Objective To prepare monoclonal antibodies against bovine CD4 and identify their basic biological characteristics. Methods Recombinant bovine CD4 (rHis-BoCD4 and rGST-BoCD4) was successfully expressed and purified by constructing a prokaryotic plasmid of bovine CD4 gene. The bovine CD4 monoclonal antibody was produced using hybridoma technology. The subtype and potency of the monoclonal antibody were identified and analyzed by ELISA, while specificity was analyzed through indirect immunofluorescence assay (IFA) and Western-blot. Results Four hybridoma cell lines, namely, 1H4, 6A10, 3F9 and 4G10, stably secreting monoclonal antibodies against BoCD4 were successfully obtained. The subclasses of the monoclonal antibodies subclass 6A10 was IgG2b and the rest of the monoclonal antibodies were of IgM type. Western-blot results showed that the four anti-bovine CD4 mAb strains were able to specifically bind to the bovine CD4 protein expressed in vitro. Indirect immunofluorescence assay showed that four monoclonal antibodies were able to specifically recognize the natural bovine CD4 protein. Flow cytometry assay showed that 3F9 was best to recognize bovine natural CD4 molecules. Conclusion Four monoclonal antibody strains with high specificity to natural bovine CD4 protein were successfully prepared, which lays the foundation for the subsequent studies on the function of bovine CD4 and diagnosis and treatment of bovine T-lymphocyte diseases.
Animals ; Antibodies, Monoclonal/isolation & purification* ; Cattle ; CD4 Antigens/genetics* ; Hybridomas/immunology* ; Antibody Specificity/immunology* ; Mice ; Mice, Inbred BALB C ; Enzyme-Linked Immunosorbent Assay ; Fluorescent Antibody Technique, Indirect

Objective: To investigate the role of CD4⁺CD25⁺regulatory cell (CD4⁺CD25⁺Treg) in auditory neuropathy (AN) using a rat model of autoimmune auditory neuropathy. Methods: The SD rats were immunized with P0 protein emulsified in complete Freunds adjuvant for 8 weeks. The number of CD4⁺CD25⁺Treg in peripheral blood and cochlea and the expression of Foxp3 gene in cochlea were detected respectively 2, 4, 6 and 8 weeks after the immunization with P0 protein in rats. Then CD4⁺CD25⁺Treg were transferred intravenously to the AN rats at 2, 4, 6 and 8 weeks of the immunization, respectively. The change of auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) were detected, and the morphological changes in the inner ear were investigated. Results: The number of CD4⁺CD25⁺Treg in the peripheral blood of AN rats decreased gradually after 2, 4, 6 and 8 weeks of P0 protein immunization. The number of CD4⁺CD25⁺Treg in cochlea gradually increased with the prolongation of immunization time, but the expression of Foxp3 gene in cochlea gradually decreased over time. After intravenous transplantation of CD4⁺CD25⁺Treg in AN rats, the threshold of ABR response decreased, and DPOAE had no significant change. The number of spiral ganglion neurons in cochlea increased, and hair cells had no significant change under electron microscope. Conclusions: The decrease in the number and function of CD4⁺CD25⁺Treg reduces its inhibitory effect on autoimmune response and promotes the occurrence of autoimmune auditory neuropathy in AN rats. Adoptive transfer of CD4⁺CD25⁺Treg can reduce the autoimmune response and promote the recovery of autoimmune auditory neuropathy.
Animals ; Rats ; Forkhead Transcription Factors ; Myelin P0 Protein ; Rats, Sprague-Dawley ; T-Lymphocytes, Regulatory ; CD4 Antigens/immunology* ; Interleukin-2 Receptor alpha Subunit/immunology*

OBJECTIVETo study the relationship between the expression of peripheral blood HLA-DR, CD4CD25 regulatory T cells, IL-17 and IL-27 with liver damage in children with human cytomegalovirus (HCMV) infection.

METHODSTwenty-one HCMV children with liver damage and twenty-one HCMV children without liver damage were enrolled in this study. The expression of peripheral blood HLA-DR and CD4CD25 regulatory T cells was detected by flow cytometry. Plasma levels of IL-17 and IL-27 were measured using ELISA.

RESULTSThe plasma levels of IL-17 and IL-27 in children with liver damage were significantly higher than in those without liver damage, while the expression of peripheral blood CD4CD25 regulatory T cells was lower than in those without liver damage (P<0.05). Plasma IL-17 and IL-27 levels were negatively correlated with the expression of peripheral blood CD4CD25 regulatory T cells (P<0.01).

CONCLUSIONSImmune imbalance mediated by CD4CD25 regulatory T cells and over-expression of IL-17 and IL-27 may be involved in the pathogenesis of liver damage in children with HCMV infection.

