T cell accumulation in B cell follicles is regulated by dendritic cells and is independent of B cell activation

We investigated the mechanism of CD4 T cell accumulation in B cell follicles after immunization. Follicular T cell numbers were correlated with the number of B cells, indicating B cell control of the niche that T cells occupy. Despite this, we found no role for B cells in the follicular migration of T cells. Instead, T cells are induced to migrate into B cell follicles entirely as a result of interaction with dendritic cells (DCs). Migration relies on CD40-dependent maturation of DCs, as it did not occur in CD40-deficient mice but was reconstituted with CD40(+) DCs. Restoration was not achieved by the activation of DCs with bacterial activators (e.g., lipopolysaccharide, CpG), but was by the injection of OX40L-huIgG1 fusion protein. Crucially, the up-regulation of OX40L (on antigen-presenting cells) and CXCR-5 (on T cells) are CD40-dependent events and we show that T cells do not migrate to follicles in immunized OX40-deficient mice.     

Actinic keratosis is squamous cell carcinoma

BACKGROUND: The prevalence of nonmelanoma skin cancer in the United States is alarming. It can be most appropriately treated if the earliest manifestation of cutaneous squamous cell carcinoma is recognized. METHODS: Clinical, histologic, and molecular biology, considerations were reviewed to determine whether actinic keratosis is the earliest clinical manifestation of cutaneous squamous cell carcinoma. RESULTS: The clinical, histologic, and molecular parameters of actinic keratosis are those of squamous cell carcinoma. CONCLUSION: Actinic keratosis does not transform, convert, or progress into cutaneous squamous cell carcinoma but is the earliest clinically recognizable manifestation of this malignancy.     

The B cell antigen CD75 is a cell surface sialytransferase

In this work we have isolated a cDNA clone encoding the B cell antigen CD75. The amino acid sequence of CD75 is shown to be identical to that of human alpha 2,6 sialyltransferase, believed to be primarily associated with the Golgi complex. This is the first demonstration of cell surface expression of sialytransferase which, in B cells, may play an important role in intercellular adhesion and antigen presentation events.     

Caspase activation is required for T cell proliferation

Triggering of Fas (CD95) by its ligand (FasL) rapidly induces cell death via recruitment of the adaptor protein Fas-associated death domain (FADD), resulting in activation of a caspase cascade. It was thus surprising that T lymphocytes deficient in FADD were reported recently to be not only resistant to FasL-mediated apoptosis, but also defective in their proliferative capacity. This finding suggested potentially dual roles of cell growth and death for Fas and possibly other death receptors. We report here that CD3-induced proliferation and interleukin 2 production by human T cells are blocked by inhibitors of caspase activity. This is paralleled by rapid cleavage of caspase-8 after CD3 stimulation, but no detectable processing of caspase-3 during the same interval. The caspase contribution to T cell activation may occur via TCR-mediated upregulation of FasL, as Fas-Fc blocked T cell proliferation, whereas soluble FasL augmented CD3-induced proliferation. These findings extend the role of death receptors to the promotion of T cell growth in a caspase-dependent manner.     

Beta cell replication is the primary mechanism for maintaining postnatal beta cell mass

The endocrine pancreas undergoes major remodeling during neonatal development when replication of differentiated beta cells is the major mechanism by which beta cell mass is regulated. The molecular mechanisms that govern the replication of terminally differentiated beta cells are unclear. We show that during neonatal development, cyclin D2 expression in the endocrine pancreas coincides with the replication of endocrine cells and a massive increase in islet mass. Using cyclin D2-/- mice, we demonstrate that cyclin D2 is required for the replication of endocrine cells but is expendable for exocrine and ductal cell replication. As a result, 14-day-old cyclin D2-/- mice display dramatically smaller islets and a 4-fold reduction in beta cell mass in comparison to their WT littermates. Consistent with these morphological findings, the cyclin D2-/- mice are glucose intolerant. These results suggest that cyclin D2 plays a key role in regulating the transition of beta cells from quiescence to replication and may provide a target for the development of therapeutic strategies to induce expansion and/or regeneration of beta cells.     

