The role of natural killer group 2D (NKG2D) in peripheral T cells as a costimulatory receptor is well established. However, its contribution to T cell thymic education and functional imprint is unknown. Here, we report significant changes in development, receptor signaling, transcriptional program, and function in T cells from mice lacking NKG2D signaling. In C57BL/6 (B6) and OT-I mice, we found that NKG2D deficiency results in Vβ chain usage changes and stagnation of the double-positive stage in thymic T cell development. We found that the expression of CD5 and CD45 in thymocytes from NKG2D deficient mice were reduced, indicating a direct influence of NKG2D on the strength of T cell receptor (TCR) signaling during the developmental stage of T cells. Depicting the functional consequences of NKG2D, peripheral OT-I NKG2D-deficient cells were unresponsive to ovalbumin peptide stimulation. Paradoxically, while αCD3/CD28 agonist antibodies led to phenotypic T cell activation, their ability to produce cytokines remained severely compromised. We found that OT-I NKG2D-deficient cells activate STAT5 in response to interleukin-15 but were unable to phosphorylate ERK or S6 upon TCR engagement, underpinning a defect in TCR signaling. Finally, we showed that NKG2D is expressed in mouse and human thymic T cells at the double-negative stage, suggesting an evolutionarily conserved function during T cell development. The data presented in this study indicate that NKG2D impacts thymic T cell development at a fundamental level by reducing the TCR threshold and affecting the functional imprint of the thymic progeny. In summary, understanding the impact of NKG2D on thymic T cell development and TCR signaling contributes to our knowledge of immune system regulation, immune dysregulation, and the design of immunotherapies.

Wall shear stress (WSS) is a critical factor in vascular biology, and both high and low WSS are implicated in atherosclerosis. Fibronectin (FN) is a key extracellular matrix protein that plays an important role in cell activities. Under high shear stress, plasma FN undergoes fibrillogenesis; however, its behavior under low shear stress remains unclear. This study aimed to investigate the formation ofin vitrocell-free fibrillar FN (FFN) under low shear rate conditions and its effect on bovine aortic endothelial cell behavior. FN (500µg ml-1) was perfused through slide chambers at three flow rates (0.16 ml h-1, 0.25 ml h-1, and 0.48 ml h-1), corresponding to low shear rates of 0.35 s-1, 0.55 s-1, and 1.05 s-1, respectively, for 4 h at room temperature. The formed FN matrices were observed using fluorescence microscopy and scanning electron microscopy. Under low shear rates, distinct FN matrix structures were observed. FFN0.48 formed immense fibrils with smooth surfaces, FFN0.25 formed a matrix with a rough surface, and FFN16 exhibited nodular structures. FFN0.25 supported cell activities to a greater extent than native FN and other FFN surfaces. Our study suggests that abnormally low shear conditions impact FN structure and function and enhance the understanding of FN fibrillogenesis in vascular biology, particularly in atherosclerosis.

Graft-versus-host disease (GVHD) is a primary and often lethal complication of allogenic hematopoietic stem cell transplantation (HSCT). Prophylactic regimens for GVHD are given as standard pretransplantation therapy; however, up to 50% of these patients develop acute GVHD (aGVHD) and require additional immunosuppressive intervention. Using a mouse GVHD model, we previously showed that injecting mice with exopolysaccharide (EPS) from Bacillus subtilis prior to GVHD induction significantly increased 80-day survival after transplantation of complete allogeneic major histocompatibility complex-mismatched cells. To ask whether EPS might also inhibit GVHD in humans, we used humanized NSG-HLA-A2 mice and induced GVHD by i.v. injection of A2neg human peripheral blood mononuclear cells (PBMCs). Because we could not inject human donors with EPS, we transferred EPS-pretreated dendritic cells (DCs) to inhibit aGVHD. We derived these DCs from CD34+ human cord blood cells, treated them with EPS, and then injected them together with PBMCs into the NSG-HLA-A2 mice. We found that all mice that received untreated DCs were dead by day 35, whereas 25% of mice receiving EPS-treated DCs (EPS-DCs) survived. This DC cell therapy could be readily translatable to humans, because we can generate large numbers of human EPS-DCs and use them as an “off the shelf” treatment for patients undergoing HSCT.