CD4 Antigens ; immunology ; Cytomegalovirus ; physiology ; Cytomegalovirus Infections ; blood ; complications ; genetics ; Female ; Flow Cytometry ; HLA-DR Antigens ; genetics ; immunology ; Humans ; Infant ; Interleukin-17 ; blood ; genetics ; Interleukin-2 Receptor alpha Subunit ; immunology ; Interleukins ; blood ; genetics ; Liver ; injuries ; metabolism ; Liver Diseases ; blood ; etiology ; immunology ; Male ; T-Lymphocytes, Regulatory ; immunology

OBJECTIVE: To identify differentially expressed genes in peripheral blood mononuclear cells between patients with continuous mild-to-moderate asthma and healthy controls using mRNA microarray in order to explore the underlying signaling pathways and clarify the roles of CD4+ T cells in the pathogenesis of asthma. METHODS: Global transcriptomic profiles of the CD4+ T cells were defined by using Agilent Sure Print G3 Human GE 8×60K microarray. Enrichment pathways were analyzed with Ingenuity Pathway Analysis (IPA) software. RESULTS: Compared with controls, 805 genes were up-regulated, 192 were down-regulated in asthma patients. Among these, the expression of 38 annotated genes have varied by 4 times or more. Expression of CD300A was inversely proportional to the absolute value of eosinophils (r=-0.89, P=0.02) as well as the proportion of eosinophils (r=-0.94, P=0.004), while CSF1R was inversely proportional to PD20 (r=-0.83, P=0.04) and AQLQ (r=-0.88, P=0.02) by correlation analysis. CONCLUSION Numerous pathophysiological pathways may be involved in the pathogenesis of asthma. Above findings have provided a basis for the delineation the pathogenesis of asthma.
Antigens, CD ; genetics ; Asthma ; immunology ; CD4-Positive T-Lymphocytes ; cytology ; Case-Control Studies ; Eosinophils ; Gene Expression Profiling ; Humans ; Leukocytes, Mononuclear ; Oligonucleotide Array Sequence Analysis ; Receptors, Granulocyte-Macrophage Colony-Stimulating Factor ; genetics ; Receptors, Immunologic ; genetics ; Transcriptome

To study the relationship between acute graft-versus-host disease (aGVHD) and the SIRT1 expression in peripheral blood CD4+T cells from patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT).
 Methods: We collected 40 patients who underwent allo-HSCT from human leukocyte antigen (HLA)-identical sibling donors. SIRT1 expression level in CD4+T cells was measured by real-time PCR and Western blot. Acetylation and phosphorylation of STAT3 in CD4+T cells were detected by Western blot. The binding level between SIRT1 and STAT3 in CD4+T cells was analyzed by co-immunoprecipitation and Western blot. Over-expression of SIRT1 in aGVHD CD4+T cells, as well as STAT3 acetylation and phosphorylation were measured by Western blot. The mRNA levels of RORγt, IL-17A, IL-17F related to Th17 were detected by real-time PCR.
 Results: SIRT1 expression was significantly down-regulated, while STAT3 expression, acetylation and phosphorylation levels were significantly up-regulated in patients with aGVHD compared with patients without aGVHD. The STAT3 acetylation was positively correlated with STAT3 phosphorylation (r=0.69, P<0.01). Less SIRT1-STAT3 complexes were found in CD4+T cells from patients with aGVHD compared with patients without aGVHD. After SIRT1 over-expression in aGVHD CD4+T cells, the STAT3 acetylation and phosphorylation, and the expression of RORγt, IL-17A, and IL-17F related to Th17 were significantly down-regulated (P<0.05).
 Conclusion: SIRT1 deficiency in CD4+T cells plays a crucial role in up-regulation of STAT3 acetylation and phosphorylation, the increase of Th17 related gene expression, and induction of aGVHD after allogeneic hematopoietic stem cell transplantation.
Acute Disease ; CD4-Positive T-Lymphocytes ; metabolism ; Down-Regulation ; Graft vs Host Disease ; etiology ; metabolism ; Hematopoietic Stem Cell Transplantation ; Histocompatibility Antigens Class I ; Humans ; Interleukin-17 ; metabolism ; Nuclear Receptor Subfamily 1, Group F, Member 3 ; metabolism ; STAT3 Transcription Factor ; metabolism ; Sirtuin 1 ; deficiency ; metabolism ; Transplantation, Homologous ; Up-Regulation

