White cell and platelet content affects the release of bioactive factors in different blood-derived scaffolds.

Platelet-derived factors are biomaterials that might accelerate healing process in oral, maxillofacial, and several other applications. Release of specific factors by platelet concentrates is critical to achieving a successful outcome. Here, we have shown that platelet-rich fibrin (PRF) clots were beneficial sources of leukocytes, which may directly affect the release of chemokines and growth factors. When compared with the standard leukocyte-PRF (L-PRF), the experimental low-force modified procedure [defined as advanced-PRF (A-PRF)] entrapped the same content of viable leukocytes, released a similar amount of inflammatory cytokines, but secreted 3-, 1.6-, 3-, and 1.2-fold higher levels of Eotaxin, CCL5, platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF), respectively. A leukocyte-free scaffold, such as plasma rich in growth factors (PRGF), released only platelet-specific factors and, in particular, the F3 fraction, the richest in growth factors, secreted higher amount of CCL5 and PDGF compared to F1 and F2 fractions. In conclusion, different procedures and leukocyte content affect cytokine, chemokines, and growth factor release from platelet derivatives, which may be helpful in different clinical settings.

T follicular helper (Tfh ) cells in normal immune responses and in allergic disorders.

Follicular helper T cells (Tfh ) are located within germinal centers of lymph nodes. Cognate interaction between Tfh , B cells, and IL-21 drives B cells to proliferate and differentiate into plasma cells thereby leading to antibody production. Tfh cells and IL-21 are involved in infectious and autoimmune diseases, immunodeficiencies, vaccination, and cancer. Human peripheral blood CXCR5(+) CD4(+) T cells comprise different subsets of Tfh -like cells. Despite the importance of the IgE response in the pathogenesis of allergic disorders, little is known about the role of follicular and blood Tfh cells and IL-21 in human and experimental allergic disease. Here, we review recent advances regarding the phenotypic and functional characteristics of both follicular and blood Tfh cells and of the IL-21/IL-21R system in the context of allergic disorders.

Elevated plasma levels of vascular permeability factors in C1 inhibitor-deficient hereditary angioedema.

BACKGROUND: Hereditary angioedema with C1 inhibitor deficiency (C1-INH-HAE) is a rare inherited genetic disease characterized by recurrent swelling episodes of the skin, gastrointestinal tract, and upper airways. Angioedema attacks result from increased vascular permeability due to the release of bradykinin from high molecular weight kininogen. Currently, there are no biomarkers predicting the frequency of angioedema attacks. Vascular permeability is modulated by several factors, including vascular endothelial growth factors (VEGFs) and angiopoietins (Angs). As increased circulating levels of VEGFs and Angs have been observed in diseases associated with higher vascular permeability (e.g., systemic capillary leak syndrome and sepsis), we sought to analyze plasma concentrations of VEGFs and Angs in patients with C1-INH-HAE. METHODS: Sixty-eight healthy controls and 128 patients with C1-INH-HAE were studied. Concentrations of angiogenic (VEGF-A, Ang1, Ang2), anti-angiogenic (VEGF-A165b ) and lymphangiogenic (VEGF-C) factors were evaluated by ELISA. C1-INH functional activity was assessed by EIA. RESULTS: Plasma concentrations of VEGF-A, VEGF-C, Ang1, and Ang2 were higher in patients with C1-INH-HAE in remission than in healthy controls. Concentration of VEGF-A was further increased in patients with lower C1-INH functional activity. Patients with C1-INH-HAE experiencing more than 12 angioedema attacks per year were characterized by higher plasma levels of VEGF-A, VEGF-C, and Ang2 compared with the other patients. CONCLUSIONS: We hypothesize that VEGFs and Angs induce a state of 'vascular preconditioning' that may predispose to angioedema attacks. In addition, the identification of increased plasma levels of VEGFs and Angs in patients with C1-INH-HAE may prompt the investigation of VEGFs and Angs as biomarkers of C1-INH-HAE severity.

Low expression level but potent antigen presenting function of CD1d on monocyte lineage cells.