Activation and growth of colony-stimulating factor-dependent cell lines is cell cycle stage dependent

Hematopoietic cell development is regulated by a series of growth factors that are progressively restricted in their biological activity. IL-3 is a multi-lineage growth factor that supports the growth and differentiation of progenitor cells belonging to multiple lineages. However, the mechanism by which IL-3 induces proliferation and differentiation of these cells is not completely understood. In this report, we have used two IL-3-dependent cell lines, FDC-P1 (a myeloid progenitor) and F15.12 (a lymphoid progenitor) to investigate IL-3-mediated growth and differentiation. When either FDC-P1 or FL5.12 cells are deprived of IL-3, greater than 90% of all cells accumulate in the G0 phase of the cell cycle. Upon readdition of IL-3, the cells will reenter the active phases of the cell cycle. Therefore, IL-3 can act as both a competence (G0----G1) factor, and a progression (G1----M) factor for hematopoietic precursor clones. FDC-P1 cells can also proliferate in response to granulocyte/macrophage colony-stimulating factor (G/M-CSF) and IL-4 (B cell stimulatory factor 1 [BSF-1]). However, resting (G0) FDC-P1 cells have lost their ability to grow in response to both G/M-CSF and IL-4, even though both factors can induce a G0----G1 transition. Therefore, G/M-CSF or IL-4 behave as progression factors among certain IL-3-responsive clones, and in those cases only in defined points in the cell cycle. Both IL-4 and G/M-CSF can maintain long-term growth of FDC-P1 cells. Upon removal of factor for 24 h, these clones accumulate in the G1 phase of the cell cycle and do not appear to enter G0 even after 36 h of factor deprivation. Therefore, cells maintained in G/M-CSF or IL-4 have altered growth requirements compared with the IL-3-dependent lines from which they were derived. The ability of various hematopoietic growth factors to regulate cell cycle progression in IL-3-dependent cell lines is dependent not only upon the lineage from which these cells were derived, but also the phase of the cell cycle in which those cells reside. The consequences of these interactions dictate the manner by which various clones will respond to CSFs and whether the cells will grow and/or differentiate.     

P53 mutation in acute T cell lymphoblastic leukemia is of somatic origin and is stable during establishment of T cell acute lymphoblastic leukemia cell lines.

Samples donated by patients with T cell acute lymphoblastic leukemia (T-ALL) were screened for mutations of the p53 tumor suppressor gene. Peripheral blood cells of T-ALL relapse patient H.A. were found to possess a heterozygous point mutation at codon 175 of the p53 gene. To determine whether this was an inherited mutation, a B cell line (HABL) was established. Leukemic T cell lines (HATL) were concurrently established by growing peripheral blood leukemic T cells at low oxygen tension in medium supplemented with IGF-I. Previously we had shown that > 60% of leukemic T cell lines possessed mutations in the p53 gene (Cheng, J., and M. Hass. 1990. Mol. Cell. Biol. 10:5502), mutations that might have originated with the donor's leukemic cells, or might have been induced during establishment of the cell lines. To answer whether establishment of the HATL lines was associated with the induction of p53 mutations, cDNAs of the HATL and HABL lines were sequenced. The HATL lines retained the same heterozygous p53 mutation that was present in the patient's leukemic cells. The HABL line lacked p53 mutations. Immunoprecipitation with specific anti-p53 antibodies showed that HATL cells produced p53 proteins of mutant and wild type immunophenotype, while the HABL line synthesized only wild-type p53 protein. The HATL cells had an abnormal karyotype, while the HABL cells possessed a normal diploid karyotype. These experiments suggest that (a) p53 mutation occurred in the leukemic cells of relapse T-ALL patient HA; (b) the mutation was of somatic rather than hereditary origin; (c) the mutation was leukemia associated; and (d) establishment of human leukemia cell lines needs not be associated with in vitro induction of p53 mutations. It may be significant that patient HA belonged to a category of relapse T-ALL patients in whom a second remission could not be induced.     

