Enhanced Ex Vivo Generation of Erythroid Cells from Human Induced Pluripotent Stem Cells in a Simplified Cell Culture System with Low Cytokine Support.

Red blood cell (RBC) differentiation from human induced pluripotent stem cells (hiPSCs) offers great potential for developmental studies and innovative therapies. However, ex vivo erythropoiesis from hiPSCs is currently limited by low efficiency and unphysiological conditions of common culture systems. Especially, the absence of a physiological niche may impair cell growth and lineage-specific differentiation. We here describe a simplified, xeno- and feeder-free culture system for prolonged RBC generation that uses low numbers of supporting cytokines [stem cell factor (SCF), erythropoietin (EPO), and interleukin 3 (IL-3)] and is based on the intermediate development of a "hematopoietic cell forming complex (HCFC)." From this HCFC, CD43+ hematopoietic cells (purity >95%) were continuously released into the supernatant and could be collected repeatedly over a period of 6 weeks for further erythroid differentiation. The released cells were mainly CD34+/CD45+ progenitors with high erythroid colony-forming potential and CD36+ erythroid precursors. A total of 1.5 × 107 cells could be harvested from the supernatant of one six-well plate, showing 100- to 1000-fold amplification during subsequent homogeneous differentiation into GPA+ erythroid cells. Mean enucleation rates near 40% (up to 60%) further confirmed the potency of the system. These benefits may be explained by the generation of a niche within the HCFC that mimics the spatiotemporal signaling of the physiological microenvironment in which erythropoiesis occurs. Compared to other protocols, this method provides lower complexity, less cytokine and medium consumption, higher cellular output, and better enucleation. In addition, slight modifications in cytokine addition shift the system toward continuous generation of granulocytes and macrophages.

CD43 and CD49d from the B-Cell Chronic Lymphoproliferative Disorders Diagnostic Panel Are Useful to Detect Erythroid Dysplasia.

BACKGROUND: Despite bone marrow (BM) immunophenotyping by flow cytometry has progressively been recognized as an important tool for the diagnosis of myelodysplastic syndromes (MDS), the sparse knowledge about normal erythroid maturation and the lack of markers for erythroid characterization is a major shortcoming. METHODS: Here, we analyzed the expression of CD43 and CD49d, two markers included in the diagnostic panel for B-cell chronic lymphoproliferative disorders (B-CLPD), in the CD34+ compartment of normal BM and along the normal and dysplastic erythroid maturation. For this, 13 normal BM aspirates and 18 BM aspirates from MDS patients were studied by flow cytometry. RESULTS: Normal BM presented a higher expression of CD43 and CD49d among CD34+ erythroid precursors, compared to CD34+ cells committed to the remaining hematopoietic cell lineages. CD43 expression progressively decreased along the normal erythroid maturation, whereas CD49d levels increased from Stage I to Stage II, were maintained in Stages II and III, and then decreased until the last stage of maturation. In MDS, the expression of CD43 and CD49d followed a similar pattern, but with decreased expression levels for both markers, observed in all erythroid maturation stages (P < 0.05). CONCLUSIONS: Our results point to the usefulness of CD43 and CD49d, two markers commonly present in B-CLPD diagnosis panels, in the identification of dysplastic phenotypic features in the erythroid lineage. This allows a feasible and inexpensive way to identify patients who would benefit from a more extensive study to evaluate the presence of MDS, during the processing of suspected B-CLPD samples. © 2019 International Clinical Cytometry Society.

Differential Expression of CD43, CD81, and CD200 in Classic Versus Variant Hairy Cell Leukemia.