Fibronectin (FN) is an important adhesive extracellular matrix protein. It forms a fibrillar matrix on cell surface which controls cell morphology, adhesion, migration and proliferation. This study aimed to analyze the effect of in vitro cell-free formed fibrillar fibronectin with different morphologies on mouse fibroblasts L929 cells migration. To evaluate fibrils morphology on cell function, fibroblast L929 were cultured on surface pre-coated with FN and FN fibrils at concentration of 0.25 and 0.75 mg/mL at 4 °C overnight, then cell migration assay were performed. Migration speed of cells monolayers grown on different fibronectin surfaces was measured by using snapshot pictures with a regular inverted microscope at 0, 12, 48 and 72 h. Our cell-free FN fibrillogenesis indicated that at different concentrations, fibronectin organized into various structural matrix. Our data showed that large fibronectin fibrils decreased the rate of cell migration while small fibronectin fibrils (0.25 mg/mL) did not, compared with native plasma fibronectin. These data demonstrate that there is a strong relationship between structure and function of fibronectin. Taken together, our data draws new attention towards controlling biological function of fibronectin by its fibrillar structure.

Plasma fibronectin (FN) is synthesized by hepatocytes and secreted into the circulation in a soluble, compact and non-fibrillar form. However, intrinsic functions of FN in the body are prevalent to the multimeric FN fibrils. Plasma FN is assembled by cells or adherent platelets into functional fibrils. Reports have indicated that the process to incorporate FN into multimer fibrils can also occur in cell-free models in vitro by incubation with denaturants, reducing agents, or anastellin (FN peptidic fragment). Here, we report on (1) the formation of insoluble fibrillar-like supramolecules of plasma FN (FN fibrils) by exposing the molecules to increasing shear rates and (2) the functional characterization in platelet adhesion and aggregation.

Introduction: Insoluble fibrils are the active form of fibronectin (FN) with exposed functional domains capable of interacting with a variety of biomolecules and cells to support many cellular processes. Recent studies show that the formation of FN fibrils can occur in cell-free systems. Currently, the characteristic of FN fibrils and their potential in wound healing application are not fully understood. Our project aims at (i) using urea to induce the formation of FN fibrils with different conformations and (ii) to characterize their conformation-dependent effect on cellular adhesion and spreading.

In insoluble fibrillar form, fibronectin exerts its most significant biological activities. Inducing assembly of fibrillar FN on scaffold prior to cell seeding provides controllable early interactions between cells and the biomaterial. This study aimed to synthesize fibronectin fibrils under cell-free conditions, evaluate the conformation of induced fibrils and their effect on platelet adhesion. To induce fibrillogenesis, purified fibronectin were dialyzed against urea 2 M in 16 h followed by dialysis against PBS pH 7.3. Microscopic images revealed that urea induced heterogeneous formation of fibronectin fibrils with various morphologies ranging from aggregation form to fibrillar form. Fibrillar fibronectin with diameter in range of 30–130 µm tend to assembly into matrix, whereas fibronectin aggregates with size around 50 µm suspend in solution. Our data showed that among 4 tested concentrations, 1 mg/ml, as it yields the most aggregated fibronectin and fibrillar matrix, is the optimal condition for fibronectin polymerization. At lower concentrations, less fibronectin fibrils were formed and they did not link together to form a matrix. Platelet adhesion assay indicated a stronger platelet adhesion on surfaces coated with fibronectin fibrils than those with untreated fibronectin. Adherent platelets gathered into small groups around aggregated fibronectin and separate fibrils while on fibrillar matrix, they adhere into networks of larger clusters. Denaturation of plasma fibronectin by urea induces irreversible heterogeneous formation of fibronectin fibrils and improves platelet adhesion.

Platelet thrombi at site of vascular injury require resistance to shear stress generated by the flowing blood. Fibronectin assembly can contribute to platelet adhesion and aggregation. Here, we examined the effect of shear stress and the role of αIIbβ3 and αvβ3 in fibronectin assembly by adherent platelets under flow dynamic conditions in vitro. Platelets were placed onto immobilized fibronectin (50 µg/ml) with fluorescently labeled fibronectin. Shear rates (500 s−1 or 5000 s−1) were generated by a viscometer (Diamed) for 2 or 10 min. Adherent platelets were lysed with 2

