BACKGROUNDAspergillosis infection is common in the patients with insufficient immunity. The role of mammalian target of rapamycin (mTOR), T-box expressed in T-cells (T-bet), and eomesodermin (EOMES) in mediating T lymphocytes differentiation in response to Aspergillus fumigatus infection in immunocompromised rats was investigated in this study.

METHODSInvasive pulmonary aspergillosis (IPA) of immunosuppressive twenty male rats were established and sacrificed at 24 h (n = 5), 48 h (n = 5), 72 h (n = 5), and 96 h (n = 5) after A. fumigatus infection. In addition, control (n = 5), cyclophosphamide (CTX) (n = 5), and aspergillosis (n = 5) group were also established the tissues and pathology of lung tissue was examined by hematoxylin and eosin staining. CD8+ T-cells was sorted by flow cytometry. Serum mTOR, S6K, T-bet, and EOMES were quantified by enzyme-linked immunosorbent assay.

RESULTSHistology of lung tissue indicated severe lung tissue injury including infiltration of inflammatory cells, alveolar wall damage or degradation, blood congestion, and hemorrhage in the CTX, IPA, and CTX + IPA rats. Hyphae were seen in the IPA, and CTX + IPA groups. The proportion of CD8+ T-cells was significantly increased in the animals of CTX + IPA. Memory CD8+ T-cells was significantly increased in early stage (24 h and 48 h, P < 0.001), but decreased in the late phase of fungal infection (72 h and 96 h) in the animals of CTX + IPA. In addition, at early stage of fungal infection (24 h and 48 h), serum mTOR (P < 0.001), S6K (P < 0.001), and T-bet (P < 0.05) was significantly higher, while EOMES was significantly lower (P < 0.001), in CTX + IPA group than that in control, CTX alone or IPA alone group. Conversely, serum mTOR, S6K, T-bet, and EOMES showed opposite changed in the late stage (72 h and 96 h). Pearson's correlation analysis indicated that mTOR and S6K were significantly correlated with T-bet (r = 0.901 and 0.91, respectively, P < 0.001), but negatively and significantly correlated with EOMES (r = -0.758 and -0.751, respectively, P < 0.001).

CONCLUSIONSmTOR may regulate transcription factors of EOMES and T-bet, and by which mechanism, it may modulate lymphocytes differentiation in animals with immune suppression and fungal infection.

Animals ; CD8-Positive T-Lymphocytes ; cytology ; metabolism ; Cell Differentiation ; genetics ; physiology ; Invasive Pulmonary Aspergillosis ; metabolism ; pathology ; Lung ; metabolism ; pathology ; Lymphocytes ; cytology ; immunology ; Male ; Rats ; Rats, Wistar ; T-Box Domain Proteins ; genetics ; metabolism ; TOR Serine-Threonine Kinases ; genetics ; metabolism ; Tissue Culture Techniques

Heat-shock protein (Hsp) 70 potentiates specific immune responses to some antigenic peptides fused to it. Here, the prokaryotic plasmids harboring the envelope glycoprotein E0 gene of classical swine fever virus (CSFV) and/or the Hsp70 gene of Haemophilus parasuis were constructed and expressed in Escherichia coli Rosseta 2(R2). The fusion proteins were then purified. Groups of Balb/c mice were immunized with these fusion proteins, respectively, and sera collected 7 days after the third immunization. Immune effects were determined via an enzyme-linked immunosorbent assay and flow cytometric analyses. E0-Hsp70 fusion protein and E0+Hsp70 mixture significantly improved the titer of E-specific antibody, levels of CD4+ and CD8+ T cells, and release of interferon-γ. These findings suggested that Hsp70 can significantly enhance the immune effects of the envelope glycoprotein E0 of CSFV, thereby laying the foundation of further application in pigs.
Animals ; Antibodies, Viral ; blood ; CD4-Positive T-Lymphocytes ; cytology ; immunology ; CD8-Positive T-Lymphocytes ; cytology ; immunology ; Cell Proliferation ; Classical swine fever virus ; genetics ; Female ; HSP70 Heat-Shock Proteins ; genetics ; immunology ; Haemophilus parasuis ; genetics ; Immunization ; Interferon-gamma ; metabolism ; Mice ; Mice, Inbred BALB C ; Plasmids ; genetics ; Recombinant Fusion Proteins ; genetics ; immunology ; Viral Envelope Proteins ; genetics

