Abnormalities of cells and extracellular matrix of T/T embryos

Abstract. The dominant mutation T , (Brachyury), of the T/t -complex in the mouse causes severe disorganization in neural tube, notochord, and somites in homozygotes. The use of scanning electron microscopy to investigate the relationships of cells to one another and to the extracellular matrix in the three axial organs and in the head mesenchyme reveals that cells in all areas examined are abnormal in size, shape, and arrangement in T/T embryos. Cells of T/T head mesenchyme and somites are arrayed in flat sheets of broadened cells with fewer cytoplasmic processes than those of normal littermates. The notochord is discontinuous and its surface is exposed rather than covered by a dense matrix as in the normal. Likewise the sheath of the T/T neural tube is less dense than normal. Cell size and shape are very irregular whereas normal neural tube cells are all about the same size. Extracellular matrix in T/T embryos is greatly decreased in all areas.     

Maturation of cytotoxic T cells within sponge matrix allografts

Two parallel events may provide cytotoxic effector cells at the site of allograft challenge: 1) the migration of effector cells to the graft, and 2) the local maturation of effector cells. Various experimental conditions were manipulated to ascertain the relative role of local maturation of precytotoxic cells at the site of alloantigen challenge. A C57BL/6 allogeneic-coated sponge matrix allograft was placed in a BALB/c animal. The entire animal with the sponge was subjected to sublethal irradiation, or alternatively, just the animal was subjected to the same dose of irradiation, and the sponge was shielded. The data demonstrate that by day 5, precytotoxic cells directed against donor alloantigen appears within the sponge matrix allograft. These precytotoxic cells are in themselves x-ray sensitive, but in the absence of irradiation and in the absence of further contributions from the host can develop into mature cytotoxic cells with specificity limited to that of donor alloantigen. The relative role of local maturation vs continued influx of mature cytotoxic cells cannot be determined from these sets of experiments. These data, however, show that there is potential for local development of mature cytotoxic cells in the absence of other host influences.     

Extracellular matrix conditions T cells for adhesion to tissue interstitium

The activation and differentiation of peripheral blood T cells (PBT) are known to correlate with increased surface expression and adhesive capacity of beta(1) integrins, which mediate adhesion to the extracellular matrix (ECM). However, little is known about the regulation of integrin expression, affinity, and avidity on tissue T cells after they are embedded in the interstitial ECM. In this study we show that tissue T cells, freshly isolated from their residence in the interstitial ECM of the intestinal lamina propria, express a distinct subset of functionally active integrins that contribute to enhanced adhesion to purified collagen, fibronectin, and cell-derived ECM when compared with freshly isolated, short term activated, and long term cultured PBT. Furthermore, integrin usage is distinct between circulating and tissue-derived T cells, in that lamina propria T cells prefer to bind to collagen, while PBT lymphoblasts choose fibronectin when presented with a complex, three-dimensional, cell-derived matrix. To identify the extrinsic factors that regulate the conversion from a nonadhesive PBT to highly adhesive tissue T cell, we demonstrate that activation of PBT in the presence of fibronectin or collagen rapidly generates a surface integrin expression profile, an integrin usage pattern, and adhesive capacity mirroring that of a tissue T cell. These results indicate that the tissue ECM microenvironment instructs newly arrived T cells for further interactions with the underlying matrix and thereby imprints them with a signature tissue adhesive phenotype.     

