In allogeneic transplantation, including the B6 anti-BALB.B settings, H60 and H4 are two representative dominant minor histocompatibility antigens that induce strong CD8 T-cell responses. With different distribution patterns, H60 expression is restricted to hematopoietic cells, whereas H4 is ubiquitously expressed. H60-specific CD8 T-cell response has been known to be dominant in most cases of B6 anti-BALB.B allo-responses, except in the case of skin transplantation. To understand the mechanism underlying the subdominance of H60 during allogeneic skin transplantation, we investigated the dynamics of the H60-specific CD8 T cells in B6 mice transplanted with allogeneic BALB.B tail skin. Unexpectedly, longitudinal bioluminescence imaging and flow cytometric analyses revealed that H60-specific CD8 T cells were not always subdominant to H4-specific cells but instead showed a brief dominance before the H4 response became predominant. H60-specific CD8 T cells could expand in the draining lymph node and migrate to the BALB.B allografts, indicating their active participation in the anti-BALB.B allo-response. Enhancing the frequencies of H60-reactive CD8 T cells prior to skin transplantation reversed the immune hierarchy between H60 and H4. Additionally, H60 became predominant when antigen presentation was limited to the direct pathway. However, when antigen presentation was restricted to the indirect pathway, the expansion of H60-specific CD8 T cells was limited, whereas H4-specific CD8 T cells expanded significantly, suggesting that the temporary immunodominance and eventual subdominance of H60 could be due to their reliance on the direct antigen presentation pathway. These results enhance our understanding of the immunodominance phenomenon following allogeneic tissue transplantation.
Animals ; Antigen Presentation ; Antigen-Presenting Cells/immunology/metabolism ; CD8-Positive T-Lymphocytes/*immunology ; Epitopes, T-Lymphocyte/*immunology ; Female ; Graft Rejection/*immunology ; Interferon-gamma ; Lymphocyte Activation/immunology ; Lymphocyte Count ; Mice ; Minor Histocompatibility Antigens/*immunology/metabolism ; *Skin Transplantation ; Transplantation, Homologous

OBJECTIVEAntigen-presenting cells such as monocytes and dendritic cells (DCs) stimulate T-cell proliferation and activation during adaptive immunity. This cellular interaction plays a role in the growth of atherosclerotic plaques. Tanshinone II A (TSN) had been shown to decrease the growth of atherosclerotic lesions. We therefore investigated the ability of TSN to inhibit human monocyte-derived DCs and their T-cellstimulatory capacity.

METHODSDCs derived from human monocytes cultured with recombinant human interleukin (IL)-4 and recombinant human granulocyte-macrophage colony-stimulating factor were co-cultured with TSN and lipopolysaccharide for 48 h. Phosphate-buffered saline was used as a negative control. Activation markers and the capacity of DCs for endocytosis were measured by flow cytometry, and proinflammatory cytokines were measured by enzyme-linked immunosorbent assays. DCs were co-cultured with lymphocytes to measure T-cell proliferation and IL-2 secretion by mixed lymphocyte reactions.

RESULTSTSN dose-dependently attenuated DC expression of costimulatory molecules (CD86), and decreased expression of major histocompatibility complex class II (human loukocyte antigen-DR) and adhesion molecules (CD54). Moreover, TSN reduced secretion of the proinflammatory cytokines IL-12 and IL-1 by human DCs, and restored the capacity for endocytosis. Finally, TSN-preincubated DCs showed a reduced capacity to stimulate T-cell proliferation and cytokine secretion.

CONCLUSIONSTSN inhibits DC maturation and decreases the expression of proinflammatory cytokines, while impairing their capacity to stimulate T-cell proliferation and cytokine secretion. These effects may contribute to the influence of TSN on the progression of atherosclerotic lesions.

Antigen-Presenting Cells ; drug effects ; Atherosclerosis ; immunology ; pathology ; B7-2 Antigen ; metabolism ; Cell Membrane ; drug effects ; metabolism ; Cytokines ; secretion ; Dendritic Cells ; drug effects ; immunology ; secretion ; Diterpenes, Abietane ; pharmacology ; Endocytosis ; drug effects ; Flow Cytometry ; Humans ; Immunity, Cellular ; drug effects ; Inflammation Mediators ; metabolism ; Lymphocyte Activation ; drug effects