BACKGROUND: Hairy cell leukemia (HCL) and hairy cell leukemia variant (HCLv) are rare diseases with overlapping clinicopathological features. Features distinguishing HCL from HCLv include expression of CD25, CD123, CD200, annexin-A1, and the presence of BRAF V600E mutation. HCLv typically lacks these markers, but they may occur in a subgroup of HCL patients with an aggressive clinical course. We examined CD43, CD81, CD79b, and CD200 expression in HCL and HCLv. METHODS: Multiparametric flow cytometry (FCM) was performed on blood from 59 HCL and 15 HCLv patients for protocol entry. Mean fluorescent intensity (MFI) of CD43, CD79b, CD81, and CD200 was determined (for CD200, n = 17 and 7, respectively). RESULTS: Median MFI of HCL vs HCLv was 545 vs 272 for CD43, 602 vs 2,450 for CD81, 4,962 vs 1,969 for CD79b, and 11,652 vs 1,405 for CD200, respectively. Analysis of the median differences, HCL minus HCLv (and their 95% confidence intervals and P-values) indicated that CD43 MFI (estimated median difference (95% CI): 212 [72-413; P = 0.0027) and CD200 MFI (9,883 [3,514-13,434]; P < 0.0001) were higher in HCL than in HCLv, while CD81 MFI (-1,858 [-2,604 to -1,365]; P < 0.0001) was lower in HCL than in HCLv. CD79b MFI HCL median was more than double that of HCLv, but the observed difference (1,571 [-739 to 4,417]) was consistent with the null hypothesis of no difference (P = 0.13). CONCLUSIONS: CD200, CD43, and CD81 are likely differentially expressed between HCL and HCLv, reflecting their differing disease biology. Inclusion of these markers in FCM is potentially informative. © 2019 International Clinical Cytometry Society.

Glycoproteomic Analysis of MGL-Binding Proteins on Acute T-Cell Leukemia Cells.

C-type lectins are a diverse group of proteins involved in many human physiological and pathological processes. Most C-type lectins are glycan-binding proteins, some of which are pivotal for innate immune responses against pathogens. Other C-type lectins, such as the macrophage galactose-type lectin (MGL), have been shown to induce immunosuppressive responses upon the recognition of aberrant glycosylation on cancer cells. MGL is known to recognize terminal N-acetylgalactosamine (GalNAc), such as the Tn antigen, which is commonly found on malignant cells. Even though this glycan specificity of MGL is well described, there is a lack of understanding of the actual glycoproteins that bind MGL. We present a glycoproteomic workflow for the identification of MGL-binding proteins, which we applied to study MGL ligands on the human Jurkat leukemia cell line. In addition to the known MGL ligands and Tn antigen-carrying proteins CD43 and CD45 on these cells, we have identified a set of novel cell-surface ligands for MGL. Importantly, for several of these, O-glycosylation has hitherto not been described. Altogether, our data provide new insight into the identification and structure of novel MGL ligands that presumably act as modulatory molecules in cancer immune responses.

Differential inhibition of mucin-type O-glycosylation (MTOG) induced by peracetyl N-thioglycolyl-d-galactosamine (Ac5GalNTGc) in myeloid cells.

Investigations on the structure and functional roles of glycosylation - an intricate, complex, and dynamic post translational modification on proteins - in biological processes has been a challenging task. Glycan modifications vary depending on the specific cell type, its developmental stage, and resting or activated state. In the present study, we aim to understand the differences between the mucin-type O-glycosylation (MTOG) of two functionally divergent human cell lines, K562 (chronic myeloid leukemia) and U937 (histiocytic lymphoma), having myeloid origins. MTOG is initiated by the addition of N-acetyl-α-d-galactosamine (GalNAc) to Ser/Thr of glycoproteins. We exploited the metabolic glycan engineering (MGE) strategy using the peracetyl N-thioglycolyl-d-galactosamine (Ac5GalNTGc), a synthetic GalNAc analogue, to engineer the glycoconjugates. Ac5GalNTGc was metabolized and incorporated as N-thioglycolyl-d-galactosamine (GalNTGc) in cell surface glycoproteins in both the cell lines with varying degrees of efficiency. Notably, metabolic incorporation of GalNTGc resulted in differential inhibition of MTOG. It was observed that endogenous glycosylation machinery of K562 is relatively more stringent for selecting GalNTGc whereas U937 is flexible towards this selection. Additionally, we studied how the glycan modifications vary on a given CD antigen in these cell lines. Particularly, MTOG on CD43 was differentially inhibited in K562 and U937 as revealed by glycan-dependent and glycan-independent antibodies. It was observed that the effect of MGE on CD43 was similar to global effects on both cell lines. Consequences of MGE using GalNAc analogues depend on the expression and activity of various glycosyl transferases which determine global glycosylation on cell surface as well as on specific glycoproteins.

