Expression In Antigen-presenting Cells Research Articles

Differential regulation of CD36 expression in antigen-presenting cells: Oct-2 dependence in B lymphocytes but not dendritic cells or macrophages.

In mice, three antigen-presenting cell types [B lymphocytes, macrophages and dendritic cells (DC)] express the scavenger receptor CD36. This molecule has been implicated in many important functions, including DC maturation and antigen presentation. In murine B cells, the CD36 gene requires the Oct-2 transcription factor for its expression. We previously found that B cells from Oct-2-null mice display defects in maturation, survival and proliferation. Here we have looked for a possible role for CD36 in B cells, but found that CD36 is dispensable for all responses tested. Although loss of CD36 did not directly affect B cell function, it did modulate slightly the isotype and level of IgG produced in vivo in naive mice, and IgM in Leishmania-infected mice. We also show that in DC and macrophages, CD36 expression is independent of Oct-2. We conclude that CD36 does not play a major role in B cell function, but that CD36 may contribute indirectly to humoral immunity through cells of the innate immune system.

Lentiviral vectors containing the human immunodeficiency virus type-1 central polypurine tract can efficiently transduce nondividing hepatocytes and antigen-presenting cells in vivo

AbstractHigh-titer self-inactivating human immunodeficiency virus type-1 (HIV-1)–based vectors expressing the green fluorescent protein reporter gene that contained the central polypurine and termination tract and the woodchuck hepatitis virus posttranscriptional regulatory element were constructed. Transduction efficiency and biodistribution were determined, following systemic administration of these improved lentiviral vectors. In adult severe combined immunodeficiency (SCID) mice, efficient stable gene transfer was achieved in the liver (8.0% ± 6.0%) and spleen (24% ± 3%). Most transduced hepatocytes and nonhepatocytes were nondividing, thereby obviating the need to induce liver cell proliferation. In vivo gene transfer with this improved lentiviral vector was relatively safe since liver enzyme concentration in the plasma was only moderately and transiently elevated. In addition, nondividing major histocompatibility complex class II–positive splenic antigen-presenting cells (APCs) were efficiently transduced in SCID and normal mice. Furthermore, B cells were efficiently transduced, whereas T cells were refractory to lentiviral transduction in vivo. However, in neonatal recipients, lentiviral transduction was more widespread and included not only hepatocytes and splenic APCs but also cardiomyocytes. The present study suggests potential uses of improved lentiviral vectors for gene therapy of genetic blood disorders resulting from serum protein deficiencies, such as hemophilia, and hepatic disease. However, the use of liver-specific promoters may be warranted to circumvent inadvertent transgene expression in APCs. In addition, these improved lentiviral vectors could potentially be useful for genetic vaccination and treatment of perinatal cardiac disorders.

Designing gene therapy vectors: avoiding immune responses by using tissue-specific promoters.

Attempts to correct genetic disorders by gene therapy have been hindered by various problems including unwanted immune responses against the gene product. It has been shown that immune responses with DNA vaccines after i.m. injection of antigen-encoding plasmid DNA are primed solely by professional antigen-presenting cells (APC), even though myocytes are the primary type of cell transfected. This possibly involves direct transfection of some APC in regional lymph nodes draining the injected muscle. Here we have used plasmid DNA vaccines that express hepatitis B surface antigen (HBsAg) to evaluate the possibility of abrogating these immune responses by use of a tissue-specific promoter that does not drive expression in APC. We show that HBsAg-specific humoral or cell-mediated responses are not induced in mice when the muscle-specific human muscle creatine kinase promoter is used in place of the ubiquitous cytomegaloviral promoter to drive expression of HBsAg. This may have significance in the field of gene therapy where one aims to achieve stable expression of the desired gene product without interference from the host immune response.

Binding Properties of the Mannose Receptor

A comprehensive approach to the study of mannose receptor (MR) biology has unveiled an unexpected level of complexity and stresses the importance of post-translational modifications and gene regulation in the analysis of protein function. The existence of endogenous tissue ligands for the MR highlights the need to reduce MR expression in antigen presenting cells and/or to regulate T cell stimulation after presentation of MR ligands, in order to avoid autoimmunity. This regulation might be achieved by down modulation of the antigen presenting cell stimulatory capacity upon MR ligation. In macrophages there are conflicting evidence regarding the outcome of MR recognition. These results are not unexpected if endogenous mannosylated and sulphated self-antigens, that need to be shielded from the immune system, are being eliminated through this receptor. The presence of counter receptors for the cysteine rich (CR) domain of the MR in specialized myeloid cells in lymphoid organs adds a new dimension to this system. It opens the possibility for a delivery pathway for MR carbohydrate recognition domains (CRDs) ligands that needs to be investigated further.

