Human splenic sinusoidal lining cells express antigens associated with monocytes, macrophages, endothelial cells, and T lymphocytes

The antigenic and functional properties of splenic sinusoidal lining cells (SLC) have not been studied extensively. Some investigators have suggested that SLC are actively phagocytic, and thus part of the mononuclear phagocyte system. Others dispute this and assign the functions of endothelial cells to the SLC. During studies in situ of phenotypic subpopulations of human splenic macrophages (M phi), we found that SLC share membrane antigens (HLA-DR and OKM5), an enzyme (lysozyme), and histochemical properties (nonspecific esterases) with monocytes and M phi. In addition, we showed that LSC, like endothelial cells, synthesize factor VIII of the clotting system and also bear the receptor for transferrin. Our previous studies found that SLC express the antigens found on helper/inducer (OKT4, Leu-3a,b) and suppressor/cytotoxic (OKT8, Leu-2a) T lymphocyte subsets. We have confirmed these observations, and have shown by means of preincubation with soluble complexes of anti-human IgG-human IgG that the detection of T cell and other antigens on SLC is not due to nonspecific binding of antibodies by Fc receptors. By using techniques designed to isolate and purify splenic M phi, we were able to obtain SLC in suspension and to demonstrate that they retain the antigens detected in situ. Thus, the human splenic SLC expresses a unique combination of antigens, histochemical properties, and cell products in common with monocytes, M phi, and T lymphocytes.     

Specificity, phenotype, and precursor frequency of primary cytolytic T lymphocytes specific for class II major histocompatibility antigens

Most cytolytic T lymphocytes (CTL) recognize class I rather than class II MHC determinants, and relatively little is known about those CTL that do recognize class II MHC determinants. The present study was undertaken to document the specificity, phenotype, and precursor frequency of primary class II allospecific CTL. It was found that class II-allospecific CTL could be consistently generated in vitro from unprimed spleen or thymus populations in the presence of exogenously added helper factors. The class II MHC specificity of both the precursor and CTL effectors activated in primary cultures by Ia-disparate stimulator cells was documented both by blocking experiments with anti-Ia mAb and by the use of L cell transfectants. The mechanism by which primary allospecific CTL effectors lysed their targets appeared to involve direct cell-cell contact, because they failed to lyse bystander target cells. The frequency in unprimed spleen populations of precursor CTL specific for class II alloantigens was examined by limiting dilution analysis and was found to be as high as 1/15,000 splenocytes and approximately 10% of the frequency reported for primary class I allospecific CTL. Finally, the Lyt phenotype of primary class II allospecific CTL precursors and effectors was determined. It was found that anti-class II CTL derive from at least two distinct precursor subpopulations that are either L3T4+Lyt-2- or L3T4-Lyt-2+, and that the Lyt phenotype expressed by the CTL effectors are concordant with that of their precursors. No correlation was found between the I subregion gene products recognized by CTL effectors and the Lyt phenotype they expressed in that both I-A- and I-E-specific CTL were both L3T4+Lyt-2- and L3T4-Lyt-2+.     

Rapamycin-conditioned dendritic cells are poor stimulators of allogeneic CD4+ T cells, but enrich for antigen-specific Foxp3+ T regulatory cells and promote organ transplant tolerance