MerTK is required for apoptotic cell-induced T cell tolerance

Self-antigens expressed by apoptotic cells (ACs) may become targets for autoimmunity. Tolerance to these antigens is partly established by an ill-defined capacity of ACs to inhibit antigen-presenting cells such as dendritic cells (DCs). We present evidence that the receptor tyrosine kinase Mer (MerTK) has a key role in mediating AC-induced inhibition of DC activation/maturation. Pretreatment of DCs prepared from nonobese diabetic (NOD) mice with AC blocked secretion of proinflammatory cytokines, up-regulation of costimulatory molecule expression, and T cell activation. The effect of ACs on DCs was dependent on Gas6, which is a MerTK ligand. NOD DCs lacking MerTK expression (NOD.MerTK(KD/KD)) were resistant to AC-induced inhibition. Notably, autoimmune diabetes was exacerbated in NOD.MerTK(KD/KD) versus NOD mice expressing the transgenic BDC T cell receptor. In addition, beta cell-specific CD4(+) T cells adoptively transferred into NOD.MerTK(KD/KD) mice in which beta cell apoptosis was induced with streptozotocin exhibited increased expansion and differentiation into type 1 T cell effectors. In both models, the lack of MerTK expression was associated with an increased frequency of activated pancreatic CD11c(+)CD8alpha(+) DCs, which exhibited an enhanced T cell stimulatory capacity. These findings demonstrate that MerTK plays a critical role in regulating self-tolerance mediated between ACs, DCs, and T cells.     

Antigen-specific immunosuppression in visceral leishmaniasis is cell mediated.

Visceral leishmaniasis is associated with an antigen-specific immunosuppression during the acute disease. Patients become responsive to Leishmania antigen in both in vivo and in vitro assays after successful antimony therapy. The cell type involved in the suppression of lymphocyte reactivity to Leishmania antigen was studied by selective depletion of mononuclear cell (MNC) populations and in co-cultivation experiments. Adherent cells were depleted on plastic and by passage on nylon wool columns. High-avidity Fc+ cells were depleted by adherence to BSA-anti-BSA complexes and OKT4+ and OKT8+ cells were depleted by treatment with monoclonal antibody (anti-OKT4+ and OKT8+) and complement. Depletion of MNC preparations of adherent cells, high-avidity Fc+ cells, OKT4+ cells and OKT8+ cells failed to restore the lymphocyte reactivity to Leishmania antigen. Antimony therapy was associated with restoration of the proliferative responses of unseparated MNC (before treatment 460 +/- 76 cpm and after treatment 4,293 +/- 1,442 cpm). Co-culture of frozen cells obtained before chemotherapy with autologous MNC obtained after treatment reduced the response of posttreatment cells to Leishmania antigen by 80%. We conclude that the antigenic specific suppression of lymphocyte proliferation in visceral leishmaniasis is cell mediated.     

Splenic T zone development is B cell dependent.

The factors regulating growth and patterning of the spleen are poorly defined. We demonstrate here that spleens from B cell-deficient mice have 10-fold reduced expression of the T zone chemokine, CCL21, a threefold reduction in T cell and dendritic cell (DC) numbers, and reduced expression of the T zone stromal marker, gp38. Using cell transfer and receptor blocking approaches, we provide evidence that B cells play a critical role in the early postnatal development of the splenic T zone. This process involves B cell expression of lymphotoxin (LT)alpha1beta2, a cytokine that is required for expression of CCL21 and gp38. Introduction of a B cell specific LTalpha transgene on to the LTalpha-deficient background restored splenic CCL21 and gp38 expression, DC numbers, and T zone size. This work also demonstrates that the role of B cells in T zone development is distinct from the effect of B cells on splenic T cell numbers, which does not require LTalpha1beta2. Therefore, B cells influence spleen T zone development by providing: (a) signals that promote T cell accumulation, and: (b) signals, including LTalpha1beta2, that promote stromal cell development and DC accumulation. Defects in these parameters may contribute to the immune defects associated with B cell deficiency in mice and humans.     