Sustained expression of human apo A-I following adenoviral gene transfer in mice.

Elevation of HDL cholesterol, following adenoviral apolipoprotein A-I (apo A-I) gene transfer, may delay or revert ischemic cardiovascular disease, provided transgene expression is persistent. The choice of promoter may have significant impact on persistence of transgene expression. Human apo A-I expression was compared after adenoviral gene transfer with a cytomegalovirus promoter (CMV) driven construct (AdCMV/A-I.gA-I) and with a construct (AdA-I.gA-I.4xapoE) containing the endogenous 256 bp apo A-I promoter (A-I), the genomic human apo A-I DNA (gA-I) and 4 human apo E enhancers (4xapoE) in three different mouse strains: C57BL/6, Balb/c and Fvb. After gene transfer with 5 x 10(8) p.f.u. of AdCMV/A-I.gA-I, human apo A-I expression was observed for 35 days in C57BL/6 mice, but declined below 1 mg/dl within 14 days both in Balb/c and Fvb mice, due to a strong humoral immune response against human apo A-I. In contrast, after transfer with AdA-I.gA-I.4xapoE, human apo A-I expression persisted for 6 months in all three strains and no antibodies against human apo A-I occurred in Fvb or Balb/c mice. Human apo A-I transgene DNA level 35 days after transfer with AdA-I.gA-I.4xapoE was 4.6- to 5.5-fold higher than with AdCMV/A-I.gA-I. CMV promoter attenuation occurred in all three strains, but promoter attenuation was not observed in any strain after transfer with AdA-I.gA-I.4xapoE. In conclusion, gene transfer with AdA-I.gA-I.4xapoE is associated with absence of an immune response against human apo A-I, improved transgene DNA persistence and absence of promoter shut-off, resulting in human apo A-I expression for up to 6 months in three different mouse strains. Possibly, the absence of human apo A-I expression in antigen-presenting cells with the liver-specific apo A-I promoter containing construct abrogated the immune response against human apo A-I in Balb/c and Fvb mice.

Specific transgene expression in antigen presenting cells derived from lentivirally transduced hematopoietic stem/progenitor cells

We previously demonstrated that we could achieve sustained transgene expression in dendritic cells (DCs) differentiated in vitro from retrovirally transduced human CD34+ hematopoietic stem/progenitor cells (HSPC). We report here our attempt to specifically express a transgene in DCs and other antigen presenting cells (APC) derived from transduced HSPC. We used self-inactivating lentiviral vectors, in which the GFP reporter expression in transduced cells is solely controlled by the promoter of either human HLA-DRα (MHC II) gene or a housekeeping gene (PGK or EF1α). The three vectors (DR.GFP, PGK.GFP and EF.GFP) were pseudotyped with the VSV-G envelope using 293T cells, and they can transduce a variety of human and murine cells efficiently. The levels of transgene expression by EF.GFP or PGK.GFP (3–8 fold lower) were equivalent in both MHC II+ and MHC II− cells. In contrast, transgene expression mediated by DR.GFP is 10–20 fold lower in human MHC II− cells (K562 and U937) than in MHC II+ cells (TF1). Similarly, the expression by DR.GFP vector was strong inthe murine MHC II+ cells (A20) but reduced drastically in murine MHC II− cells (RENCA and CT26). We also monitored transgene expression in differentiated DC derived from mouse bone marrow (BM) cells in vitro. Fresh BM cells from normal mice were transduced by either DR.GFP or EF.GFP, and then cultured for 8 days to allow DC differentiation. GFP expression was observed exclusively in the MHC II+ cell population derived from DR.GFP transduced BM cells, whereas transgene expression was observed equally in both MHC II+ and MHC II− cell populations derived from EF.GFP transduced BM cells. Thus, we have developed a gene transduction system to allow stable and specific transgene expression in APC, which has many applications for immunobiology and immunotherapy.

IN VITRO ANTIGEN PRESENTING CELL FUNCTION IS DEFICIENT IN STABLE LUNG TRANSPLANT RECIPIENTS.