The ability of dendritic cells (DC) to regulate Ag-specific immune responses via their influence on T regulatory cells (Treg) may be key to their potential as therapeutic tools or targets for the promotion/restoration of tolerance. In this report, we describe the ability of maturation-resistant, rapamycin (RAPA)-conditioned DC, which are markedly impaired in Foxp3(-) T cell allostimulatory capacity, to favor the stimulation of murine alloantigen-specific CD4(+)CD25(+)Foxp3(+) Treg. This was distinct from control DC, especially following CD40 ligation, which potently expanded non-Treg. RAPA-DC-stimulated Treg were superior alloantigen-specific suppressors of T effector responses compared with those stimulated by control DC. Supporting the ability of RAPA to target effector T and B cells, but permit the proliferation and suppressive function of Treg, an infusion of recipient-derived alloantigen-pulsed RAPA-DC followed by a short postoperative course of low-dose RAPA promoted indefinite (>100 day) heart graft survival. This was associated with graft infiltration by CD4(+)Foxp3(+) Treg and the absence of transplant vasculopathy. The adoptive transfer of CD4(+) T cells from animals with long-surviving grafts conferred resistance to rejection. These novel findings demonstrate that, whereas maturation resistance does not impair the capacity of RAPA-DC to modulate Treg, it profoundly impairs their ability to expand T effector cells. A demonstration of this mechanism endorses their potential as tolerance-promoting cellular vaccines.     

Role of class II histocompatibility antigens in Staphylococcus aureus protein A-induced activation of human T lymphocytes

The capacity of peripheral blood monocytes and B lymphocytes to support staphylococcal protein A (SpA)-induced proliferation of autologous and allogeneic T cells, as well as the role of major histocompatibility complex (MHC) class I and II molecules in this activation process, were investigated. Highly purified peripheral T lymphocytes did not proliferate in response to SpA, but their response was reconstituted by both irradiated (or mitomycin C-treated) monocytes and B lymphocytes. The effect of B cells on the SpA-induced T-cell response could not be explained by a contamination of residual accessory cells because long-term continuous B-cell lines restored SpA-induced T-cell DNA synthesis as effectively as did monocytes. Support of SpA responsiveness by B cells could not be accounted for by polyclonal binding of SpA to cell surface immunoglobulins, since the ability of SpA-unreactive and SpA-reactive B cells was comparable. The cells from two human leukemic lines--K562 and Raji--showed the same ability in supporting the pokeweed mitogen-induced T-cell response, but the class II-positive Raji cells were much more effective than class II-negative K562 cells in restoring the T-cell responsiveness to SpA. Monoclonal antibodies specific for monomorphic determinants of MHC class II antigens, as well as their F(ab')2 fragments, consistently inhibited the SpA-induced proliferative response, whereas antibodies specific for MHC class I antigens were without effect. The antibodies specific for class II antigens appeared to act at the level of accessory cell, since pretreatment with these antibodies inhibited the ability of SpA-pulsed monocytes or Raji cells to present SpA to autologous or allogeneic T lymphocytes, respectively. These data indicate that either monocytes or normal and lymphoblastoid B cells can act as accessory cells for the proliferative response of human T cells to soluble SpA and that monomorphic determinants of MHC class II molecules play an important role in this activation process.     

Liver sinusoidal endothelial cells are insufficient to activate T cells

Liver sinusoidal endothelial cells (LSEC) have been reported to express MHC class II, CD80, CD86, and CD11c and effectively stimulate naive T cells. Because dendritic cells (DC) are known to possess these characteristics, we sought to directly compare the phenotype and function of murine LSEC and DC. Nonparenchymal cells from C57BL/6 mice were obtained by collagenase digestion of the liver followed by density gradient centrifugation. From the enriched nonparenchymal cell fraction, LSEC (CD45(-)) were then isolated to 99% purity using immunomagnetic beads. Flow cytometric analysis of LSEC demonstrated high expression of CD31, von Willebrand factor, and FcgammaRs. However, unlike DC, LSEC had low or absent expression of MHC class II, CD86, and CD11c. LSEC demonstrated a high capacity for Ag uptake in vitro and in vivo. Although acetylated low-density lipoprotein uptake has been purported to be a specific function of LSEC, we found DC captured acetylated low-density lipoprotein to a similar extent in vivo. Consistent with their phenotype, LSEC were poor stimulators of allogeneic T cells. Furthermore, in the absence of exogenous costimulation, LSEC induced negligible proliferation of CD4(+) or CD8(+) TCR-transgenic T cells. Thus, contrary to previous reports, our data indicate that LSEC alone are insufficient to activate naive T cells.     