Coexpression of CD5 and CD43 predicts worse prognosis in diffuse large B-cell lymphoma.

Both CD5 and CD43 are expressed on the surface of B lymphocytes of definite phase and associated with the adverse outcome in diffuse large B-cell lymphoma (DLBCL). However, the relationship between CD5 and CD43 expression and the prognostic value of CD5/CD43 coexpression in DLBCL are unknown. We herein determined the correlation between CD5 and CD43 expression, as separate factors or in combination, with the clinicopathological features and survival of 200 patients with DLBCL receiving standard chemotherapy with or without rituximab. Among these DLBCL patients, CD5 expression, CD43 expression, and CD5/CD43 coexpression were detected in 18 (9%), 57 (27%), and 10 (5%) patients, respectively, and all were positively correlated with advanced age and nongerminal cell type. CD5-positive and CD43-positive DLBCL patients had poorer event-free survival (EFS, P < 0.001) and overall survival (OS, P < 0.001) than CD5-negative and CD43-negative patients, respectively. CD5/CD43 coexpression was correlated with a significantly worse prognosis than CD5 or CD43 expression alone. Univariate analysis showed that CD5 expression, CD43 expression, and CD5/CD43 coexpression were all adverse prognostic factors for DLBCL patient survival, and CD5/CD43 coexpression was associated with a greater relative risk for recurrence and death than either CD5 or CD43 expression alone. Multivariate analysis demonstrated that CD5/CD43 coexpression was an independent prognostic factor for EFS (P < 0.001) and OS (P < 0.001) in DLBCL. In conclusion, our data indicate that DLBCL patients with CD5/CD43 coexpression represent a specific subgroup with a significantly worse prognosis than those expressing either marker alone.

Optimization of Synthetic mRNA for Highly Efficient Translation and its Application in the Generation of Endothelial and Hematopoietic Cells from Human and Primate Pluripotent Stem Cells.

Identification of transcription factors that directly convert pluripotent stem cells (PSCs) into endothelial and blood cells and advances in the chemical modifications of messenger RNA (mRNA) offer alternative nucleic acid-based transgene-free approach for scalable production of these cells for drug screening and therapeutic purposes. Here we evaluated the effect of 5' and 3' RNA untranslated regions (UTRs) on translational efficiency of chemically-modified synthetic mRNA (modRNA) in human PSCs and showed that an addition of 5'UTR indeed enhanced protein expression. With the optimized modRNAs expressing ETV2 or ETV2 and GATA2, we are able to produce VE-cadherin+ endothelial cells and CD34+CD43+ hematopoietic progenitors, respectively, from human PSCs as well as non-human primate (NHP) PSCs. Overall, our findings provide valuable information on the design of in vitro transcription templates being used in PSCs and its broad applicability for basic research, disease modeling, and regenerative medicine.

Functional Heterogeneity of Endothelial Cells Derived from Human Pluripotent Stem Cells.