Urothelial cell senescence is not linked with telomere shortening

The success of regenerative medicine relies in part on the quality of the cells implanted. Cell cultures from cells isolated from bladder washes have been successfully established, but molecular changes and cell characteristics have not been explored in detail. In this work, we analysed the role of telomere shortening in relation to the regenerative potential and senescence of cells isolated from bladder washes and expanded in culture. We also analysed whether bladder washes would be a potential source for attaining stem cells or promoting stem cell proliferation by using two different substrates to support their growth: a feeder layer of growth‐arrested murine fibroblasts J2 3T3 cells and a xeno‐free human recombinant laminin‐coated surface. We found no association between telomere shortening and senescence in urothelial cells in vitro. Urothelial cells had a stable telomere length and expressed mesenchymal stem cells markers but failed to differentiate into bone or adipocytes. Feeder layer showed an advantage to laminin‐coated surfaces in respect to proliferative capacity with the expense of risking that feeder layer cells could persist in later passages. This emphasizes the importance of using carefully controlled culture conditions and molecular quality controls before autotransplantation in future clinical settings. In conclusion, urothelial cells isolated by bladder washes show regenerative potential that need further understanding. Senescence in vitro might be due to cellular stress, and if so, further improvements in culture conditions may lead to longer cell life and higher proliferative capacity.     

Human HTm4 is a hematopoietic cell cycle regulator

Proper control of cell cycle progression is critical for the constant self-renewal, differentiation, and homeostasis of the hematopoietic system. Cells of all types share the common cell cycle regulators. The different expression patterns of common regulators, in a broad sense, define cell-type or lineage specificity. However, there remains the possibility of hematopoietic cell cycle regulators tailored to the demands of the hematopoietic system. Here we describe a novel protein, HTm4, which serves as a hematopoietic cell cycle regulator. Our data indicate that HTm4 is expressed in hematopoietic tissues and is tightly regulated during the differentiation of hematopoietic stem cells. It binds to cyclin-dependent kinase-associated (CDK-associated) phosphatase-CDK2 (KAP-CDK2) complexes, and the three proteins demonstrate similar patterns of cellular expression in human lymphoid tissues. HTm4 stimulates the phosphatase activity of KAP, and its C-terminal region is required for binding to KAP-CDK2 complexes and the modulation of KAP activity. Overexpression of HTm4 can cause cell cycle arrest at the G(0)/G(1) phase. Thus, HTm4 is a novel hematopoietic modulator for the G(1)-S cell cycle transition.     

Cytolytic T cell reactivity to Epstein-Barr virus is lost during in vitro T cell expansion

In the setting of allogeneic hematopoietic stem cell transplantation, ex vivo culturing of donor T lymphocytes is a necessary step for processes such as gene modification. Often the aim is to enable control of undesired alloreactivity after in vivo administration of the cultured cells. However, it is not fully understood how T cell reactivity against donor and third-party targets is affected by the ex vivo cell culturing process. We have assessed how the activity of anti-Epstein Barr virus (EBV)-specific T lymphocytes from healthy EBV-seropositive donors is affected by in vitro cell culturing. Peripheral blood mononuclear cells (PBMCs) were expanded in X-VIVO 15 culture medium supplemented with 5% human serum. The cells were stimulated by either OKT3 (10 ng/ml) and interleukin (IL)-2 (500 U/ml) or by using anti-CD3/CD28-coated immunomagnetic beads and IL-2 (100 U/ml). Induction of polyclonal EBV-specific cytotoxic T lymphocyte cultures was attempted by stimulation of the in vitro-expanded cells at different time points during the cell expansion process, with pre-established autologous EBV-transformed lymphoblastoid cell lines (LCLs). While EBV-specific cytotoxic T lymphocytes (CTL) were generated from untreated PBMCs of 5 healthy donors, EBV-specific cytotoxicity was significantly decreased or absent in CTL cultures established from in vitro-expanded PBMCs. Our results indicate that the ex vivo cell expansion process itself significantly reduces the activity and/or the number of EBV-specific T cells. Additional stimulation with CD28 antibodies could not prevent this effect. Because T cell depleted bone marrow or stem cell grafts are known to contribute to the development of post transplant lymphoproliferative disorders, this should be taken into consideration if one considers expanding and administering PBMCs in conjunction with a T cell-depleted stem cell grafts.     