CD1d is a key antigen-presenting molecule involved in the selection and activation of a highly conserved T cell subset known as NK T cells. In this study, we analyzed the expression, regulation and function of human CD1d by various antigen-presenting cells (APC) of myeloid origin, including circulating monocytes, monocyte-derived dendritic cells and macrophages. CD1d was expressed as a mature glycoprotein by these cells, and unlike the other members of the human CD1 family its expression was constitutive and was not strongly up-regulated by GM-CSF and IL-4 or a range of other cytokines. Despite their remarkably low surface expression of CD1d, all myeloid lineage cells tested were extremely potent APC for responses of NK T cell clones to the synthetic glycolipid antigen, alpha-galactosyl ceramide. Prominent localization of CD1d to the endocytic system of monocyte lineage cells was observed, and functional studies suggested that this was important for achieving efficient antigen loading onto CD1d. Overall, these results support the view that monocyte lineage cells are important stimulators of CD1d-restricted immune responses, while also underscoring the unique regulation of CD1d expression by these cells.

Distinct roles for B7 costimulation in contact hypersensitivity and humoral immune responses to epicutaneous antigen.

Productive interactions between B7-1 and B7-2 costimulatory molecules on dendritic cells (DC) and CD28 on T cells are thought to be critical for successful antigen presentation. Epicutaneous application of haptens induces both contact hypersensitivity (CHS), an inflammatory cutaneous response mediated by CD8+ T cells, and an anti-hapten antibody response mediated by CD4+ helper T cells. The role of B7 costimulation in the immune response to oxazolone (Ox) was analyzed using mice lacking either B7-1 (B7-1-/-), B7-2 (B7-2-/-), or both (Db-/-) of these costimulatory molecules. The absence of both B7-1 and B7-2 results in diminished CHS. This inhibition is largely overcome at higher hapten sensitizing doses indicating the presence of compensatory pathways. In contrast, anti-Ox IgG1 and IgG2a responses were not detected in the absence of both B7-1 and B7-2, even at high sensitizing doses, indicating an obligatory role of B7 costimulation in IgG class switching. B7-1 and B7-2 have overlapping functions in both CHS responses and anti-hapten response. B7-2-/- mice demonstrated a modestly reduced CHS response only at very low doses of Ox (0.05%), but responded normally at higher Ox doses, and B7-1-/- mice had CHS responses indistinguishable from those of wild-type mice. Similarly, anti-Ox IgG responses were comparable in wild-type, B7-1-/- and B7-2-/- mice. Taken together, these studies reveal distinct roles for B7 costimulation in response to epicutaneous antigens with an obligatory role for IgG class switching and an important, but nonessential role for CHS responses.

Characterization and localization of Mox2, the gene encoding the murine homolog of the rat MRC OX-2 membrane glycoprotein.

MRC OX-2 is a rat type I membrane glycoprotein and a member of the immunoglobulin gene superfamily that has recently been shown to be able to costimulate murine T cell proliferation (Borriello et al. J. Immunol, 158, 4548, 1997). We now report the genomic organization for the gene encoding the murine homolog of MRC OX-2 (Mox2). The gene is composed of 6 exons and 5 introns spanning a minimum of 13.7 kb. Exon 1 encodes the amino terminal four amino acids and is located in the center of a 500-bp CpG island, suggestive of the presence of a promoter. Analysis of the sequences immediately upstream of exon 1, however, do not reveal a TATA or CAAT box. We also demonstrate that in addition to the canonical transcript, composed of exons 1 through 6, this gene gives rise to an additional nonproductive transcript resulting from the absence of exon 2, which leads to a frameshift and premature translation termination. The ratio of these alternative transcripts is not regulated by mitogenic stimulation with ConA or LPS. The Mox2 gene maps to Chr 16, telomeric to the clustered T cell costimulatory molecules Cd80 and Cd86 (Reeves et al. Genomics in press).

The role of donor and recipient B7-1 (CD80) in allograft rejection.

Blockade of CD28-mediated T cell costimulatory signals produces effective immunosuppression of a variety of T cell-dependent in vivo immune responses, including autoimmune disorders and transplant rejection. The soluble fusion protein CTLA4Ig, which competitively blocks CD28 ligands B7-1 and B7-2, can prevent allograft and xenograft rejection and in some circumstances induce transplantation tolerance. To determine the relative roles of B7-1 and B7-2 in graft rejection, we have performed islet and cardiac allografts with normal and B7-1(-/-) mice in conjunction with selective blocking reagents. We found that the absence of B7-1 on donor or recipient tissues leads to a slight prolongation of islet allograft survival, but has minimal or no effect on cardiac allograft survival. Allograft function is further prolonged in the islet model when both donor and recipient lack B7-1, although cardiac allograft survival is not prolonged. In the cardiac model, treatment with CTLA4Ig induces long term survival in B7-1(-/-) recipients regardless of donor status. In contrast, anti-B7-2 mAb leads to indefinite allograft survival only when the recipient and donor both lack B7-1, indicating that even in the absence of available B7-2, B7-1 molecules on the donor or recipient cells alone are sufficient to induce graft rejection. These data also indicate that B7-1 and B7-2 are the only CD28 ligands relevant to cardiac allograft rejection in mice.