Dual function of the extracellular matrix: stimulatory for cell cycle progression of naive T cells and antiapoptotic for tissue-derived memory T cells

Tissue T cells encounter Ag in a distinct microenvironment, where they are embedded in the interstitial extracellular matrix (ECM). In contrast, while naive T cells are exposed to Ag in the lymph node, immediately after naive T cells are activated they must extravasate into the ECM to function effectively. Because integrin-mediated adhesion to the ECM modulates cell cycle progression and survival in adherent nonimmune cells, we hypothesize that blood and tissue-derived T cells have similarly adapted their behavior to their first or continued encounter with ECM. T cells from peripheral blood (PBT) and tissue (the intestinal lamina propria T cell (LPT)) were stimulated with anti-CD3-coated beads in the presence or absence of native ECM derived from intestinal fibroblasts, plate-immobilized fibronectin, or collagen type I. Native ECM and collagen, but not fibronectin, induced in anti-CD3 activated PBT a 4- to 5-fold increase in the entry, progression, and completion of the cell cycle over that triggered by anti-CD3 alone. Neutralizing beta1 integrin Abs abrogated this increase. None of these ECM proteins stimulated cell cycle progression in LPT. In contrast, anti-CD3 activation of LPT in the presence of native ECM and fibronectin reduced activation-induced cell death by 40%. These results demonstrate that naive and effector/memory T cells respond differently upon exposure to specific ECM components. When naive PBT encounter Ag in the context of ECM, their progression through the cell cycle is enhanced, favoring clonal expansion; while tissue T cell longevity may be mediated by interactions with the ECM.     

Extracellular matrix proteins of human epidermal keratinocytes and feeder 3T3 cells

Cultures of human epidermal keratinocytes obtained from adult epidermis were initiated using irradiated BALB/3T3 cells as feeder layers. At different stages of confluence of the epidermal islands, feeder cells were removed and the extracellular matrix proteins of both pure component cells and cocultures were analyzed biochemically and by immunochemical methods and compared to those of skin fibroblasts of the same donors. The keratinocytes synthesized and secreted fibronectin and small amounts of laminin and type IV collagen. In addition, a nondisulfide-linked collagenous polypeptide (Mr = 120,000) was synthesized by the keratinocytes and was confined to the cell layers. Collagenous polypeptides with Mr = 120,000 were also synthesized by organ cultures of epidermal tissue and were detected in its acid or detergent extracts but again no secretion to culture medium was found. The Mr = 120,000 collagen had biochemical and immunological properties distinct from those of types I-V collagens. In immunofluorescence of keratinocyte cultures, fibronectin staining was prominent in the lining marginal cells of the expanding periphery of the epidermal cell islands but was not detected in the terminally differentiating cells in the upper layers of stratified colonies. Very little type IV collagen was found deposited in pericellular matrix form by the keratinocytes. In contrast, the mouse 3T3 feeder cells were found to produce both type IV collagen and laminin in addition to the previously identified connective tissue glycoproteins of fibroblasts, interstitial procollagens, and fibronectin. Basement membrane collagen of the 3T3 cells was found deposited as apparently unprocessed procollagen alpha 1(IV) and alpha 2(IV) chains. The production in culture conditions of basal lamina glycoproteins by the fibroblastic feeder cells may promote the attachment and growth of the cocultured keratinocytes.     

Activated T cells complicate the identification of regulatory T cells in rheumatoid arthritis

Most cell surface markers for CD4⁺CD25⁺ regulatory T cells (Tregs) are also expressed by activated non-regulatory T cells. Recently, CD127 down-regulation was found to identify functional Tregs in healthy individuals, but there are no data from patients with inflammatory conditions. We examined peripheral blood mononuclear cells (PBMC) from rheumatoid arthritis patients with active inflammation and from healthy controls, and found that CD4⁺ T cells contained an equal proportion of CD25⁺CD127⁻/low cells in both groups. In patients, not all these cells expressed intracellular FOXP3. Upon activation by anti-CD3/anti-CD28, PBMC rapidly down-regulated CD127, while FOXP3 up-regulation was transitory and occurred in fewer cells. The activated cells were not anergic to restimulation and had no suppressive effects. The distinct kinetics indicate that the FOXP3⁻CD127⁻/low cells in rheumatoid arthritis patients most likely represent activated non-regulatory T cells. This complicates the use of CD127 for identification of Tregs in inflammatory diseases.     