Human urinary disorders are generally studied in rodent models due to limitations of functional in vitro culture models of primary human urothelial cells (HUCs). Current HUC culture models are often derived from immortalized cancer cell lines, which likely have functional characteristics differ from healthy human urothelium. Here, we described a simple explant culture technique to generate HUCs and assessed their in vitro functions. Using transmission electron microscopy, we assessed morphology and heterogeneity of the generated HUCs and characterized their intercellular membrane structural proteins relative to ex vivo urothelium tissue. We demonstrated that our cultured HUCs are free of fibroblasts. They are also heterogeneous, containing cells characteristic of both immature basal cells and mature superficial urothelial cells. The cultured HUCs expressed muscarinic receptors (MR1 and MR2), carnitine acetyltransferase (CarAT), immunoregulatory cytokines IL7, IL15, and IL23, as well as the chemokine CCL20. HUCs also expressed epithelial cell-specific molecules essential for forming intercellular structures that maintain the functional capacity to form the physiological barrier of the human bladder urothelium. A subset of HUCs, identified by the high expression of CD44, expressed the Toll-like receptor 4 (TLR4) along with its co-receptor CD14. We demonstrated that HUCs express, at the mRNA level, both forms of the IL22 receptor, the membrane-associated (IL22RA1) and the secreted soluble (IL22RA2) forms; in turn, IL22 inhibited expression of MR1 and induced expression of CarAT and two antimicrobial peptides (S100A9 and lipocalin-2). While the cellular sources of IL22 have yet to be identified, the HUC cytokine and chemokine profiles support the concept that IL22-producing cells are present in the human bladder mucosa tissue and that IL22 plays a regulatory role in HUC functions. Thus, the described explant technique is clearly capable of generating functional HUCs suitable for the study of human urinary tract disorders, including interactions between urothelium and IL22-producing cells.

Foxn1 is essential for thymic organogenesis and T lymphopoiesis. While reduced Foxn1 expression results in a decline in T lymphopoiesis, overexpression of Foxn1 in the thymus of a transgenic mouse model (Foxn1Tg) attenuates the age-associated decline in T lymphopoiesis. T lymphopoiesis begins with ETP, derived from MPP in the BM. A decline in MPP and ETP numbers with age is thought to contribute to reduced T lymphopoiesis. Previously, we showed that reduced ETP number with age is attenuated in Foxn1Tg; whether the effect is initiated in the BM with MPP is not known. Here, we report that Foxn1 is expressed in Wt BM and over expressed in Foxn1Tg. With age, the number of MPP in Foxn1Tg was not reduced, and Foxn1Tg also have a larger pool of HSC. Furthermore, the Foxn1Tg BM is more efficient in generating MPP. In contrast to MPP, CLP and B lineage cell numbers were significantly lower in both young and aged Foxn1Tg compared to Wt. We identified a novel population of lineageneg/low, EpCAMpos, CD138neg cells as Foxn1-expressing BM cells that also express Delta-like 4. Thus, Foxn1 affects both T lymphopoiesis and hematopoiesis, suggesting that the Foxn1 BM niches function in skewing MPP development toward T lineage progenitors.

Foxn1 is essential for thymic organogenesis and T lymphopoiesis. Whereas reduced Foxn1 expression results in a decline in T lymphopoiesis, overexpression of Foxn1 in the thymus of a transgenic mouse model (Foxn1Tg) attenuates the age-associated decline in T lymphopoiesis. T lymphopoiesis begins with early T cell progenitors (ETP), derived from multipotent progenitors (MPP) in the bone marrow (BM). A decline in MPP and ETP numbers with age is thought to contribute to reduced T lymphopoiesis. Previously, we showed that reduced ETP number with age is attenuated in Foxn1 transgenic (Tg); whether the effect is initiated in the BM with MPP is not known. In this study, we report that Foxn1 is expressed in wild-type BM and overexpressed in Foxn1Tg. With age, the number of MPP in Foxn1Tg was not reduced, and Foxn1Tg also have a larger pool of hematopoietic stem cells. Furthermore, the Foxn1Tg BM is more efficient in generating MPP. In contrast to MPP, common lymphoid progenitors and B lineage cell numbers were significantly lower in both young and aged Foxn1Tg compared with wild type. We identified a novel population of lineageneg/low, CD45pos EpCAMpos, SCA1pos, CD117neg, CD138neg, MHCIIneg cells as Foxn1-expressing BM cells that also express Delta-like 4. Thus, Foxn1 affects both T lymphopoiesis and hematopoiesis, and the Foxn1 BM niche may function in skewing MPP development toward T lineage progenitors.