CTLA4Ig prevents lymphoproliferation and fatal multiorgan tissue destruction in CTLA-4-deficient mice.

Mice lacking CTLA-4 develop a fatal spontaneous lymphoproliferative disease with massive lymphocytic infiltrates and tissue destruction in many organs. CTLA-4-deficient (-/-) splenocytes and lymph node cells proliferate without added stimuli in vitro. We report here that CTLA4Ig treatment of CTLA-4 -/- mice prevents lymphoproliferation and fatal multiorgan tissue damage in vivo and proliferation of CTLA-4 -/- splenocytes and lymph node cells in vitro. Therefore, stimulation via CD28-B7 interactions appears necessary for CTLA-4 -/- T cell proliferation and the production of lymphoproliferative disease in vivo. When CTLA4Ig treatment is terminated, CTLA-4 -/- T cells become activated and lymphoproliferative disease develops. The lack of long term protective effects of CTLA4Ig treatment suggests that CTLA-4 is needed for the induction and or maintenance of tolerance.

MRC OX-2 defines a novel T cell costimulatory pathway.

T cell activation requires the engagement of the TCR as well as a second, costimulatory signal. In this study, we demonstrate that MRC OX-2 (OX-2) mediates a previously unrecognized T cell costimulatory signal leading to enhanced T cell proliferation. One extensively studied costimulatory pathway, the B7/CD28 pathway, delivers its signal when CD28 is engaged by either of two ligands, B7-1 or B7-2, expressed on APC. Recent data have suggested that an additional ligand may exist in this pathway. This possibility prompted us to search previously cloned genes with both structural and expression characteristics similar to B7-1 and B7-2. Our search yielded OX-2, a rat lymphocyte activation marker, as a promising candidate gene. We now report that Chinese hamster ovary cell transfectants expressing the OX-2 protein can costimulate murine CD4+ T cells to proliferate in an Ag-independent fashion using anti-CD3, as well as an Ag-dependent fashion using peptide. In contrast to B7-1-mediated costimulation, OX-2 does not result in detectable levels of IL-2, IL-4, or IFN-gamma. In addition, OX-2 transfectants do not bind the soluble receptor reagents of the B7/CD28 pathway (CD28-Ig and CTLA4Ig). Furthermore, OX-2 costimulation is not inhibited by CTLA4Ig, as is B7-1-mediated costimulation, but is readily inhibited with an anti-OX-2 mAb. Thus, OX-2 is a T cell costimulatory ligand that acts through a non-B7/CD28 pathway, which leads to functionally distinct consequences, as reflected by the resulting cytokine profile.

Role of costimulators in T cell differentiation: studies using antigen-presenting cells lacking expression of CD80 or CD86.

For T cells to be optimally activated, recognition of Ag/MHC complexes by the TCR must be accompanied by a second, costimulatory signal that can be provided efficiently by the related costimulatory molecules CD80 (B7-1) and CD86 (B7-2). Recently, CD80 and CD86 have been implicated as differential determinants of Th1- vs Th2-type cytokine profiles. However, this remains a controversial issue since conflicting results have been obtained in different experimental models both in vivo and in vitro. To investigate the role of CD80 and CD86 in Th subset differentiation, we have examined the cytokine profiles induced in TCR transgenic T cells stimulated by peptide in association with splenic APCs obtained from knockout mice that selectively lack expression of either the CD80 or the CD86 molecule. Our data suggest that CD86, and to a lesser extent CD80, can make significant contributions to the production of both IL-4 and IFN-gamma. However, neither molecule plays an obligatory role in priming for the production of either effector cytokine. Furthermore, CD80 and CD86 contribute to the magnitude of T cell activation, but do not appear to selectively regulate Th1 vs Th2 differentiation.

B7-1 and B7-2 have overlapping, critical roles in immunoglobulin class switching and germinal center formation.

Humoral immune responses were characterized in mouse strains lacking either or both B7 molecules. Mice deficient in both B7-1 and B7-2 failed to generate antigen-specific IgG1 and IgG2a responses and lacked germinal centers when immunized by a number of routes and even in the presence of complete Freund's adjuvant. These results demonstrate that B7-mediated signaling plays a critical role in germinal center formation and immunoglobulin class switching in vivo. Mice lacking only B7-1 or B7-2 mounted high-titer antigen-specific IgG responses when immunized in complete Freund's adjuvant, indicating that B7-1 and B7-2 can have overlapping, compensatory functions for IgG responses. When immunized intravenously without adjuvant, B7-2-deficient mice failed to switch antibody isotypes or form germinal centers, whereas B7-1-deficient mice gave antibody responses comparable with wild-type mice. Thus, B7-2 has an important role in initiating antibody responses in the absence of adjuvant, but the induction of B7-1 by adjuvant in B7-2-deficient mice can compensate for the absence of B7-2.