Specification of endothelial cells (ECs) into arterial, venous, and lymphatic cells is a crucial process of vascular development, and expanding our knowledge about EC specification from human pluripotent stem cells (hPSCs) will aid the design of optimal strategies for producing desired types of ECs for therapies. In our prior studies, we revealed that hPSC-derived VE-cadherin(V)+CD31+CD34+ ECs are heterogeneous and include at least three major subsets with distinct hemogenic properties: V+CD43/235a-CD73- hemogenic endothelial progenitors (HEPs), V+CD43loCD235a+73- angiogenic hematopoietic progenitors (AHPs), and V+CD43/235a-73+ non-HEPs. In this study, using angiogenesis assays, we demonstrated that ECs within these subsets have distinct endothelial colony- and tube-forming properties, proliferative and migratory properties, and endothelial nitric oxide synthase and inflammatory cytokine production potentials. Culture of isolated subsets in arterial, venous, and lymphatic conditions revealed that AHPs are skewed toward lymphatic, HEPs toward arterial, and non-HEPs toward venous differentiation in vitro. These findings suggest that selection and enhancement of production of a particular EC subset may aid in generating desirable EC populations with arterial, venous, or lymphatic properties from hPSCs.

Dysfunctional immunoregulation in human liver allograft rejection associated with compromised galectin-1/CD7 pathway function.

Regulatory T cells in rejected allograft patients display an inability to control responder T cells. Galectin-1 (Gal1) inhibits responder T cells through binding CD7. We investigated whether the dysfunctional immunoregulation in liver allograft rejection patients results from reduced regulatory T-cell Gal1 expression and/or responder T-cell CD7 expression. Circulating regulatory T cells and responder T cells were profiled from 31 acute rejection transplant patients, 85 transplant patients in remission, and 40 healthy controls. CD7+ and CD7- responder T cells were co-cultured with regulatory T cells to assess regulatory T-cell suppressor function. Gal1-small interfering RNA was used to silence regulatory T-cell Gal1. The CD7+ cell percentage was inversely correlated with AST, ALT, and GGT levels. The proportions of CD7+ responder T cells and Gal1+ regulatory T cells were higher in healthy controls than in transplant patients in remission and lowest in acute rejection transplant patients. Notably, CD7+ responder T-cell susceptibility to Gal1+ regulatory T-cell control was ranked in the same manner. Silencing Gal1 expression in regulatory T cells reduced their ability to suppress CD7+ (but not CD7-) responder T cells. Additionally, the proportions of CD43+ and CD45+ responder T cells were higher in healthy controls than in acute rejection transplant patients. CD43 co-expression (but not CD45 co-expression) on CD7+ responder T cells promoted their apoptosis in a Gal1-dependent manner. In sum, dysfunctional immunoregulation in liver allograft rejection patients can be partly attributed to reduced regulatory T-cell Gal1 expression and reduced responder T-cell CD7 expression. Responder T-cell CD43 downregulation in acute rejection patients may further contribute to reduced responder T-cell responsiveness to regulatory T-cell control.

A human VE-cadherin-tdTomato and CD43-green fluorescent protein dual reporter cell line for study endothelial to hematopoietic transition.

Human embryonic stem cell line WA01 was genetically modified using zinc-finger nucleases and the PiggyBac/transponson system to introduce a fluorescence reporter for VE-cadherin (VEC; tdTomato) and CD43 (eGFP). Phenotypic and functional assays for pluripotency revealed the modified hES cell reporter lines remained normal. When the cells were differentiated into hematoendothelial lineages, either by directed differentiation or direct reprogramming, flow cytometric and fluorescence microscopy showed that VEC+ endothelial cells express tdTomato and CD43+ hematopoietic progenitors express eGFP.

Cluster of differentiation 43 deficiency in leukocytes leads to reduced atherosclerosis--brief report.