143 In vitro peripheral blood mononuclear cells (PBMCs) of stable transplant recipients respond to polyclonal activation, but not to soluble protein antigens (ag), which have to be processed and presented by self antigen presenting cells (APCs). The aim of the present study was to assess APC function in stable lung transplant (LT) recipients and to elucidate whether defective response to soluble ag is due to T cell or APC dysfunction. We obtained PBMCs from 29 stable LT recipients with normal pulmonary function a mean of 3 years after transplantation. Patients received a triple CsA-based immunosuppressive regimen. We also obtained PBMCs from 29 healthy volunteers, which served as controls. LT recipients' and control PBMCs were stimulated with a) allogeneic PBMCs, b) anti-CD3 monoclonal antibody (mAb), c) tetanus toxoid (TT) ± anti-CD28 mAb. APC function of patients' PBMCs was tested in a mixed leukocyte reaction (MLR) using patients' PBMCs as stimulators and control PBMCs as responders. The readout for these experiments was IL-2 production in culture supernatants. APC expression of MHC class II, B7.1, B7.2 and CD40 molecules was determined by flow cytometry. Finally, APC production of IL-10 and IL-12 in response to stimulation with CD40-L transfected fibroblasts was assessed. Patients' T cells produced normal amounts of IL-2 in response to allogeneic PBMCs (500 ± 259 vs 397 ± 236 pg/ml in controls; P = NS) and anti-CD3 mAb (2685 ± 1380 vs 1817 ± 1380 pg/ml in controls; P = NS). Response to TT was completely blunted (203 ± 270 vs 604 ± 574 pg/ml in controls; P < 0.0001) and could not be restored by providing costimulation with anti-CD28 mAb. Patients' APCs were poor stimulators in MLR and this was correlated with a drastic downregulation of MHC class II expression (median fluorescence intensity: 275 ± 66 vs 931 ± 163 in controls; P < 0.0001). Patients' APCs also produced significantly less IL-10 and IL-12 than control APCs subsequent to CD40 engagement. In conclusion, stable LT recipients' T cells cannot be activated in vitro by soluble ag such as TT. Deficient ag presentation by dysfunctional APCs probably contributes to this defect.

A Novel Role for the Major Histocompatibility Complex Class II Transactivator CIITA in the Repression of IL-4 Production

Class II transactivator (CIITA) is known as a coactivator for MHC class II gene expression in antigen-presenting cells. Surprisingly, when CIITA−/− CD4 T cells were stimulated in the presence of IL-12, they produced not only IFNγ but also high levels of IL-4. The IL-4 production is due to the accumulation of IL-4 gene transcripts in Th1 cells. This transcriptional control is observed in T cells differentiating to the Th1 but not Th2 lineage, consistent with induction of expression of the CIITA gene in T cells by IFNγ. Thus, in addition to its role in transactivation of genes involved in antigen presentation, CIITA plays a critical role during the T cell differentiation by negatively regulating the IL-4 gene transcription.

Regulation of HLA-DR and costimulatory B7 molecules in human thyroid carcinoma cells: differential binding of transcription factors to the HLA-DRalpha promoter.

The consequence of autoantigen presentation by thyroid cells is dependent on the magnitude of expression of both HLA class II antigens (mainly HLA-DR) and costimulatory molecules, such as B7 (CD80 and CD86). Autoimmune thyrocytes are induced to express HLA-DR by interferon-gamma (IFN-gamma). The costimulatory signal leading to autoantibody production or cytotoxic T-cell immune response could be provided by antigen presenting cells (APCs) attracted to the thyroid by the primary autoimmune stimulus. Malignant thyrocytes can express HLA-DR antigens either constitutively, as a result of a nonimmunologic stimulus, or on induction with IFN-gamma after triggering of an immune response. However, their ability to express B7 molecules, which may determine enhanced antitumoral immune response mainly in the absence of intrathyroidal macrophages, has not yet been studied. The regulation of HLA-DR gene expression in APCs, such as B cells, is mediated by a series of short DNA consensus sequences located in the promoter, termed the W, X, and Y boxes, which bind several known transcription factors. We have previously characterized the expression of HLA-DR in four human thyroid carcinoma cell lines and found differences between constitutive and high- or moderate-induced expression of the protein and mRNA. Evaluation of B7 expression on the surface of thyroid cancer cells and understanding the mechanisms of HLA-DR gene expression may help in designing efficient immune response to thyroid tumors. Using the electrophoretic mobility shift assay (EMSA), we have demonstrated differences between the four thyroid cell lines in the binding of transcription factors to each of the three boxes. The binding to the promoter in each of the cell lines resulted in a single band, probably representing a complex of proteins formed via protein-protein interactions. Using flow cytometry we have shown that the B7 molecule was absent in the four thyroid cell lines and could not be induced by IFN-gamma. The absence of surface B7 molecules from the malignant thyroid cells may lead to either suppression of antitumoral cytotoxic T cell response or demand the cooperation of infiltrating APCs to favor immune response. Differences previously found in HLA-DR expression in the four human malignant thyroid cell lines may be explained by the variation in the binding of transcription factors to the boxes in the HLA-DRalpha promoter. The binding patterns of nuclear proteins derived from the four thyroid cell lines or from the B lymphocyte cell line--Raji--to each of the boxes or to the whole promoter exhibit similarities, thus suggesting similar DNA-protein interactions.