As a member of the HSP90 family, heat shock protein (HSP) Gp96 is one of the most abundant proteins in the endoplasmic reticulum (ER), which displayed important molecular chaperones function in cells. Gp96 can stimulate the production of cytokines by activating the antigen presentation cells (such as dendritic cell, et al) in innate immunity. It is capable of eliciting an antigen-specific cytotoxic T lymphocyte (CTL) immune response to eliminate pathogens and tumors by facilitating antigen cross-presentation in adaptive immunity. Gp96 is also an ideal adjuvant in many recent researches. Here, we review the progress that addresses the role of biological characteristics, immunogenic mechanism that may be involved in the induction of anti-infection immune response and antitumor immunity, which may guide the new vaccine strategies with the knowledge of Gp96-antigen complexes.
Adjuvants, Immunologic ; genetics ; metabolism ; Antigen-Presenting Cells ; physiology ; Communicable Diseases ; immunology ; Dendritic Cells ; immunology ; Endoplasmic Reticulum ; immunology ; Humans ; Membrane Glycoproteins ; immunology ; Neoplasms ; immunology ; T-Lymphocytes, Cytotoxic ; immunology

This study was aimed to investigate the effect of regulatory T (Treg) cells on the T cell lymphoma EL4 cells and its mechanism in vitro. C57BL/6 mouse Treg cells were isolated by magnetic cell sorting (MACS). The purity of Treg cells and their expression of Foxp3 were identified by flow cytometry (FCM) and PT-PCR respectively. The suppression of Treg cells on EL4 cells was detected by 3H-TdR method. At the same time, enzyme-linked immunosorbent assay (ELISA) was used to detect the secretion of cytokine TGF-β1 and IL-10. The results showed that CD4+CD25+ T cells could be successfully isolated by MACS with the purity reaching 94.52% and the expression of Foxp3 reaching 84.72%. After sorting, the expression of Foxp3 mRNA could be detected by RT-PCR. 3H-TdR assay confirmed that regulatory T cells could suppress the proliferation of EL4 cells with or without antigen presenting cells (APC) or dendritic cells (DC), APC or DC might effectively enhance the suppression. In addition, DC alone also suppressed the proliferation. TGF-β1 and IL-10 could be detected in the supernatant by ELISA. It is concluded that the Treg cells can obviously suppress the proliferation of T cell lymphoma cells in vitro, APC or DC can enhance this suppressive effect, while the DC alone also can suppress the proliferation of EL4 cells, the TGF-β1 and IL-10 cytokine pathway may be one of the mechanisms of suppression.
Animals ; Antigen-Presenting Cells ; immunology ; Cell Line, Tumor ; Cell Proliferation ; Dendritic Cells ; immunology ; Female ; Forkhead Transcription Factors ; metabolism ; Interleukin-10 ; secretion ; Interleukin-2 Receptor alpha Subunit ; immunology ; Lymphoma, T-Cell ; immunology ; pathology ; Mice ; Mice, Inbred C57BL ; T-Lymphocytes, Regulatory ; immunology ; Transforming Growth Factor beta1 ; secretion

BACKGROUNDRapamycin (RAPA) is a relatively new immunosuppressant drug that functions as a serine/threonine kinase inhibitor to prevent rejection in organ transplantation. RAPA blocks activation of T-effector (Teff) cells by inhibiting the response to interleukin-2. Recently, RAPA was also shown to selectively expand the T-regulator (Treg) cell population. To date, no studies have examined the mechanism by which RAPA converts Teff cells to Treg cells.

METHODSPeripheral CD4(+)CD25(-) naive T cells were cultivated with RAPA and B cells as antigen-presenting cells (APCs) in vitro. CD4(+)CD25(-) T cells were harvested after 6 days and analyzed for expression of forkhead box protein 3 (Foxp3) using flow cytometry. CD4(+)CD25(+)CD127(-) subsets as the converted Tregs were isolated from the mixed lymphocyte reactions (MLR) with CD127 negative selection, followed by CD4 and CD25 positive selection using microbeads and magnetic separation column (MSC). Moreover, mRNA was extracted from converted Tregs and C57BL/6 naive CD4(+)CD25(+) T cells and Foxp3 levels were examined by quantitative real-time polymerase chain reaction (rt-PCR). A total of 1 x 10(5) carboxyfluorescein succinimidyl ester (CFSE)-labeled naive CD4(+)CD25(-) T cells/well from C57BL/6 mice were cocultured with DBA/2 or C3H maturation of dendritic cells (mDCs) (0.25 x 10(5)/well) in 96-well round-bottom plates for 6 days. Then 1 x 10(5) or 0.25 x 10(5) converted Treg cells were added to every well as regulatory cells. Cells were harvested after 6 days of culture and analyzed for proliferation of CFSE-labeled naive CD4(+)CD25(-) T cells using flow cytometry. Data were analyzed using CellQuest software.