OBJECTIVE: The aim of this study was to investigate the role of cluster of differentiation 43 (CD43), an integral membrane glycoprotein with both proadhesive and antiadhesive activities, in atherosclerosis. APPROACH AND RESULTS: Low-density lipoprotein receptor-deficient mice were lethally irradiated and reconstituted with either bone marrow from CD43(-/-) mice or from wild-type controls. We found that mice lacking the CD43 on their leukocytes had significantly less severe atherosclerosis and that, contrary to our expectation, macrophage infiltration into the vessel wall was not affected by the lack of CD43 in the leukocytes. However, we found that CD43 mediates cholesterol homeostasis in macrophages by facilitating cholesterol efflux. This resulted in a significant reduction in storage of cholesterol in the aorta of mice lacking CD43 in the leukocytes. CONCLUSIONS: CD43 may be an important mediator of macrophage lipid homeostasis, thereby affecting macrophage foam cell formation and ultimately atherosclerotic plaque development.

Tracing the origin of the HSC hierarchy reveals an SCF-dependent, IL-3-independent CD43(-) embryonic precursor.

Definitive hematopoietic stem cells (HSCs) develop in the aorta gonad mesonephros (AGM) region in a stepwise manner. Type I pre-HSCs express CD41 but lack CD45 expression, which is subsequently upregulated in type II pre-HSCs prior to their maturation into definitive HSCs. Here, using ex vivo modeling of HSC development, we identify precursors of definitive HSCs in the trunk of the embryonic day 9.5 (E9.5) mouse embryo. These precursors, termed here pro-HSCs, are less mature than type I and II pre-HSCs. Although pro-HSCs are CD41(+), they lack the CD43 marker, which is gradually upregulated in the developing HSC lineage. We show that stem cell factor (SCF), but not interleukin-3 (IL-3), is a major effector of HSC maturation during E9-E10. This study extends further the previously established hierarchical organization of the developing HSC lineage and presents it as a differentially regulated four-step process and identifies additional targets that could facilitate the generation of transplantable HSCs from pluripotent cells for clinical needs.

Identification of multipotent progenitors that emerge prior to hematopoietic stem cells in embryonic development.

Hematopoiesis in the embryo proceeds in a series of waves, with primitive erythroid-biased waves succeeded by definitive waves, within which the properties of hematopoietic stem cells (multilineage potential, self-renewal, and engraftability) gradually arise. Whereas self-renewal and engraftability have previously been examined in the embryo, multipotency has not been thoroughly addressed, especially at the single-cell level or within well-defined populations. To identify when and where clonal multilineage potential arises during embryogenesis, we developed a single-cell multipotency assay. We find that, during the initiation of definitive hematopoiesis in the embryo, a defined population of multipotent, engraftable progenitors emerges that is much more abundant within the yolk sac (YS) than the aorta-gonad-mesonephros (AGM) or fetal liver. These experiments indicate that multipotent cells appear in concert within both the YS and AGM and strongly implicate YS-derived progenitors as contributors to definitive hematopoiesis.

Utilization of the AAVS1 safe harbor locus for hematopoietic specific transgene expression and gene knockdown in human ES cells.

Human pluripotent stem cells offer a powerful system to study human biology and disease. Here, we report a system to both express transgenes specifically in ES cell derived hematopoietic cells and knockdown gene expression stably throughout the differentiation of ES cells. We characterize a CD43 promoter construct that when inserted into the AAVS1 "safe harbor" locus utilizing a zinc finger nuclease specifically drives GFP expression in hematopoietic cells derived from a transgenic ES cell line and faithfully recapitulates endogenous CD43 expression. In addition, using the same gene targeting strategy we demonstrate that constitutive expression of short hairpin RNAs within a microRNA backbone can suppress expression of PU.1, an important regulator of myeloid cell development. We show that PU.1 knockdown cell lines display an inhibition in myeloid cell formation and skewing towards erythroid development. Overall, we have generated a powerful system to track hematopoietic development from pluripotent stem cells and study gene function through hematopoietic specific gene expression and constitutive gene knockdown.

Distinct B-cell populations contribute to vaccine antigen-specific antibody production in a transgenic mouse model.