Liver sinusoidal endothelial cells that endocytose allogeneic cells suppress T cells with indirect allospecificity

A portal venous injection of allogeneic donor cells is known to prolong the survival of subsequently transplanted allografts. In this study, we investigated the role of liver sinusoidal endothelial cells (LSECs) in immunosuppressive effects induced by a portal injection of allogeneic cells on T cells with indirect allospecificity. To eliminate the direct CD4+ T cell response, C57BL/6 (B6) MHC class II-deficient C2tatm1Ccum (C2D) mice were used as donors. After portal injection of irradiated B6 C2D splenocytes into BALB/c mice, the host LSECs that endocytosed the irradiated allogeneic splenocytes showed enhanced expression of MHC class II molecules, CD80, and Fas ligand (FasL). Due to transmigration across the LSECs from BALB/c mice treated with a portal injection of B6 C2D splenocytes, the naive BALB/c CD4+ T cells lost their responsiveness to stimulus of BALB/c splenic APCs that endocytose donor-type B6 C2D alloantigens, while maintaining a normal response to stimulus of BALB/c splenic APCs that endocytose third-party C3H alloantigens. Similar results were not observed for naive BALB/c CD4+ T cells that transmigrated across the LSECs from BALB/c FasL-deficient mice treated with a portal injection of B6 C2D splenocytes. Adaptive transfer of BALB/c LSECs that had endocytosed B6 C2D splenocytes into BALB/c mice via the portal vein prolonged the survival of subsequently transplanted B6 C2D hearts; however, a similar effect was not observed for BALB/c FasL-deficient LSECs. These findings indicate that LSECs that had endocytosed allogeneic splenocytes have immunosuppressive effects on T cells with indirect allospecificity, at least partially via the Fas/FasL pathway.     

Generation of major histocompatibility complex class I antigens

Presentation of antigenic peptides by major histocompatibility complex (MHC) class I molecules on the surface of antigen-presenting cells is an effective extracellular representation of the intracellular antigen content. The intracellular proteasome-dependent proteolytic machinery is required for generating MHC class I-presented peptides. These peptides appear to be derived mainly from newly synthesized defective ribosomal products, ensuring a rapid cytotoxic T lymphocyte-mediated immune response against infectious pathogens. Here we discuss the generation of MHC class I antigens on the basis of the currently understood molecular, biochemical and cellular mechanisms.     

白假丝酵母激活的巨噬细胞自噬促进MHCⅡ抗原提呈

目的 探究白假丝酵母（Candida albicans）激活的Raw264.7细胞自噬在主要组织相容性复合体Ⅱ类分子（MHCⅡ）抗原提呈以及协同刺激分子表达中的作用。方法 C. albicans刺激Dectin-1单克隆抗体封闭或白皮杉醇阻断的Raw264.7细胞,Western blot法检测LC3Ⅱ表达量,RT-PCR检测CD80与CD86的表达。免疫荧光实验观察有无3-MA预处理的GFP-LC3-Raw264.7细胞与C. albicans共孵育后MHCⅡ与LC3在胞浆的分布,ELISA法检测有无封闭Dectin-1或3-MA预处理的Raw264.7细胞与C. albicans共孵育不同时间段后IL-6的分泌。结果 阻断Dectin-1或Sky后C. albicans诱导的LC3Ⅱ表达降低。LC3、MHCⅡ与胞内C. albicans存在显著的共定位关系,阻断自噬后C. albicans与MHCⅡ的共定位明显减弱。C. albicans引发Raw264.7细胞表达CD80与CD86 mRNA,封闭Dectin-1或阻断自噬后二者转录水平降低。C. albicans通过Dectin-1引发Raw264.7细胞分泌IL-6,阻断自噬对IL-6分泌无显著影响。结论 C. albicans通过Dectin-1/Sky通路激活巨噬细胞自噬,自噬体的构建促进MHCⅡ招募至胞内C. albicans,并促进协同刺激分子的表达。     