Vaccination of mice with attenuated mutants ofListeria monocytogenes: requirement for induction of macrophage la expression

Isogenic mutant strains ofListeria monocytogenesdemonstrating graded attenuation in mice were used to analyse the correlation between bacterial virulence, ability to induce class II MHC (la) molecules in antigen presenting cells and ability to vaccinate against secondary infection. The mutants used differed only in the amino acid sequence of the thiol–activated hemolysin, Listeriolysin O (LLO). The results indicate thatL. monocytogenesmutants of reduced virulence have the potential to act as vaccines only if they are sufficiently persistant to induce la expression in antigen presenting cells. The findings also suggest that specific mutagenesis of virulence factors, including LLO, could provide an approach for creatingListeria monocytogenesstrains with potential for use as attenuated live vaccines.

Distribution of the alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein in human tissues.

The hepatic alpha 2-macroglobulin receptor (alpha 2MR)/low density lipoprotein receptor-related protein (LRP) binds and endocytoses alpha 2-macroglobulin-proteinase complexes in plasma. In addition, it binds lipoproteins, a novel 40 kDa protein, and complexes between plasminogen activators and plasminogen activator inhibitor type-1. This study shows, for the first time, the tissue distribution of alpha 2MR/LRP as determined by immunohistochemistry with specific monoclonal antibodies. The analysis revealed alpha 2MR/LRP-expression in a restricted spectrum of cell types, including neurons and astrocytes in the central nervous system, epithelial cells of the gastrointestinal tract, smooth muscle cells, fibroblasts, Leydig cells in testis, granulosa cells in ovary, and dendritic interstitial cells of kidney. Monocyte-derived cells displayed marked alpha 2MR/LRP expression in the phagocytes of liver, lung and lymphoid tissues, but no or low expression in antigen-presenting cells including Langerhans' cells of the skin. The high abundance of alpha 2MR/LRP in certain cell types of most organs suggests two main routes for alpha 2MR/LRP-mediated ligand clearance: (1) systemic removal in liver of circulating ligands, and (2) non-hepatic interstitial removal in different organs, including the brain.

Prostaglandins and transplantation

Prostaglandins are important in the allograft response. Allograft antigenic stimulation causes release of prostaglandins both in vivo and in vitro. Macrophages and monocytes are the cells that produce the prostaglandins from arachidonic acid via the cyclooxygenase pathway. Prostaglandins exert immunosuppressive effects at several steps on both afferent and efferent phases in the cell-mediated immune system: reduction of Ia expression in antigen-presenting cells, inhibition of IL-1 and IL-2 production, and activation of suppressor cells. The immunosuppressive effects of blood transfusions and essential fatty acids appear to be mediated by PGE.

T helper cell subsets require the expression of distinct costimulatory signals by antigen-presenting cells.

We examined the ability of macrophages and B cells to function as antigen-presenting cells (APCs) for murine TH1 and TH2 cloned T helper cell lines. Antigen presented by concanavalin A-elicited peritoneal macrophages or resting splenic B cells stimulated antigen-dependent proliferation of both T helper subsets. Paraformaldehyde fixation of the APCs following different conditions of activation indicated differential requirements for costimulatory signals by TH1 and TH2 cells. TH2 proliferative responses were strictly dependent on APC expression of IL-1. TH1 proliferation was dependent on APC expression of a non-IL-1 costimulatory signal present on freshly isolated macrophages and on splenic B cells activated with anti-immunoglobulin plus interferon gamma.

Prostaglandins and surgical diseases: Part I

Prostaglandins are important in the allograft response. Allograft antigenic stimulation causes release of prostaglandins both in vivo and in vitro. Macrophages and monocytes are the cells that produce the prostaglandins from arachidonic acid via the cyclooxygenase pathway. Prostaglandins exert immunosuppressive effects at several steps on both afferent and efferent phases in the cell-mediated immune system: reduction of Ia expression in antigen-presenting cells, inhibition of IL-1 and IL-2 production, and activation of suppressor cells. The immunosuppressive effects of blood transfusions and essential fatty acids appear to be mediated by PGE.