clarified in terms of the cytokine and transcription factor requirement, the CD4 CTL differentiation mechanism remains elusive. These cells are thought to be most closely related to Th1 cells secreting IFNγ and regulated by eomesodermin and/or T-bet transcription factors for their differentiation. However, our studies and those of others have identified CD4 CTLs within other CD4+ T cell subsets, including naïve T cells. We have identified class I-restricted T cell-associated molecule as a marker of CD4 CTL and, by using this marker, we detected a subset of naïve T cells that have the potential to differentiate into CD4 CTL. CD4 CTL develops at sites of infections as well as inflammation. In this review, we summarize recent findings about the generation of CD4 CTL and propose a model with several differentiation pathways.

Targeting melanoma with front-line therapy does not abrogate Nodal-expressing tumor cells

Metastatic melanoma is a highly aggressive skin cancer with a poor prognosis. It is the leading cause of skin cancer deaths with a median overall survival for advanced-stage metastatic disease of <6 months. Despite advances in the field with conventional and targeted therapies, the heterogeneity of melanoma poses the greatest ongoing challenge, ultimately leading to relapse and progression to a more drug-resistant tumor in most patients. Particularly noteworthy are recent findings, indicating that these therapies exert selective pressure on tumors resulting in the activation of pathways associated with cancer stem cells that are unresponsive to current therapy. Our previous studies have shown how Nodal, an embryonic morphogen of the transforming growth factor-beta superfamily, is one of these critical factors that is reactivated in aggressive melanoma and resistant to conventional chemotherapy, such as dacarbazine. In the current study, we sought to determine whether BRAF inhibitor (BRAFi) therapy targeted Nodal-expressing tumor cells in uniquely matched unresectable stage III and IV melanoma patient samples before and after therapy that preceded their eventual death due to disease. The results demonstrate that BRAFi treatment failed to affect Nodal levels in melanoma tissues. Accompanying experiments in soft agar and in nude mice showed the advantage of using combinatorial treatment with BRAFi plus anti-Nodal monoclonal antibody to suppress tumor growth and metastasis. These data provide a promising new approach using front-line therapy combined with targeting a cancer stem cell-associated molecule—producing a more efficacious response than monotherapy.

Class I-Restricted T Cell-Associated Molecule Is a Marker for IFN-γ-Producing iNKT Cells in Healthy Subjects and Patients with Type 1 Diabetes.

Class I-restricted T cell-associated molecule (CRTAM) is an activation marker expressed on the cell surface of activated invariant natural killer T (iNKT) cells, CD8+ T cells, and a small subset of CD4+ T cells. CRTAM has also been associated with a proinflammatory profile in murine CD4+ T cells. However, CRTAM has not been thoroughly explored in human cells. This work focused on evaluating CRTAM expression in human iNKT lymphocytes after activation with α-galactosylceramide, its widely used specific glycolipid antigen. We also analyzed the involvement of costimulatory molecules in CRTAM expression and whether CRTAM expression is associated with a specific effector cytokine profile. We found that the signal produced by invariant T cell receptor (iTCR) engagement with α-galactosylceramide is sufficient to trigger CRTAM expression on human iNKT cells after 18 h of stimulation. Moreover, we observed a clear association between CRTAM expression and IFN-γ production in iNKT cells from healthy subjects and patients with type 1 diabetes. However, blocking the engagement of costimulatory molecules, such as CD40, CD80, and CD86, did not modify CRTAM expression. These results indicate that CRTAM may also play a role in triggering the production of IFN-γ in human iNKT cells and that CRTAM could be used as a marker to identify these inflammatory cells.

Proteasome degradative activity regulates CD8+ T lymphocyte metabolism and fate specification

Abstract During an immune response to microbial infection, CD8+ T lymphocytes can undergo asymmetric division, giving rise to daughter cells that exhibit distinct tendencies to adopt the terminal effector and memory fates. Here we show that ‘pre-effector’ and ‘pre-memory’ cells arising from the first CD8+ T cell division in vivo exhibit low and high rates of proteasome degradative activity, respectively. Pharmacologic inhibition of proteasome activity in activated CD8+ T cells resulted in increased inflammatory cytokine production and effector cell-associated molecule expression in vitro, as well as a loss of survival capacity and an impaired recall response to secondary infection in vivo. By contrast, increasing proteasome activity using a novel small molecule proteasome activator resulted in augmented memory cell-associated characteristics in vitro and an increased proportion of central memory cells with enhanced recall capabilities in vivo. Transcriptomic and proteomic analyses revealed that modulating proteasome activity in activated CD8+ T cells affected processes associated with cellular metabolism. These proteasome-induced metabolic consequences were mediated, in part, through asymmetric segregation of Myc, a transcription factor that has been shown to control glycolysis and proliferation, during cell division. Taken together, these results suggest proteasome degradative activity as a key regulator of CD8+ T lymphocyte fate specification by virtue of effects on cellular metabolism.

Gata1 overexpression in neurons increases the expression of cell-mediated cytotoxicity-related genes

ABSTRACTCell-mediated immune activation and inflammation have been suggested as key pathophysiological mediators of illness caused by stress and major depressive disorder (MDD). Previous studies have shown that MDD is an inflammatory disorder because plasma levels of pro-inflammatory cytokines are increased in the depressed patients. We have conducted studies to characterize differentially expressed genes in postmortem prefrontal cortex (PFC) of MDD. In the current study, using Gene Ontology analysis, we have identified that ‘natural killer cell-mediated cytotoxicity' pathway genes are overrepresented in the set of up-regulated genes in the PFC of MDD. To determine if altered gene expression is relevant to Gata1, we tested whether several natural killer (NK) cell cytotoxicity genes are up-regulated in cultured neurons by Gata1 overexpression. We found that the mRNA expression of class-I restricted T cell associated molecule (Crtam), nuclear factor, interleukin 3 regulated (Nfil3), and granzyme B (Gzmb) were significantly increased by the Gata1 overexpression in the cultured cortical neurons. Crtam is an immune receptor that is primarily expressed on activated cytotoxic lymphocytes, including NK cells and CD8 + T cells. Nfil3 is a transcription factor that affects the development of NK cells, and Gzmb is a serine protease that is produced by cytotoxic T cells and NK cells. These results suggest that cell-mediated immune activation is involved in the pathological mechanisms of MDD, and that Gata1 may be associated with immune activation in the central nervous system.