The generation of memory B cells by vaccination plays a critical role in maintaining antigen-specific antibodies and producing antibody responses upon re-exposure to a pathogen. B-cell populations contributing to antibody production and protection by vaccination remain poorly defined. We used influenza virus-like particle (VLP) vaccine in a transgenic mouse model that would identify germinal centre-derived memory B cells with the expression of yellow fluorescent protein (YFP(+) cells). Immunization with influenza VLP vaccine did not induce significant increases in YFP(+) cells although vaccine antigen-specific antibodies in sera were found to confer protection against a lethal dose of influenza A virus (A/PR8). In addition, CD43(+)  B220(-) populations with low YFP(+) cells mainly contributed to the production of vaccine antigen-specific IgG isotype-switched antibodies whereas CD43(-)  B220(+) populations with high YFP(+) cells were able to produce vaccine antigen-specific IgM antibodies. Challenge infection of immunized transgenic mice with live influenza A virus resulted in significant increases in YFP(+) cells in the B220(-) populations of spleen and bone marrow cells. These results suggest that CD43(+)  B220(-) B cells generated by vaccination are important for producing influenza vaccine antigen-specific antibodies and conferring protection.

Intracellular patterns of sialophorin expression define a new molecular classification of breast cancer and represent new targets for therapy.

BACKGROUND: Sialophorin is a transmembrane sialoglycoprotein. Normally, the molecule is only produced by white blood cells where it regulates functions such as intercellular adhesion, intracellular signalling, apoptosis, migration and proliferation. METHODS: Normal breast tissue and primary breast tumours were analysed by immunohistochemistry for sialophorin expression. The sialophorin-positive breast cancer cell line MCF7 was engineered to stably express either non-targeted or sialophorin-targeted small interfering RNA (siRNA). Assays were then performed in vitro to assess apoptosis, intracellular adhesion, transendothelial migration and cytotoxicity. An orthotopic mouse model assayed ability to produce tumours in vivo. RESULTS: Normal breast epithelial cells exhibit expression of the N-terminal domain of sialophorin in the cytoplasm but not the nucleus. The majority of these normal cells are also negative for expression of the C-terminal domain. In contrast, malignant breast epithelial cells exhibit N-terminal expression both in the cytoplasm and nucleus and the majority express the C-terminus in the nucleus. Using differential patterns of intracellular expression of the N and C termini of sialophorin, we define six subtypes of breast cancer that are independent of histological and receptor status classification. Targeting sialophorin with siRNA resulted in the MCF7 breast cancer cell line exhibiting increased homotypic adhesion, decreased transendothelial migration, increased susceptibility to apoptosis, increased vulnerability to lysis by natural killer cells and decreased ability to produce tumours in mice. CONCLUSION: Our results indicate that intracellular patterns of sialophorin expression define a new molecular classification of breast cancer and that sialophorin represents a novel therapeutic target.

Cancer-associated CD43 glycoforms as target of immunotherapy.

CD43 is a sialoglycosylated membrane protein that is involved in cell proliferation and differentiation. CD43 glycoforms that are recognized by the UN1 monoclonal antibody (mAb) were expressed in lymphoblastoid T-cell lines and solid tumors, such as breast, colon, gastric, and squamous cell lung carcinomas, while unexpressed in the normal counterparts. The cancer association of UN1/CD43 epitope suggested the possibility to use the UN1 mAb for tumor diagnosis and therapy. In this study, we show that the UN1 mAb was endowed with antitumor activity in vivo because its passive transfer inhibited the growth of UN1-positive HPB-ALL lymphoblastoid T cells in mice. Furthermore, we demonstrate that tumor inhibition was due to UN1 mAb-dependent natural killer-mediated cytotoxicity. By screening a phage-displayed random peptide library, we identified the phagotope 2/165 as a mimotope of the UN1 antigen, as it harbored a peptide sequence that was specifically recognized by the UN1 mAb and inhibited the binding of the UN1 mAb to UN1-positive tumor cells. On the basis of sequence homology with the extracellular region of CD43 (amino acids 64 to 83), the 2/165 peptide sequence was likely mimicking the protein core of the UN1/CD43 epitope. When used as vaccine in mice, the 2/165 phagotope raised antibodies against the UN1/CD43 antigen, indicating that the 2/165 phagotope mimicked the UN1 antigen structure, and could represent a novel immunogen for cancer immunotherapy. These findings support the feasibility of using monoclonal antibodies to identify cancer-associated mimotopes for immunotherapy.