Phagocytic processing of antigens for presentation by class II major histocompatibility complex molecules

Microbes and other particulate antigens (Ags) are internalized by phagocytosis and then reside in plasma membrane-derived phagosomes. The contribution of phagosomes to the degradation of Ags has long been appreciated. It has been unclear, however, whether peptides derived from these degraded antigens bind class II major histocompatibility complex (MHC-II) molecules within phagosomes or within endocytic compartments that receive Ag fragments from phagosomes. Recent experiments have demonstrated that phagosomes containing Ag- conjugated latex beads express a full complement of Ag-processing molecules, e.g. MHC-II molecules, invariant chain, H2-DM and proteases sufficient to degrade bead- associated Ag. These phagosomes mediate the formation of peptide–MHC-II complexes, which are transported to the cell surface and presented to T cells. Phagosomes acquire both newly synthesized and plasma membrane-derived MHC-II molecules, but the formation of peptide–MHC-II complexes in phagosomes primarily involves newly synthesized MHC-II molecules. The content and traffic of phagosomal proteins vary considerably with the type of Ag ingested. Pathogenic microbes can alter phagosome composition and function to reduce Ag processing. For example, Mycobacterium tuberculosis blocks the maturation of phagosomes and reduces the ability of infected cells to present exogenous soluble protein Ags.     

Cytotoxic T lymphocytes of the rat are predominantly restricted by RTL1.A and Not RT1.C-determined major histocompatibility class I antigens

Two types of biochemically defined class I major histocompatibility complex (MHC) antigens are found in the rat, RTLA antigens that are ubiquitously expressed and RTLC antigens which so far are detectable only on certain cell types, notably B and T lymphocytes. It is shown that the cytotoxic T lymphocyte response to minor H antigens of the LEW strain, including the H-Y antigen, and to TNP-modified syngeneic lymphoid cells is restricted by RTLA but not RTLC gene products. This conclusion is based on bulk culture assays including cold target inhibition tests and limiting dilution experiments using recombinants between the RT1 ^a and RT1 ^u haplotypes. The possibility that class I MHC antigens exist which have no major restriction function is discussed.     

Differential phosphorylation of murine class I major histocompatibility antigens

Recent studies have shown that the H-2K and H-2D transplantation antigens are expressed differentially in different tissues of mouse. Our previous investigations also established that in thioglycolate-stimulated peritoneal macrophages the H-2D^k antigen exists in distinct cell surface and intracellular forms. These two forms are glycosylated differently. In this report, we have found that (1) H-2D^k antigen is phosphorylated whereas H-2K^k antigen is not, and (2) only the cell surface form of H-2D^k antigen is phosphorylated in thioglycolate-stimulated macrophages derived from C3H/Heha mice. This differential phosphorylation of H-2 antigens will provide a model system for further studies on the molecular mechanism and function of phosphrrylation of H-2 antigens.     

Extrathymic selection of TCR gamma delta + T cells by class II major histocompatibility complex molecules

The influence of MHC antigens on TCR gamma delta usage in CD8+ intraepithelial lymphocytes (IELs) was examined using a pan-reactive and V delta 4 region-specific MAb. While an average of 30% of IELs from the majority of mice of various MHC haplotypes were V delta 4+, a 2-fold or greater percentage of IELs from H-2k mice were V delta 4+. Analysis of IELs from F1 mice indicated that the increase in TCRs using V delta 4 was likely to be the result of positive selection. The V delta 4 usage patterns of IELs from recombinant inbred strains and from mice recombinant within H-2 revealed that the increase in V delta 4 usage mapped to H-2 and required I-E expression. Moreover, selection of TCRs using V delta 4 occurred in chimeric mice in the absence of a thymus. The results demonstrate an extrathymic selective mechanism for gamma delta TCRs of CD8+ IELs and suggest that these cells may exhibit MHC class II-restricted antigen recognition.     