We evaluated the effectiveness of rhamnogalacturonan II (RG-II)-stimulated bone marrow-derived dendritic cells (BMDCs) vaccination on the induction of antitumor immunity in a mouse lymphoma model using EG7-lymphoma cells expressing ovalbumin (OVA). BMDCs treated with RG-II had an activated phenotype. RG-II induced interleukin (IL)-12, IL-1beta, tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) production during dendritic cell (DC) maturation. BMDCs stimulated with RG-II facilitate the proliferation of CD8+ T cells. Using BMDCs from the mice deficient in Toll-like receptors (TLRs), we revealed that RG-II activity is dependent on TLR4. RG-II showed a preventive effect of immunization with OVA-pulsed BMDCs against EG7 lymphoma. These results suggested that RG-II expedites the DC-based immune response through the TLR4 signaling pathway.
Acute-Phase Proteins/metabolism ; Adaptor Proteins, Vesicular Transport/metabolism ; Animals ; Antigens, CD14/metabolism ; Bone Marrow Cells/cytology/drug effects ; CD8-Positive T-Lymphocytes/*immunology ; Carrier Proteins/metabolism ; Cell Differentiation/drug effects ; Cell Nucleus/drug effects/metabolism ; Cell Proliferation/drug effects ; Cytokines/biosynthesis ; Dendritic Cells/cytology/drug effects/enzymology/*immunology ; Enzyme Activation/drug effects ; Lymphocyte Activation/*drug effects ; Membrane Glycoproteins/metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Mitogen-Activated Protein Kinases/metabolism ; Myeloid Differentiation Factor 88/metabolism ; NF-kappa B/metabolism ; Neoplasms/immunology/*pathology ; Pectins/*pharmacology ; Phenotype ; Protein Transport/drug effects ; Receptors, Chemokine/metabolism ; Signal Transduction/drug effects ; T-Lymphocytes, Cytotoxic/cytology/drug effects ; Toll-Like Receptor 4/*agonists/metabolism

BACKGROUNDIt has been reported that CD8(+) regulatory cells could be induced upon oral tolerance. The purpose of this study was to investigate the changes of CD8α(+) T cells in dextran sulfate sodium (DSS)-induced colitis mice pretreated by oral immune regulation.

METHODSThe effects of five low oral doses of colitis-extracted proteins (CEP) on colitis were evaluated by clinical manifestation and histological lesions. The percentages of CD8α(+) T cells gating on CD3(+) T cells were evaluated in the gut-associated lymphoid tissues (GALT) and the spleens by flow cytometry. Differences between the two groups were compared by Student's t test or Mann-Whitney U test.

RESULTSCompared to bovine serum albumin (BSA)-fed control mice, administration of CEP resulted in marked alleviation of colitis. The proportion of CD8α(+) T cells, not only in intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs) of the large intestine (LI) but also in spleen from CEP-fed colitis mice, was significantly higher than that from BSA-fed colitis mice (LI-IELs: (71.5 ± 5.4)% vs. (60.1 ± 4.3)%, P < 0.01; LI-LPLs: (60.7 ± 5.2)% vs. (51.9 ± 4.7)%, P < 0.01; spleen: (24.1 ± 3.6)% vs. (20.3 ± 4.1)%, P < 0.05; n = 8). Mucosal repair in repair-period mice five days after termination of DSS treatment was also accompanied by an increase of CD8α(+) T cells in large intestinal mucosal lymphocytes (LI-IELs: (72.1 ± 3.7)% vs. (61.5 ± 4.5)%, P < 0.01; LI-LPLs: (62.1 ± 5.7)% vs. (52.7 ± 3.6)%, P < 0.01; n = 8). The proportion of CD3(+) T cells increased in Peyer's patches (PPs) and decreased in mesenteric lymph nodes (MLNs) from colitis mice compared to untreated mice, whereas the change pattern of CD3(+) T cells in PPs and MLNs from CEP-fed colitis mice was just on the contrary.

CONCLUSIONImprovement of DSS-induced colitis resulted from oral immune regulation is associated with an increase in CD8α(+) T cells in spleen and large intestinal mucosa.

Administration, Oral ; Animals ; CD8-Positive T-Lymphocytes ; metabolism ; Colitis ; chemically induced ; complications ; Dextran Sulfate ; toxicity ; Flow Cytometry ; Lymphocytes ; cytology ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Proteins ; administration & dosage ; immunology ; Spleen ; cytology ; metabolism