Reduced iNKT cells numbers in type 1 diabetes patients and their first-degree relatives.

Type 1 diabetes (T1D) is an autoimmune disease that is characterized by the specific destruction of insulin-producing pancreatic β cells. Invariant natural killer T (iNKT) cells have been associated with development of T1D. Class I MHC-restricted T cell-associated molecule (CRTAM) is expressed on activated iNKT, CD8(+), and CD4(+) T cells, and it is associated with the pro-inflammatory profiles of these cells. Crtam gene expression in CD3(+) lymphocytes from non-obese diabetic (NOD) mice is associated with T1D onset. However, expression of CRTAM on T cells from patients with T1D has not yet been evaluated. We compared iNKT cell (CD3(+)Vα24(+)Vβ11(+)) numbers and CRTAM expression in a Mexican population with recent-onset T1D and their first-degree relatives with control families. Remarkably, we found lower iNKT cell numbers in T1D families, and we identified two iNKT cell populations in some of the families. One iNKT cell population expressed high iTCR levels (iNKT(hi)), whereas another expressed low levels (iNKT(lo)) and also expressed CRTAM. These findings support a probable genetic determinant of iNKT cell numbers and a possible role for these cells in T1D development. This study also suggests that CRTAM identifies recently activated iNKT lymphocytes.

CRTAM is negatively regulated by ZEB1 in T cells

T cell activation leads to the induction of genes that are required for appropriate immune responses. This includes CRTAM (Class-I MHC-restricted T cell associated molecule), a protein that plays a key role in T cell development, proliferation, and generating cell polarity during activation. We previously characterized the CRTAM promoter and described how AP-1 family members are important for inducing CRTAM expression upon antigenic activation. Here, we show that CRTAM is a molecular target for ZEB1 (zinc finger E-box-binding protein), a homeodomain/Zn finger transcription factor. Overexpression of ZEB1 repressed CRTAM promoter activity, as well as endogenous CRTAM levels in human T cells. ZEB1-mediated transcriptional repression was abolished when E-box-like elements in the CRTAM promoter are mutated. In summary, ZEB1 functions as a transcriptional repressor for the CRTAM gene in both non-stimulated and stimulated T cells, thereby modulating adaptive immune responses.

Egr2-dependent gene expression profiling and ChIP-Seq reveal novel biologic targets in T cell anergy

T cell anergy is one of the mechanisms contributing to peripheral tolerance, particularly in the context of progressively growing tumors and in tolerogenic treatments promoting allograft acceptance. We recently reported that early growth response gene 2 (Egr2) is a critical transcription factor for the induction of anergy in vitro and in vivo, which was identified based on its ability to regulate the expression of inhibitory signaling molecules diacylglycerol kinase (DGK)-α and -ζ. We reasoned that other transcriptional targets of Egr2 might encode additional factors important for T cell anergy and immune regulation. Thus, we conducted two sets of genome-wide screens: gene expression profiling of wild type versus Egr2-deleted T cells treated under anergizing conditions, and a ChIP-Seq analysis to identify genes that bind Egr2 in anergic cells. Merging of these data sets revealed 49 targets that are directly regulated by Egr2. Among these are inhibitory signaling molecules previously reported to contribute to T cell anergy, but unexpectedly, also cell surface molecules and secreted factors, including lymphocyte-activation gene 3 (Lag3), Class-I-MHC-restricted T cell associated molecule (Crtam), Semaphorin 7A (Sema7A), and chemokine CCL1. These observations suggest that anergic T cells might not simply be functionally inert, and may have additional functional properties oriented towards other cellular components of the immune system.

Advances in adult asthma diagnosis and treatment in 2012: Potential therapeutics and gene-environment interactions

In the Journal of Allergy and Clinical Immunology in 2012, research reports related to asthma in adults clustered around mechanisms of disease, with a special focus on their potential for informing new therapies. There was also consideration of the effect of the environment on health from pollution, climate change, and epigenetic influences, underlining the importance of understanding gene-environment interactions in the pathogenesis of asthma and response to treatment.

Advances in pediatric asthma in 2012: Moving toward asthma prevention

Last year's "Advances in pediatric asthma: moving forward" concluded the following: "Now is also the time to utilize information recorded in electronic medical records to develop innovative disease management plans that will track asthma over time and enable timely decisions on interventions in order to maintain control that can lead to disease remission and prevention." This year's summary will focus on recent advances in pediatric asthma on modifying disease activity, preventing asthma exacerbations, managing severe asthma, and risk factors for predicting and managing early asthma, as indicated in Journal of Allergy and Clinical Immunology publications in 2012. Recent reports continue to shed light on methods to improve asthma management through steps to assess disease activity, tools to standardize outcome measures in asthma, genetic markers that predict risk for asthma and appropriate treatment, and interventions that alter the early presentation of asthma to prevent progression. We are well on our way to creating a pathway around wellness in asthma care and also to use new tools to predict the risk for asthma and take steps to not only prevent asthma exacerbations but also to prevent the early manifestations of the disease and thus prevent its evolution to severe asthma.