The thymus requires continuous seeding of progenitors from hematopoietic stem cells (HSC) in bone marrow (BM) for naïve T cell production. HSC give rise to multipotent progenitors (MPP) and their progeny common lymphoid progenitors (CLP); however, MPP are the predominant source of early T cell progenitors (ETP) in the thymus. ETP, HSC, MPP and CLP decline with age, culminating in reduced naïve T cell production. We reported that the age-associated decline in ETP is abrogated in K14-Foxn1 transgenic mice (Foxn1Tg). Here, we show that Foxn1 is expressed in BM and its expression was 98-fold higher in Foxn1Tg. Foxn1 transgene expression increased endogenous Foxn1 mRNA levels by 18- to 24-fold; Foxn1 positive cells were readily detected in the BM. We found that HSC number was 2-fold higher in young Foxn1Tg versus wild type (Wt). HSC and MPP numbers declined in aged Wt but not in Foxn1Tg. A higher percent of HSC was in S,G2/M; further, HSC and MPP in subGo from Foxn1Tg were respectively 3-fold and 4-fold reduced, suggesting inhibition of cell death. In contrast, CLP frequency was 4-fold less in young Foxn1Tg. A higher number of the committed T cell progenitors (CTP) with a 2-fold increase in S,G2/M fraction were found in young Foxn1Tg versus Wt. Our data provide evidence linking Foxn1 to the development of T cell progenitors in the BM and thymus, suggesting that increased expression could limit the decline in naïve T cell production with age.

Objective. A human thymic epithelial cell (TEC) line expressing human leukocyte antigen ABC and human leukocyte antigen-DR was engineered to overexpress murine Delta-like 1 (TEC-DI1) for the purpose of establishing a human culture system that supports T lymphopoiesis from hematopoietic progenitor cells (HPCs).Materials and Methods. Cord blood or bone marrow HPCs were co-cultured with either the parental TEC line expressing low levels of the Notch ligands, Delta-like 1 and Delta-like 4, or with TEC-DI1 to determine if these cell lines support human lymphopoiesis.Results. In co-cultures with cord blood or bone marrow HPCs, TEC-DI1 cells promote de novo generation of CD7(pos)CD1a(pos) T-lineage committed cells. Most CD7(pos)CD1(hi) cells are CD4(pos)CD(pos) double-positive (DP). We found that TEC-DI1 cells are insufficient to generate mature CD3(hi)CD4(pos) or CD3(hi) CD8(pos) single-positive (SP) T cells from the CD4(pos)CD8(pos) DP T cells; however, we detected CD3(lo) cells within the DP and SP CD4 and CD8 populations. The CD(lo) SP cells expressed lower levels of interleukin-2R alpha and interleukin-7R alpha compared to CD3(lo) DP cells. In contrast to the TEC-DI1 line, the parental TEC-84 line expressing low levels of human Notch ligands permits HPC differentiation to the B-cell lineage.Conclusions. We report for the first time a human TEC line that supports lymphopoiesis from cord blood and bone marrow HPC. The TEC cell lines described herein provide a novel human thymic stroma model to study the contribution of human leukocyte antigen molecules and Notch ligands to T-cell commitment and maturation and could be utilized to promote lymphopoiesis for immune cell therapy. (C) 2011 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.

B lymphopoiesis arrests in rabbits by 4 months of age. To identify molecules that contribute to this arrest, cDNA-representational difference analysis on BM stromal cells from young and adult rabbits showed that expression of Postn that encodes for the extracellular matrix protein periostin dramatically reduced with age. Postn-small interfering RNA OP9 cells lost their capacity to support B-cell development from rabbit or murine BM cells, and reexpression of periostin restored this potential, indicating an in vitro requirement for periostin in B lymphopoiesis. In our system, we determined that periostin deficiency leads to increased cell death and decreased proliferation of B-lineage progenitors. Further, RGD peptide inhibition of periostin/α(v)β(3) interaction resulted in a marked decrease in B lymphopoiesis in vitro. Microarray analysis of the Postn-small interfering RNA OP9 cells showed decreased expression of key B-lymphopoietic factors, including IL-7 and CXCL12. In vivo, unidentified molecule(s) probably compensate periostin loss because Postn(-/-) mice had normal numbers of B-cell progenitors in BM. We conclude that the decline in periostin expression in adult rabbit BM does not solely explain the arrest of B lymphopoiesis. However, the interaction of periostin with α(v)β(3) on lymphoid progenitors probably provides both proliferative and survival signals for cells in the B-cell development pathway.