Characterization of class II histocompatibility antigens in pigs

The D region of the SLA complex in the pig has been studied by immunochemical and sequential immunoprecipitation techniques as well as the redistribution of membranous antigens (capping). The molecules identified by the anti-la sera were solubilized by NP 40, purified on lectin and precipitated. Polyacrylamide gel electrophoresis under dissociating conditions shows that these molecules are made up of two chains whose molecular weights are 32 000 and 26 000 daltons respectively. Sequential immunoprecipitation and capping experiments indicate that two distinct types of la molecules exist. At least a part of the nylon-wood-adherent lymphocyte population expresses both types of molecules.     

Production and characterization of alloantisera specific for bovine class II major histocompatibility complex antigens

Ten alloantisera defining five major histocompatibility complex (MHC) class II specificities of the bovine lymphocyte antigen (BoLA) complex were produced and characterized. Eight antisera defining four of the specificities were generated by immunizing cattle with class I compatible-class II incompatible lymphocytes. The alloantiserum defining the fifth class II specificity was produced by skin implant immunization. A pregnancy serum specific for one of the class II specificities was also identified. The class II antigens recognized by these antisera were designated 'Dx' antigens to indicate that they are BoLA-D region antigens encoded by one or more undetermined class II loci. The molecules identified by the alloantisera are heterodimers composed of a 34-kd alpha and a 26- to 28-kd beta chain, and are expressed on B-lymphocytes but not on resting T-lymphocytes. In family studies the BoLA-Dx antigens segregated in linkage with the BoLA-A locus alleles. Most of the BoLA-A alleles present in the Cornell Holstein herd at a high frequency were found to exist in gametic association with two or more serologically defined class II haplotypes. On the basis of a population study it was determined that three pairs of class I and class II alleles (w10-Dx4, w31-Dx5, and c3-Dx2) were present in the Cornell herd at significantly increased frequencies.     

Modulation of class I and class II histocompatibility antigens on human T cell lines by IFN-gamma

IFN-gamma is an immunomodulatory agent which is known to induce or enhance the expression of class II histocompatibility Ag (Ia Ag) on many lymphoid cells and cell lines of diverse origin. However, we have observed that IFN-gamma did not induce the expression of Ia Ag on Ia- human T cell lines. Neither did IFN-gamma enhance the expression of Ia Ag on Ia+ T cells. However, IFN-gamma was able to enhance the expression of class I histocompatibility Ag (HLA-A,B,C Ag) on a number of the T cell lines tested. Experiments with 125I-labeled IFN-gamma showed a relatively small degree of specific binding to these T cell lines. More extensive studies on two of the T cell lines demonstrated 1000 and 2600 IFN-gamma binding receptor sites/cell and binding affinities of 4.0 X 10(-10) M and 7.3 X 10(-10) M. Thus, although IFN-gamma can bind to human T cell lines and enhance class I histocompatibility Ag on these cells, IFN-gamma alone does not appear to regulate expression of class II histocompatibility Ag on T cell lines.     

Studies on the bovine major histocompatibility class I and class II antigens using homozygous typing cells and antigen-specific BoT4+ blast cells

Animals were identified from two sire lines as being homozygous for the class I bovine lymphocyte antigen (BoLA-A) w23. These animals were also shown to be homozygous for class II antigens (BoLA-D) which, however, differed between the two sire lines. Lymphocytes from these animals were then used either as stimulator cells in one-way mixed lymphocyte reactions (MLR) with all animals in the herd carrying the w23 antigen or as antigen presenting cells to bovine T4+ cell blasts. It was shown that, within each sire line, the genes encoding the MHC class I and class II antigens were closely linked. There were no detected recombinations between the MHC class I and class II regions nor within the BoLA-D region responsible for mixed lymphocyte reactivity. MLR typing of MHC class II antigens correlated with the results from T-lymphocyte proliferation studies. Cells from these cattle, which are homozygous at the class I and II MHC loci but differ in the class II antigen expressed, could be used to type the BoLA-D of other cattle.