RESULTSWe found that RAPA can convert peripheral CD4(+)CD25(-) naive T Cells to CD4(+)Foxp3(+) Treg cells using B cells as APCs, and this subtype of Treg can potently suppress Teff proliferation and maintain antigenic specificity.

CONCLUSIONOur findings provide evidence that RAPA induces Treg cell conversion from Teff cells and uncovers an additional mechanism for tolerance induction by RAPA.

Animals ; Antibiotics, Antineoplastic ; pharmacology ; Antigen-Presenting Cells ; drug effects ; immunology ; metabolism ; B-Lymphocytes ; drug effects ; immunology ; metabolism ; CD4-Positive T-Lymphocytes ; drug effects ; immunology ; metabolism ; Cell Proliferation ; drug effects ; Dendritic Cells ; drug effects ; immunology ; metabolism ; Forkhead Transcription Factors ; metabolism ; Interleukin-2 Receptor alpha Subunit ; metabolism ; Interleukin-7 Receptor alpha Subunit ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Inbred DBA ; Mitomycin ; pharmacology ; Polymerase Chain Reaction ; Sirolimus ; pharmacology ; T-Lymphocytes, Regulatory ; drug effects ; immunology ; metabolism

Exosomes are nanometer sized membrane vesicles, released in the extracellular milieu following the fusion of the external membrane of multivesicular body (MVB) with plasma membrane. They perform a certain function in immune regulation. Exosomes have been shown to be released by cells of hematopoietic and non-hematopoietic origin. Tumour-derived exosomes (TEX) exist in the supernatant of tumour cells, plasma and malignant effusions of tumour patients. They contain native candidate tumour associated antigen and are capable of transferring antigens to T lymphocytes, therefore efficiently promoting cytotoxic T lymphocyte (CTL) activation and producing antitumor immunity. However, recent evidence shows that tumor exosomes may induce immunologic tolerance and even activate immunosuppression which makes tumour escape from the immune surveillance of the host immune system. In addition, tumor exosomes may mediate a growth-promoting effect on tumor cells. These discrepancies are almost certainly due to differences in the phenotype of the exosomes.
Antigen-Presenting Cells ; immunology ; Antigens, Neoplasm ; immunology ; Cytoplasmic Vesicles ; immunology ; Endosomes ; immunology ; metabolism ; Exosomes ; immunology ; Humans ; Neoplasms ; immunology ; T-Lymphocytes, Cytotoxic ; immunology ; Tumor Escape

This study was aimed to investigate the effect of artificial antigen-presenting cells (aAPCs) on inducing activation and proliferation of specific T-lymphocytes through stimulating biological function of dendritic cells with aAPCs in vitro. The specific antigen of chronic myeloid leukemia CML-28 was screened as objective antigen peptide by using magnetic microbeads as vector; the CML-28 epitope sequence (Vltfaldsv) was obtained by antigen epitope prediction software; this epitope was coupled with MHC molecule and used as first signal molecule, the B7-1 molecule was used as second signal molecule; these 2 molecules simultaneously were loaded onto surface of magnetic microbeads so as to contract aAPC complex. The bone marrow mononuclear cells were derived from HLA-A2(+) healthy bone marrow donors, CD8(+) T lymphocytes were screened and co-cultured with aAPC complex. During culture the 5, 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) was added and proliferation of T-lymphocytes was detected by CPSE and proliferation level of specific T lymphocytes was detected by flow cytometry. The results showed that the proliferation level of CML-28 specific T lymphocytes obviously increased in experimental group, average level was 17.34 +/- 0.65%, while average level in control was 2.25 +/- 0.43%, there was significant difference between them (p < 0.01). It is concluded that the aAPC complex can mimic human APCs in vitro, and stimulate activation and proliferation of CD8(+) specific T lymphocytes.
Adolescent ; Adult ; Antigen Presentation ; immunology ; Antigen-Presenting Cells ; immunology ; metabolism ; Antigens, Neoplasm ; immunology ; Biomimetics ; methods ; CD8-Positive T-Lymphocytes ; immunology ; Female ; HLA-A2 Antigen ; immunology ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; immunology ; Lymphocyte Activation ; Male ; Middle Aged ; Tissue Donors ; Young Adult

Altered peptide ligand (APL), a short peptide with immune regulatory activity and substitutions of a single or multiple amino acids in an antigenic peptide, has shown potential therapeutic effect on autoimmune disease, tumor and virus infection. APL regulates immune responses by interfering the interaction between the major histocompatibility complex (MHC), antigenic peptide and T cell receptor (TCR), or by regulating the intracellular signaling of antigen presenting cells, bystander suppression and inducing heterogenous immune responses. High-specific and high-affinity APL screened from peptide laboratory by phage display, has a potential to be a new resource for drug with antigen specificity.
Animals ; Antigen-Presenting Cells ; immunology ; metabolism ; Autoimmune Diseases ; immunology ; therapy ; Humans ; Immunotherapy ; methods ; Ligands ; Major Histocompatibility Complex ; immunology ; Peptide Fragments ; biosynthesis ; immunology ; metabolism ; therapeutic use ; Peptide Library ; Receptors, Antigen, T-Cell ; immunology ; metabolism