Functional consequences of dysregulated B7-1 (CD80) and B7-2 (CD86) expression in B or T lymphocytes of transgenic mice.

T cell activation and tolerance are regulated by interactions between CD28 or CTLA-4 on T cells and B7 costimulatory molecules on APCs. We have generated transgenic mouse strains that constitutively express B7-1 (CD80) at high levels on B cells or T cells or express B7-2 (CD86) on T lymphocytes to examine the consequences of dysregulated B7 expression on T cell responses. The transgene-derived B7 molecules are functional, because B7-1 transgenic B cells are more efficient APCs than are wild-type B cells, and the activation of B7 transgenic T cells is less dependent on exogenous costimulation than that of wild-type T cells. In vivo, constitutive expression of B7 molecules leads to the elimination of immature B cells. The expression of B7 molecules on thymocytes results in the down-regulation of CD28 expression. However, B7 transgenic mice have normal numbers of mature lymphocytes and mount normal T cell responses following immunization with protein Ag. Neither anergy induction nor superantigen-mediated deletion of T cells is altered by the dysregulated expression of B7-1 or B7-2 on B or T lymphocytes in these transgenic strains. Therefore, functionally significant levels of B7 expressed constitutively on mature lymphocytes are not, by themselves, sufficient to abrogate T cell tolerance or induce autoimmune disease.

Differential expression of alternate mB7-2 transcripts.

The murine B7-2 (mB7-2) costimulatory molecule is expressed on APCs early during the course of an immune response, suggesting that it may play a pivotal role in the decision between T cell activation and anergy. Murine B7-2 mRNA displays a restricted pattern of expression; it is inducible in B cells, T cells, NK cells, and dendritic cells, but constitutively expressed in unstimulated monocytes. The constitutive and inducible expression of mB7-2 on distinct cell types indicates that mB7-2 is regulated differentially. To further characterize mB7-2 transcripts, we employed 5' rapid amplification of cDNA ends and reverse transcriptase-PCR to examine transcripts expressed in a variety of types of APCs and analyzed the genomic organization of the mB7-2 gene. We report here that the mB7-2 locus consists of 12 exons and demonstrate that exons 1 through 5 can be used in alternative fashions to produce five distinct transcripts, differing in their 5' untranslated and signal regions. The expression of these transcripts differs in distinct types of APCs and is modulated by stimuli that activate B cells. These results demonstrate that mB7-2 transcripts are differentially regulated in a tissue-specific fashion and in response to activation stimuli.

Loss of CTLA-4 leads to massive lymphoproliferation and fatal multiorgan tissue destruction, revealing a critical negative regulatory role of CTLA-4.

The B7-CD28/CTLA-4 costimulatory pathway can provide a signal pivotal for T cell activation. Signaling through this pathway is complex due to the presence of two B7 family members, B7-1 and B7-2, and two counterreceptors, CD28 and CTLA-4. Studies with anti-CTLA-4 monoclonal antibodies have suggested both positive and negative roles for CTLA-4 in T cell activation. To elucidate the in vivo function of CTLA-4, we generated CTLA-4-deficient mice. These mice rapidly develop lymphoproliferative disease with multiorgan lymphocytic infiltration and tissue destruction, with particularly severe myocarditis and pancreatitis, and die by 3-4 weeks of age. The phenotype of the CTLA-4-deficient mouse strain is supported by studies that have suggested a negative role for CTLA-4 in T cell activation. The severe phenotype of mice lacking CTLA-4 implies a critical role for CTLA-4 in down-regulating T cell activation and maintaining immunologic homeostasis. In the absence of CTLA-4, peripheral T cells are activated, can spontaneously proliferate, and may mediate lethal tissue injury.

Genomic organization of the gene coding for the costimulatory human B-lymphocyte antigen B7-2 (CD86).