Cytomegalovirus (CMV) infection is a dangerous complication in patients with chronic graft versus host disease (cGVHD). CMV-specific immunity depends on the activity of T cells. This study was aimed to investigate the effect of CMV pp65 gene modified dendritic cells (DCs) on activation of autologous T cells. Lentivirus system was utilized to introduce the CMV full-length pp65 gene into mouse DCs; CpG-DNA was used to induce mature DCs; flow cytometry and immunofluorescence were used to determine the expression of antigen and IFNgamma in T lymphocytes. The results showed that the DCs were infected with lentivirus at a multiplicity of infection (MOI) of 50 with optimal infectious efficiency of 30%-40%; mature DCs expressing pp65 gene could stimulate autologous naive T cells to express CD69 specifically; mature DCs expressing PP65 could stimulate autologous CD4+ or CD8+ T cells to produce IFNgamma. It is concluded that CMV pp65-modified and CpG-DNA-induced mature DCs can activate CMV-specific T lymphocytes in vitro.
Animals ; Antigens, CD ; genetics ; metabolism ; Antigens, Differentiation, T-Lymphocyte ; genetics ; metabolism ; Antigens, Viral ; immunology ; CD4-Positive T-Lymphocytes ; immunology ; CD8-Positive T-Lymphocytes ; immunology ; CpG Islands ; genetics ; Cytomegalovirus ; immunology ; DNA ; genetics ; Dendritic Cells ; cytology ; immunology ; metabolism ; Humans ; Interferon-gamma ; genetics ; metabolism ; Lectins, C-Type ; Lentivirus ; genetics ; metabolism ; Mice ; Phosphoproteins ; genetics ; metabolism ; Viral Matrix Proteins ; genetics ; metabolism

OBJECTIVETo investigate the effect of murine mesenchymal stem cells (mMSC) on immunoproliferative response of spleen cells.

METHODSMitogen (Con A, 20 microg/ml) or irradiated (20 Gy) allogeneic spleen cells (from BALB/c or C57BL/6 mouse depending on the responder cells) were used as stimulators. Proliferations of the responder cells were determined with MTT on day 3 after culture at 37 degrees C, 5% CO2 humidified atmosphere. The ratios of CD4+/CD8+ and CD4+CD25+ cells were analyzed with FACS assay, and the levels of cytokines in supernatants with ELISA.

RESULTS1) mMSC inhibited the response of both syngeneic and allogeneic splenic cells to ConA. At the ratio of mMSC to splenic cells being 1: 1, the inhibition rate reached 84.21%. With the ratio decreasing, the inhibition rate decreased. 2) mMSC inhibited the response of both syngeneic and allogeneic splenic cells to alloantigen. When the ratio of mMSC to responder cells was 1: 10, the inhibition rate was as high as 88.07%. 3) mMSC could increase the ratio of CD4+/ CD8+ T cells and the percentage of CD4+ CD25+ cells in splenic cells. These abilities were in a dose-dependent manner and non-MHC antigen restricted. 4) mMSC decreased interleukin (IL) -2, interferon (IFN)-gamma, while increased TGF-beta1 and IL-4 in the co-culture system.

CONCLUSIONmMSC can suppress proliferative response of splenic cells to mitogen and alloantigen, increase the ratio of CD4+/CD8+ and the proportion of CD4+ CD25+ in T cells, decrease the secretion of proinflammatory cytokines and increase the anti inflammatory cytokines in a dose-dependent and non-MHC antigen restricted manner.

Animals ; Bone Marrow Cells ; immunology ; CD4-CD8 Ratio ; CD4-Positive T-Lymphocytes ; immunology ; Cells, Cultured ; Interferon-gamma ; metabolism ; Interleukin-2 ; metabolism ; Interleukin-4 ; metabolism ; Male ; Mesenchymal Stromal Cells ; immunology ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Spleen ; cytology ; immunology ; metabolism ; Transforming Growth Factor beta ; metabolism

OBJECTIVETo evaluate of therapeutic efficacy of deoxyribouncleotidum on pulmonary tuberculosis.

METHODSEighty patients with pulmonary tuberculosis sustaining hepatic lesion after treatment with antituberculosis drugs were randomized into therapeutic group and control group. Patients in the control group received regular treatment and those in the therapeutic group had additional deoxyribouncleotidum injection.

RESULTSALT, AST, ALP and TBIL levels were significantly higher in the therapeutic group than in the control group 4 weeks after treatment. IgG, IgA, IgM levels, and CD3(+) and CD8(+) lymphocytes were significantly increased in the therapeutic group after treatment (P<0.05).

CONCLUSIONdeoxyribouncleotidum can improve hepatic function and immunity in patients with pulmonary tuberculosis.