Osteopontin is a hematopoietic stem cell niche component that negatively regulates stem cell pool size

Stem cells reside in a specialized niche that regulates their abundance and fate. Components of the niche have generally been defined in terms of cells and signaling pathways. We define a role for a matrix glycoprotein, osteopontin (OPN), as a constraining factor on hematopoietic stem cells within the bone marrow microenvironment. Osteoblasts that participate in the niche produce varying amounts of OPN in response to stimulation. Using studies that combine OPN-deficient mice and exogenous OPN, we demonstrate that OPN modifies primitive hematopoietic cell number and function in a stem cell-nonautonomous manner. The OPN-null microenvironment was sufficient to increase the number of stem cells associated with increased stromal Jagged1 and Angiopoietin-1 expression and reduced primitive hematopoietic cell apoptosis. The activation of the stem cell microenvironment with parathyroid hormone induced a superphysiologic increase in stem cells in the absence of OPN. Therefore, OPN is a negative regulatory element of the stem cell niche that limits the size of the stem cell pool and may provide a mechanism for restricting excess stem cell expansion under conditions of niche stimulation.     

Limited T cell receptor diversity of HCV-specific T cell responses is associated with CTL escape

Escape mutations are believed to be important contributors to immune evasion by rapidly evolving viruses such as hepatitis C virus (HCV). We show that the majority of HCV-specific cytotoxic T lymphocyte (CTL) responses directed against viral epitopes that escaped immune recognition in HCV-infected chimpanzees displayed a reduced CDR3 amino acid diversity when compared with responses in which no CTL epitope variation was detected during chronic infection or with those associated with protective immunity. Decreased T cell receptor (TCR) CDR3 amino acid diversity in chronic infection could be detected long before the appearance of viral escape mutations in the plasma. In both chronic and resolved infection, identical T cell receptor clonotypes were present in liver and peripheral blood. These findings provide a deeper understanding of the evolution of CTL epitope variations in chronic viral infections and highlight the importance of the generation and maintenance of a diverse TCR repertoire directed against individual epitopes.     

CD48 is a counter-receptor for mouse CD2 and is involved in T cell activation

CD2 is an intercellular adhesion molecule that has been implicated in T cell activation and differentiation both in humans and mice. Although the ligand for human CD2 has been defined as LFA-3, that for murine CD2 has not been identified yet. To identify the ligand for mouse CD2, we generated a chimeric molecule consisting of the extracellular domain of mouse CD2 and human immunoglobulin (Ig)G1 Fc (mCD2Rg). A hamster monoclonal antibody (mAb), HM48-1, was established by screening mAbs that could block the binding of mCD2Rg to T cell lines at the ligand site. The putative mouse CD2 ligand recognized by this mAb was a glycosyl phosphatidylinositol-anchored glycoprotein with an apparent molecular mass of 45 kD, which were shared characteristics with human LFA-3. However, its expression was predominantly restricted to hematopoietic cells, unlike human LFA-3. Protein microsequencing analysis for the NH2-terminal 18 amino acid residues of the affinity-purified HM48-1 antigen revealed that it is almost identical with mouse CD48. This identity was further confirmed by the reactivity of HM48-1 with a soluble recombinant CD48 (sCD48) protein and the molecule recognized by a rat mAb raised against sCD48. A rat anti-CD48 mAb blocked the mCD2Rg binding as well as HM48-1. Moreover, sCD48 also inhibited the mCD2Rg binding to the cellular ligand. Finally, like anti-CD2 mAb, HM48-1 inhibited the phytohemagglutinin response and, when crosslinked, augmented the anti-CD3 response of splenic T cells. These results indicate that CD48 is a ligand for mouse CD2 and is involved in regulating T cell activation.     

The mouse vitronectin receptor is a T cell activation antigen

In this report, we demonstrate that the T cell activation antigen, recognized by monoclonal antibody H9.2B8, is the murine homologue of the vitronectin receptor (VNR) and, thereby, we provide initial evidence that VNR is expressed on lymphoid cells. VNR is expressed on a variety of T cell lines, tumors, and Con A-activated splenocytes, but not resting T cells, and is capable of binding to the extracellular matrix proteins fibronectin, fibrinogen, and vitronectin, via the tripeptide sequence RGD. There was no evidence of novel beta chains pairing with the VNR alpha chain, as has been demonstrated in some human cells. In view of recent studies demonstrating that this molecule functions as an accessory molecule in T cell activation, the VNR may play an important role in mouse T cell functions.