Phosphatidylinositol 3-kinase/Akt activation by integrin-tumor matrix interaction suppresses Fas-mediated apoptosis in T cells

It has recently become apparent that the microenvironment made up of the extracellular matrix may affect cell signaling. In this study, we evaluated Fas-triggered apoptosis in T cells in contact with tumor cells, which resembles the cell-to-cell interactions found in tumor regions. Jurkat cells were less susceptible to the Fas-mediated apoptosis when cocultured with U118, HeLa, A549, and Huh-7 tumor cells. This was indicated by less plasma membrane alteration, an amelioration of the loss of mitochondria membrane potential, a decrease in caspase-8 and caspase-3 activation, a decrease in DNA fragmentation factor-45/35 cleavage, and a reduction in the breakage of DNA when compared with Jurkat cells cultured alone. In contrast, the tumor cell lines MCF-7 and HepG2 produced no such protective effect. This protective event was independent of the expression of Fas ligand on the tumor cells. Interrupting the beta integrins-matrix interaction diminished the coculture effect. In Jurkat cells, cell matrix contact reduced the assembly of the Fas death-inducing signaling complex and Bcl-x(L) cleavage, but enhanced the phosphorylation of ERK1/2, p38 MAPK, and Akt. Only PI3K inhibitor, but not kinase inhibitors for MEK, ERK1/2, p38 MAPK, JNK, protein kinase C, and protein kinase A, completely abolished this tumor cell contact-associated protection and in parallel restored Fas-induced Bcl-x(L) cleavage as well as decreasing the phosphorylation of Bad at serine 136. Together, our results indicate that stimulation of the beta integrin signal of T cells by contact with tumor cells may trigger a novel protective signaling through the PI3K/Akt pathway of T cells against Fas-mediated apoptosis.     

Regulatory T cells interfere with glutathione metabolism in dendritic cells and T cells

Naturally occurring CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) suppress proliferation of CD4(+)CD25(-) effector T cells (Teffs) by mechanisms that are not well understood. We have previously demonstrated a novel mechanism of Treg suppression, i.e. interference with extracellular redox remodeling that occurs during activation of T cells by dendritic cells. In this study, we demonstrate that Treg-mediated redox perturbation is antigen-dependent but not antigen-specific, is CTLA-4-dependent, and requires cell-cell contact. Furthermore, we show that Tregs use multiple strategies for extracellular redox remodeling, including diminished GSH synthesis in dendritic cells via decreased expression of γ-glutamylcysteine synthetase, the limiting enzyme for GSH synthesis. Tregs also consume extracellular cysteine and partition it more proficiently to the oxidation product (sulfate), whereas Teffs divert more of the cysteine pool toward protein and GSH synthesis. Tregs appear to block GSH redistribution from the nucleus to the cytoplasm in Teffs, which is abrogated by the addition of exogenous cysteine. Together, these data provide novel insights into modulation of sulfur-based redox metabolism by Tregs, leading to suppression of T cell activation and proliferation.     

Extracellular matrix production by mouse 3T3-F442A cells during adipose differentiation in culture

When cultured 3T3-F442A cells undergo adipose differentiation, they produce extracellular matrix (ECM) that is not present in undifferentiated cells. This ECM stains strongly with ruthenium red, tannic acid and with Alcian blue at both pH 1 and 2.5, showing histochemical characteristics similar to sulphated and non-sulphated glycosaminoglycans. Under the electron microscope, ECM was observed bound to the cell surface and in the intercellular space; it was composed of fibrils of several thicknesses with attached granules and fibrous long-spacing forms of collagen. In addition, adipocytes were observed as rounded cells interconnected with the ECM fibrils, thus giving rise to fat cell clusters similar to the adipocyte lobules found in adipose tissue. Since fat cell clusters in culture emerge by clonal expansion of one adipose precursor cell, we suggest that this ECM can keep daughter adipocytes interconnected during differentiation. ECM production by adipocytes might have some significance for the formation of fat cell lobules in vivo.     