Adjuvants, Immunologic ; administration & dosage ; therapeutic use ; Adult ; Alanine Transaminase ; metabolism ; Antitubercular Agents ; adverse effects ; therapeutic use ; Aspartate Aminotransferases ; metabolism ; CD3 Complex ; immunology ; CD8-Positive T-Lymphocytes ; cytology ; drug effects ; immunology ; Chemical and Drug Induced Liver Injury ; Deoxyribonucleotides ; administration & dosage ; therapeutic use ; Female ; Humans ; Immunoglobulin A ; blood ; Immunoglobulin G ; blood ; Immunoglobulin M ; blood ; Injections ; Liver Diseases ; blood ; drug therapy ; Male ; Middle Aged ; Treatment Outcome ; Tuberculosis, Pulmonary ; blood ; drug therapy

Blockade of signal 1 or 2 for T-cell activation by the use of anti-CD45RB and anti-CD154 monoclonal antibodies (mAb) (two-signal blockade) has been proven effective in preventing or delaying graft rejection. However, the mechanisms of its immunomodulatory effects are clearly unknown and the present studies were performed to determine how the two-signal blockade modulate allogeneic immune responses, especially T-cell mediated cellular immunity, in a murine skin allograft model. We now report on the profound inhibition of alloreactive T cells by two-signal blockade via CD4-dependent mechanisms. C57BL/6 mice of BALB/c skin allograft were treated with anti-CD45RB, anti-CD154, CTLA4-Ig, or their combinations. For depletion of CD4 or CD8 T cells, the recipients received CD4-depleting or CD8-depleting mAb. We confirmed that survival of skin allograft was markedly prolongated in the two-signal blockade-treated group. In depletion study, anti-CD45RB, anti-CD154 and CD4-depleting mAb-treated group showed acute rejection of skin allograft in contrast to CD8-depleting group treated with the two-signal blockade. In the group treated with the two-signal blockade, the proportions of CD4+CD45RB(low)and CD8+CTLA-4 regulatory T cells were increased while effector CD8+ T cells, including IFN-gamma-secreting and CD8+CD62L(low)T cells, were decreased when compared with non-treated group. In contrast, the CD4-depleted group treated with the two-signal blockade resulted in recovery from immunoregulatory effects of two-signal blockade. In addition, results of IL-4 and IL-10 production were also showed CD4-dependence. Therefore, the two-signal blockade is accompanied by CD4-dependent mechanisms in allogeneic skin transplantation.
Transplantation, Homologous ; T-Lymphocytes, Regulatory/cytology/immunology ; Skin Transplantation/*immunology ; Signal Transduction/drug effects/immunology ; Mice, Inbred C57BL ; Mice, Inbred BALB C ; Mice ; Male ; Lymphocyte Depletion ; Lymphocyte Activation/immunology ; Interleukin-4/biosynthesis ; Interleukin-10/biosynthesis ; Graft Rejection/*immunology/prevention & control ; Flow Cytometry ; Cytotoxicity, Immunologic/immunology ; CD8-Positive T-Lymphocytes/cytology/immunology/metabolism ; CD40 Ligand/*immunology ; CD4-Positive T-Lymphocytes/cytology/immunology/metabolism ; Antigens, CD45/*immunology ; Antigens, CD4/*immunology ; Antibodies, Monoclonal/administration & dosage/*pharmacology ; Antibodies, Blocking/administration & dosage/pharmacology ; Animals

This study was aimed to eveluate the role of CCR5 on donor cells in recipient models received intensive conditioning, so as provide the scientific evidence for clinical application of allo-HSCT. Lethally irradiated BALB/c mice received allogeneic bone marrow transplants from C57BL/6 mice. Mice divided into 4 groups according to receiving variant donor cells: B6 CCR5 KO group, receiving C57BL/6 CCR5(-/-) mice bone marrow cells and splenocytes; B6 WT BMC group, receiving C57BL/6 mice bone marrow cells and splenocytes; B6 CCR5 KO BMC group, receiving C57BL/6 CCR5(-/-) bone marrow cells alone; B6 WT BMC group, receiving C57BL/6 mice bone marrow cells alone. The result showed that compared to B6 WT BMC group, B6 CCR5 KO group succumbed to acute GVHD at an accelerated rate. Donor CD8(+) T cells expanded to a significantly greater extent in recipients of CCR5 KO, compared with B6 WT control cells. T cells recovered from recipients of CCR5 KO cells produced more IFN-gamma and TNF-alpha and proliferated to a T-cell at a significantly higher level than T cells from recipients of WT cells, indicating that CCR5 plays a role in downregualting donor alloreative CD8(+) T-cells expansion. Histological assessment of the mice indicated pathological lesions in the kidneys and a greater degree of liver pathological changes in mice that received CCR5 KO donor grafts. It is concluded that the knock-out of CCR5 on donor cells results in increase of GVHD and donor CD8(+) T cell expansion, as well as hepatic and renal lesions in allo-HSCT, which indicates that CCR5 is very important in allo-BMT.
Animals ; Bone Marrow Transplantation ; adverse effects ; CD8-Positive T-Lymphocytes ; cytology ; immunology ; transplantation ; Female ; Graft vs Host Disease ; metabolism ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Receptors, CCR5 ; genetics ; physiology

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