CD43 promotes cells transformation by preventing merlin-mediated contact inhibition of growth.

In normal tissues, strict control of tissue size is achieved by regulating cell numbers. The mechanism that controls total cell number is known as contact inhibition of growth and it depends on the NF2/Merlin pathway. Negative regulation of this pathway by deleterious mutations or by oncogenes results in cell transformation and tumor progression. Here we provide evidence that the CD43 sialomucin cooperates with oncogenic signals to promote cell transformation by abrogating the contact inhibition of growth through a molecular mechanism that involves AKT-dependent Merlin phosphorylation and degradation. Accordingly, inhibition of endogenous CD43 expression by RNA interference in lung, cervix and colon human cancer cells impaired tumor growth in vivo. These data underscore a previously unidentified role for CD43 in non-hematopoietic tumor progression.

CD43 expression is associated with inferior survival in the non-germinal centre B-cell subgroup of diffuse large B-cell lymphoma.

We evaluated the prognostic significance of CD43 (SPN), a membrane glycoprotein, in 140 patients with diffuse large B-cell lymphoma (DLBCL) by tissue microarray (TMA) immunostaining, and gene expression profiling (GEP) in 43 patients. CD43 protein was expressed in 19% of the cases and was strongly related to the non-germinal centre B-cell (non-GCB) subgroup by both TMA and GEP. Patients with CD43(+) DLBCL had an inferior 3-year overall survival (OS) compared to those with CD43(-) DLBCL (50% vs. 76%, P = 0·01). Within the non-GCB subgroup, patients with CD43(+) DLBCL had a particularly poor 3-year OS (32% vs. 71%, P < 0·001). Gene set enrichment analysis within the activated B-cell subgroup revealed significant enrichment in the stromal-1 signature in CD43(-) cases. We conclude that CD43 is an adverse prognostic marker in DLBCL, and is preferentially expressed in the non-GCB subgroup. The dismal outcome of CD43(+) cases in the non-GCB subgroup may be explained, at least in part, by a less favourable microenvironment.

CD43-mediated IFN-γ production by CD8+ T cells promotes abdominal aortic aneurysm in mice.

CD43 is a glycosylated surface protein abundantly expressed on lymphocytes. Its role in immune responses has been difficult to clearly establish, with evidence supporting both costimulatory and inhibitory functions. In addition, its contribution to disease pathogenesis remains elusive. Using a well-characterized murine model of elastase-induced abdominal aortic aneurysm (AAA) that recapitulates many key features of the human disease, we established that the presence of CD43 on T cells is required for AAA formation. Moreover, we found that IFN-γ-producing CD8(+) T cells, but not CD4(+) T cells, promote the development of aneurysm by enhancing cellular apoptosis and matrix metalloprotease activity. Reconstitution with IFN-γ-producing CD8(+) T cells or recombinant IFN-γ promotes the aneurysm phenotype in CD43(-/-) mice, whereas IFN-γ antagonism abrogates disease in wild-type animals. Furthermore, we showed that the presence of CD43 with an intact cytoplasmic domain capable of binding to ezrin-radixin-moesin cytoskeletal proteins is essential for optimal in vivo IFN-γ production by T cells and aneurysm formation. We have thus identified a robust physiologic role for CD43 in a relevant animal model and established an important in vivo function for CD43-dependent regulation of IFN-γ production. These results further suggest that IFN-γ antagonism or selective blockade of CD43(+)CD8(+) T cell activities merits further investigation for immunotherapy in AAA.