TNF-alpha associated with extracellular matrix fibronectin provides a stop signal for chemotactically migrating T cells

The migration of T cells into extravascular sites of inflammation is regulated by information derived from the molecular structure of the invaded tissue and from chemokine and cytokine gradients in the context of the extracellular matrix (ECM). Although recent studies have highlighted the role of particular chemoattractants in leukocyte migration, to date little is known about how specific combinations of contextual signals control the migration of leukocytes and their localization at sites of inflammation. Here we studied the interplay between a pleiotropic cytokine, TNF-alpha, and two prototypic chemoattractants, RANTES and stromal cell-derived factor-1alpha (SDF-1alpha), on human CD45RO+ T cells migrating within an ECM-like context. For this purpose, we used a newly constructed three-dimensional gel system designed to follow, in real time, the migration of individual leukocytes along chemotactic gradients in vitro. We found that TNF-alpha, which binds the ECM protein fibronectin and lacks adhesion- and migration-promoting effects of its own, can act as a proadhesive cytokine on T cells exposed to RANTES and SDF-1alpha. Furthermore, fibronectin-complexed TNF-alpha provided anchorage signals to the T cells as they moved directionally along chemoattractive gradients. This effect of TNF-alpha required an intact TNF-alpha receptor II subtype on the migrating T cells. The anchoring effect of TNF-alpha appears to be specific; IL-2, an integrin-activating proadhesive cytokine, does not transmit stoppage signals to T cell migration induced by RANTES. Thus, TNF-alpha present in the ECM at sites of inflammation may function to anchor T cells recruited to these sites by chemotactic signals.     

Intrinsic mechanical properties of the extracellular matrix affect the behavior of pre-osteoblastic MC3T3-E1 cells

Mechanical cues present in the ECM have been hypothesized to provide instructive signals that dictate cell behavior. We probed this hypothesis in osteoblastic cells by culturing MC3T3-E1 cells on the surface of type I collagen-modified hydrogels with tunable mechanical properties and assessed their proliferation, migration, and differentiation. On gels functionalized with a low type I collagen density, MC3T3-E1 cells cultured on polystyrene proliferated twice as fast as those cultured on the softest substrate. Quantitative time-lapse video microscopic analysis revealed random motility speeds were significantly retarded on the softest substrate (0.25 ± 0.01 µm/min), in contrast to maximum speeds on polystyrene substrates (0.42 ± 0.04 µm/min). On gels functionalized with a high type I collagen density, migration speed exhibited a biphasic dependence on ECM compliance, with maximum speeds (0.34 ± 0.02 µm/min) observed on gels of intermediate stiffness, whereas minimum speeds (0.24 ± 0.03 µm/min) occurred on both the softest and most rigid (i.e., polystyrene) substrates. Immature focal contacts and a poorly organized actin cytoskeleton were observed in cells cultured on the softest substrates, whereas those on more rigid substrates assembled mature focal adhesions and robust actin stress fibers. In parallel, focal adhesion kinase (FAK) activity (assessed by detecting pY397-FAK) was influenced by compliance, with maximal activity occurring in cells cultured on polystyrene. Finally, mineral deposition by the MC3T3-E1 cells was also affected by ECM compliance, leading to the conclusion that altering ECM mechanical properties may influence a variety of MC3T3-E1 cell functions, and perhaps ultimately, their differentiated phenotype. bone; focal adhesion kinase; mechanotransduction; cytoskeleton; integrins     

Association of simian virus 40 T antigen with the nuclear matrix of infected and transformed monkey cells.