The forkhead box n1 (Foxn1) transcription factor is essential for thymic organogenesis during embryonic development; however, a functional role of Foxn1 in the postnatal thymus is less well understood. We developed Foxn1 transgenic mice (Foxn1Tg), in which overexpression of Foxn1 is driven by the human keratin-14 promoter. Expression of the Foxn1 transgene increased the endogenous Foxn1 levels. In aged mice, overexpression of Foxn1 in the thymus attenuated the decline in thymocyte numbers, prevented the decline in frequency of early thymic progenitors, and generated a higher number of signal joint TCR excised circle. Histologic studies revealed that structural alterations associated with thymic involution were diminished in aged Foxn1 Tg. Total numbers of EpCAM+ MHC II+ and MHC II(hi) thymic epithelial cells were higher in young and old Foxn1Tg and more EpCAM+ MHC II(hi) TEC expressed Ki-67 in aged Foxn1Tg compared with WT. Furthermore, Foxn1Tg displayed a significant reduction in the expansion of splenic CD4+ memory compartments and attenuated the decline in CD4+ and CD8+ naive compartments. Our data indicate that manipulation of Foxn1 expression in the thymus ameliorates thymopoiesis in aged mice and offer a strategy to combat the age-associated decline in naive T-cell production and CD4 naive/memory ratios in the elderly.

Abstract The San Antonio Nathan Shock Center Conferences have attracted international speakers and participants since 1995. This annual conference, held in Bandera, Texas, USA, addresses a different topic in the biology of aging each year. The venue's intimate setting, relatively remote location, and common areas are ideal for a small conference (80–100 participants), where copious informal intellectual interchange supplements that of the formal sessions. The 2011 meeting, part of an annual series sponsored by the University of Texas Health Science Center San Antonio, TX, USA, and the Nathan Shock Center of Excellence in the Biology of Aging, addressed the causes of age-associated inflammation and its effect on age-associated diseases.

Successful cord blood (CB) transplantation in adults is hampered by low numbers of hematopoietic stem cells (HSC) and infection due to delayed establishment of immunocompetence. By performing CDR3 spectratype analysis of T and B lymphocytes from CB transplant recipients, we demonstrated that these patients do not develop a diverse repertoire until 6 months or more after transplant. To provide rapidly engrafting antigen-responsive cells to CB recipients and to increase the number of transplanted cells, we developed an in vitro system to expand lymphoid progenitors from CB HSC. CB HSC (CD34+CD45RA+CD38-) are co-cultured for one to two weeks with either HEK 293T cells or a non-tumorigenic human thymic epithelial cell line (TEC) in media supplemented with a cocktail of cytokines and growth factors. In this study, we focused on B-lineage cells (CD79a+) and found, on average, B-lineage cells expanded more than 70 fold in HEK 293T co-cultures and nearly 100 fold in TEC co-cultures. We conclude that B-lineage cells can be expanded effectively in vitro; consequently, we are testing whether these cells are functional after engraftment in NOD/SCID/IL2Rγ-/- mice. We hypothesize that compared to patients receiving HSC alone, patients receiving transplants supplemented with in vitro-generated lymphoid progenitors will become immunocompetent sooner. Funding by NIH AI068390 (KLK) and AG023809 (PTL)

The thymus is the primary site for thymopoiesis and production of naïve T cells; however, thymic function declines with age. Thymopoiesis requires continuous thymic seeding by early T cell progenitors (ETP), a progeny of the bone marrow-derived lineage marker negative, Sca1pos, c-kitpos (LSK) progenitors that express Flt3 (LSK Flt3pos). A decrease in the frequency of ETP, and in the number of LSK Flt3pos with age has been implicated as a mechanism for the decline in thymopoiesis. We have shown that a decrease in thymocyte numbers occurs as early as 3m of age and correlates with a decrease in Foxn1 expression; however, a role for Foxn1 in thymopoiesis remains unknown. Here, we show that thymi of aged Foxn1 transgenic mice (Tg) have a higher number of thymocytes and ETP compared to aged wild type mice. Forced expression of Foxn1 also rescues aged-associated alterations of thymic architecture. Unexpectedly, we found that Foxn1 is over expressed in the bone marrow (BM). While the LSK and LSK Flt3pos are reduced in aged wild type mice, these populations remain unchanged in aged Foxn1Tg. Furthermore, the aged Foxn1 Tg also have a higher number of the lineage marker negative CD90.2pos CD2pos, a population absent in the Foxn1 mutated nude mice. Thus, our data suggest that expression of Foxn1 is critical for functional thymic and BM stromal cell niches required for the survival of T cell progenitors (NIA AG023809).