In vitro large amplification of tumor-specific cytotoxic T lymphocytes (CTLs) and adoptive transfer of these cells is one of the most promising approaches to treat malignant diseases in which an effective immune response is not achieved by active immunization. However, generating sufficient numbers of tumor-specific CTLs stimulated with autologous antigen presenting cells (APCs) in vitro is one of the most problematic steps in the adoptive cell transfer (ACT) therapy. To circumvent this problem, we have developed an artificial antigen presenting complex (aAPCs) using MHC class I molecules loaded with a melanoma-specific TRP-2 peptide epitope. Our results show that TRP-2-specific CD8+ T cells elicited by immunization with recombinant adenovirus expressing the mini-gene epitope are efficiently stimulated and amplified in vitro to a greater extent by aAPCs than by natural splenic APCs. These aAPC-induced CTLs recognized endogenously processed antigens present on B16F10 melanoma cells. Efficient stimulation and proliferation of antigen- specific T cells was also confirmed using ovalbumin peptide-loaded aAPCs and OT-I TCR transgenic cells. These results demonstrate that prior in vivo immunization, which increases the precursor frequency, simplifies posterior expansion of tumor- specific CD8+ T cells, and aAPCs is superior to autologous APC for in vitro amplification. This prime and expand regimen can be an alternative method for large amplification of rare tumor-specific CTLs and aAPCs should be a useful tool for ACT immunotherapy.
Substrate Specificity ; Molecular Sequence Data ; Mice, Inbred C57BL ; Mice ; Melanoma/genetics/*immunology ; Lymphocyte Count ; Genetic Vectors/genetics ; Cell Line, Tumor ; CD8-Positive T-Lymphocytes/*cytology/*immunology/metabolism ; Biomimetics/*methods ; Antigen-Presenting Cells/immunology/metabolism ; Antigen Presentation/*immunology ; Animals ; Amino Acid Sequence ; Adoptive Transfer/methods

To examine the self-association of CD4 molecules and preliminary studies on its biological function by several indirect methods. A series of CD4 chimeras were generated including truncated CD4 lacking the short cytoplasmic tail, deleted mutantsD1/D2 devoid of D3 and D4 and D3/D4 devoid of D1 and D2 by PCR techniques, as well as another three CD4 chimeric genes by fused human Fas cytoplasmic death domain to the downstream of the above chimeras respectively. All these molecules were subcloned into pEGFP-N1, forming the corresponding expression vectors. After introducing into HEK293 cells, gene-modified cell morphological changes and target protein subcellular localization were observed and analyzed by a confocal microscopy. Moreover, stable 293/CD4 clones were obtained by transfecting the truncated CD4 recombinant plasmid into the HEK293 cell line and selected by G418. The fluorescene intensity and rosette formation of different clones was each analyzed by a confocal microscopy and cell adhesive assays. It's seen that CD4-Fas fusion gene could induce approximately 80% cell apoptosis of transfected HEK293 cells, compared to FKBP12-Fas is about 30% and CD4 gene only is 7%. Furthermore, both D1/D2-Fas and D3/D4 Fas chimeras could trigger nearly all transfected HEK293 cells to death. Cell adhesion assays showed that neither the D1/D2 nor D3/D4 chimeras when expression in HEK293 cells binds to MHC class II + Raji B cells. Interestedly, there were two type stable clones among 293/CD4. Fluorescence intensity analysis displayed that one' mean fluorescence intensity value is about twice of the other while cell-cell binding examination showed that the former is capable of forming rosette with Raji cells but the latter. All these results suggest that CD4 molecules most likely could exist as a dimer or even an oligomer on transfected HEK293 cell surface, which constitute a functional form for stable binding to MHC class II molecules.
Antigen-Presenting Cells ; immunology ; metabolism ; CD4 Antigens ; chemistry ; genetics ; metabolism ; CD4-Positive T-Lymphocytes ; immunology ; metabolism ; Cell Line ; Dimerization ; Fas Ligand Protein ; metabolism ; Histocompatibility Antigens Class II ; genetics ; immunology ; metabolism ; Humans ; Mutagenesis, Site-Directed ; Protein Binding ; genetics ; Protein Multimerization