The subnuclear distribution of simian virus 40 large T antigen within nuclei of transformed Cos and C6 monkey cells was examined. Cos cells express wild-type T antigen but lack viral sequences required for DNA replication, whereas C6 cells contain a functional viral origin but express a replication-defective mutant T antigen which is unable to bind specifically to viral DNA. Discrete subpopulations of T antigen were isolated from the soluble nucleoplasm, chromatin, and nuclear matrix of both cell lines. Although only a small quantity (2 to 12%) of the total nuclear T antigen from Cos cells was associated with the nuclear matrix, a high proportion (25 to 50%) of C6 T antigen was bound to this structure. Results obtained from lytically infected monkey cells showed that early in infection, before viral replication was initiated, a higher proportion (22%) of T antigen was found associated with the nuclear matrix compared with amounts found associated with this structure later in infection (5 to 8%). These results suggest that an increased association of T antigen with this structure is not correlated with viral replication. T antigen isolated from the C6 nuclear matrix was more highly phosphorylated than was soluble C6 T antigen and was capable of binding to the host p53 protein. C6 DNA contains three mutations: two corresponding to N-terminal changes at amino acid positions 30 and 51 and a third located internally at amino acid position 153. By analysis of the subnuclear distribution of T antigen from rat cells transformed by C6 submutant T antigens, it was determined that one or both of the mutations at the NH2 terminus are responsible for the increased quantity of C6 T antigen associated with the nuclear matrix. These results suggest that neither a functional viral DNA replication origin nor the origin binding property of T antigen is required for association of this protein with the nuclear matrix.     

Inflammasomes in T cells

The concept of non-self recognition through germ-line encoded pattern recognition receptors (PRRs) has been well-established for professional innate immune cells. However, there is growing evidence that also T cells employ PRRs and associated effector functions in response to certain non-self or damage signals. Inflammasomes constitute a special subgroup of PRRs that is hardwired to a signaling cascade that culminates in the activation of caspase-1. Active caspase-1 processes pro-inflammatory cytokines of the IL-1 family and also triggers a lytic programmed cell death pathway known as pyroptosis. An increasing body of literature suggests that inflammasomes are also functional in T cells. On the one hand, conventional inflammasome signaling cascades have been described that operate similarly to pathways characterized in innate immune cells. On the other hand, unconventional functions have been suggested, in which certain inflammasome components play a role in unrelated processes, such as cell fate decisions and functions of T helper cells. In this review, we discuss our current knowledge on inflammasome functions in T cells and the biological implications of these findings for health and disease.     

A comparative study of the calcium system in memory T cells and naive T cells

The comparative analysis of responses of memory and naive T lymphocytes to Ca2+-mobilizing agents, namely Con A, thimerosal, thapsigargin and ionomycin, was carried out. The effect of these agents on both types of T cells differed qualitatively and quantitatively. The lack of intracellular Ca2+ stores in memory T cells was shown. Ca2+-mobilizing agents did not induce influx of Ca2+ in memory T cells from outside and this was the reason for their stability to Ca2+ ionophores. It was also shown that memory T cells were resistant to the 'Ca2+ paradox'.     

Fully functional memory CD8 T cells in the absence of CD4 T cells

The role of CD4 T cells in providing help to CD8 T cells in primary and secondary responses to infection remains controversial. Using recombinant strains of virus and bacteria expressing the same Ag, we determined the requirement for CD4 T cells in endogenous CD8 T cell responses to infection with vesicular stomatitis virus and Listeria monocytogenes (LM). Depletion of CD4 T cells had no effect on the frequency of primary or secondary vesicular stomatitis virus-specific CD8 T cells in either lymphoid or nonlymphoid tissues. In contrast, the primary LM-specific CD8 T cell response was CD4 T cell dependent. Surprisingly, the LM-specific CD8 T cell recall response was also CD4 T cell dependent, which correlated with a requirement for CD40/CD40L interactions. However, concomitant inhibition of CD40L and CD4 T cell removal revealed that these pathways may be operating independently. Importantly, despite the absence of CD4 T cells during the recall response or throughout the entire response, CD8 memory T cells were functional effectors and proliferated equivalently to their "helped" counterparts. These data call into question the contention that CD4 T cells condition memory CD8 T cells during the primary response and indicate that the principal role of CD4 T cells in generating CD8 memory cells after infection is augmentation of proliferation or survival through costimulatory signals.