The generation of an antigen-specific T-cell response requires that the T lymphocyte receive two signals from the antigen presenting cell. The specificity of this response is provided by antigen presented to the T lymphocyte and involves stimulation of the T lymphocyte via the T-cell receptor (TCR)/CD3 complex. The second, or costimulatory signal, can be provided by ligation of the B-lymphocyte activation antigens B7-1 (CD80) and B7.2 (CD86) to TCR antigen CD28. The cDNAs for both CD80 and CD86 have been isolated and are predicted to encode type 1 membrane proteins of the immunoglobulin (Ig) superfamily. The predicted protein is composed of a signal peptide followed by two Ig-like extracellular domains, a transmembrane domain, and a cytoplasmic tail. Here we report that the genomic organization of CD86 reflects its functional structure, and is similar to that found for CD80. The gene is composed of eight exons which span more than 22 kilobases. The predicted protein functional domains of signal peptide, extracellular IgV- and IgC-like regions, and transmembrane domain coincide with the genomic structure. Two independent sequences had been reported for CD86 cDNA which differed in their 5'untranslated (UT) regions. We find CD86 exons 1 and 2 correspond to these alternate 5'UT sequences. Splicing of exon 1 or 2 with the signal peptide encoding exon 3 would produce mRNA transcripts complementary to the reported cDNA clones. Exons 4 and 5 correspond to IgV- and IgC-like extracellular domains, respectively. Exon 6 encodes the transmembrane region and beginning of the cytoplasmic tail. Exons 7 and 8 encode the remainder of the cytoplasmic tail and 3'UT sequences.

Reciprocal expression of co-stimulatory molecules, B7-1 and B7-2, on murine T cells following activation.

The co-stimulatory B7 molecules (B7-1 and B7-2) are expressed on professional antigen-presenting cells in mice. In this study, we demonstrate that B7-1 (CD80) and B7-2 (CD86) are also expressed on murine T cells in the absence of major histocompatibility complex class II molecules. The temporal expression of these two molecules on T cells varies with the state of activation where resting T cells express B7-2 but show little or no expression of B7-1. Following activation, B7-2 expression is down-regulated and there is a concomitant increase in the expression of B7-1 on the cell surface which peaks at about 72 h. Thus these two co-stimulatory molecules are reciprocally expressed on the T cell surface. This pattern of expression of B7-1 and B7-2 on T cells suggests that these two molecules may have different roles in the generation and regulation of immune responses.

Characterization of the murine B7-1 genomic locus reveals an additional exon encoding an alternative cytoplasmic domain and a chromosomal location of chromosome 16, band B5.

The murine B7-1 (mB7-1) molecule expressed on APCs delivers a costimulatory signal for T cell activation through its T cell counter-receptor CD28, resulting in T cell proliferation and IL-2 production. Signaling through the TCR in the absence of CD28 signaling results in T cell anergy. We have analyzed the genomic structure of mB7-1 and here describe the identification of a previously unrecognized sixth exon at the far 3' end of the locus, which encodes an alternative cytoplasmic domain. Reverse transcriptase-PCR amplification of mB7-1 transcripts demonstrates that exon 6 is functionally spliced to the transmembrane-encoding exon 4. Furthermore, using 5' rapid amplification of cDNA ends, we determined that the 5'-untranslated region extends over 1505 bp beyond the previously reported transcriptional start site. In addition, we report the chromosomal location of mB7-1 to chromosome 16, band B5.

Evaluation of gene deletions by quantitative polymerase chain reaction. Experience with the alpha-thalassemia model.

In order to evaluate the feasibility of using quantitative polymerase chain reaction (PCR) to evaluate gene dosage, we developed an assay to detect alpha-globin genes, which are frequently deleted in alpha-thalassemia patients. In this quantitative assay alpha-1 and alpha-2 globin consensus regions are coamplified by one oligonucleotide pair, along with a second primer pair targeting a single-copy reference gene, namely, tumor necrosis factor alpha, or TNF-alpha. A series of DNA samples titrating alpha-globin against TNF-alpha DNA have a strong linear relationship between template ratios and product ratios (r > 0.98). Minimal sequence divergence (91% homology) between alpha-1 and alpha-2, internal to the identical primer annealing sites, results in a lower amplification efficiency for alpha-1, to 94% of alpha-2 for each cycle. Furthermore, when applied to a variety of individual DNA samples, the signal ratios of alpha-globin to TNF-alpha were far more variable than previously observed for titrated control DNA. We conclude that DNA isolates from different individuals may have idiosyncratic changes in amplification efficiency owing to polymorphic sequence variation and/or variable presence of unidentified contaminants. Despite these potentially confounding factors, however, we were able to identify by quantitative PCR a single gene deletion later confirmed by Southern blot analysis in 20 individual DNA samples.