Objective: To study the specific killing effect of CD4 membrane protein targeted chimeric antigen receptor modified T (CAR-T) cell. Methods: The second generation CD4 targeted chimeric antigen receptor containing 4-1BB costimulation domain was insert into lentiviral vector through recombinant DNA technology. Lentivirus was prepared and packaged by 293T cells with four plasmids. Beads activated T cells were transduced with lentivirus and the transduction efficiency was checked with Protein L and flow cytometry. T cell subsets and IFN-γ concentrations were detected with probe-tagged antibody and cytometric bead assay. Results: ①The transduction efficiency of activated T cells with prepared lentivirus were 50.0%-70.0%. A subset of CD8⁺ T cell acquired dim expression of CD4 membrane protein after activation. CD4⁺T cell and CD8⁺CD4^dim T cell were gradually killed by CD4 targeted CAR-T post lentivirus transduction. ②The kill efficacy of CD4 targeted CAR-T cell and control T cell toward KARPAS 299 T cell at an E∶T ratio of 8∶1 for 24 h was (96.9±2.1)% and (11.2±3.1)%, CAR-T cell has a higher killing efficacy than control T cell (t=7.137, P=0.028). The IFN-γ concentrations in culture supernatant of CAR-T cell with K562-CD4 cell, CAR-T cell with K562 cell and CAR-T cell alone were (15 648±2 168), (1 978±354) and (1 785±268) pg/ml, CAR-T cell cocultured with K562-CD4 cell produced more IFN-γ than the other two controls (P<0.01). Conclusions: CD4 targeted CAR-T has an immunophenotype of CD8⁺CD4^-T cell. CD4 targeted CAR-T cell has killing efficacy toward normal CD4⁺T cell and CD4⁺T lymphoma cell. CD4 targeted CAR-T cell also has a killing efficacy toward CD4^dim target cell.
CD4 Antigens ; CD8-Positive T-Lymphocytes ; Cell Line, Tumor ; Humans ; Lymphoma ; Receptors, Antigen, T-Cell ; Receptors, Chimeric Antigen

No abstract available.
Adult ; Antigens, CD4/*metabolism ; Antigens, CD56/*metabolism ; Bone Marrow/metabolism/pathology ; Dendritic Cells/cytology/*metabolism ; Diagnostic Errors ; Exons ; Female ; Flow Cytometry ; Gene Rearrangement ; Hematologic Neoplasms/diagnosis ; Histone-Lysine N-Methyltransferase/genetics ; Humans ; Immunohistochemistry ; In Situ Hybridization, Fluorescence ; Leukemia, Myeloid, Acute/*diagnosis ; Male ; Middle Aged ; Myeloid-Lymphoid Leukemia Protein/genetics ; Real-Time Polymerase Chain Reaction ; Sequence Analysis, DNA ; Transcription Factors/genetics ; Translocation, Genetic

BACKGROUNDThe interaction between activated microglia and T lymphocytes can yield abundant pro-inflammatory cytokines. Our previous study proved that thymus immune tolerance could alleviate the inflammatory response. This study aimed to investigate whether intrathymic injection of myelin basic protein (MBP) in mice could suppress the inflammatory response after co-culture of T lymphocytes and BV-2 microglia cells.

METHODSTotally, 72 male C57BL/6 mice were randomly assigned to three groups (n = 24 in each): Group A: intrathymic injection of 100 μl MBP (1 mg/ml); Group B: intrathymic injection of 100 μl phosphate-buffered saline (PBS); and Group C: sham operation group. Every eight mice in each group were sacrificed to obtain the spleen at postoperative days 3, 7, and 14, respectively. T lymphocytes those were extracted and purified from the spleens were then co-cultured with activated BV-2 microglia cells at a proportion of 1:2 in the medium containing MBP for 3 days. After identified the T lymphocytes by CD3, surface antigens of T lymphocytes (CD4, CD8, CD152, and CD154) and BV-2 microglia cells (CD45 and CD54) were detected by flow cytometry. The expressions of pro-inflammatory factors of BV-2 microglia cells (interleukin [IL]-1β, tumor necrosis factor-α [TNF-α], and inducible nitric oxide synthase [iNOS]) were detected by quantitative real-time polymerase chain reaction (PCR). One-way analysis of variance (ANOVA) and the least significant difference test were used for data analysis.

RESULTSThe levels of CD152 in Group A showed an upward trend from the 3rd to 7th day, with a downward trend from the 7th to 14th day (20.12 ± 0.71%, 30.71 ± 1.14%, 13.50 ± 0.71% at postoperative days 3, 7, and 14, respectively, P < 0.05). The levels of CD154 in Group A showed a downward trend from the 3rd to 7th day, with an upward trend from the 7th to 14th day (10.00 ± 0.23%, 5.28 ± 0.69%, 14.67 ± 2.71% at postoperative days 3, 7, and 14, respectively, P < 0.05). The ratio of CD4+/CD8 + T in Group A showed a downward trend from the 3rd to 7th day, with the minimum at postoperative day 7, then an upward trend from the 7th to 14th day (P < 0.05). Meanwhile, the levels of CD45 and CD54 in Group A were found as the same trend as the ratio of CD4+/CD8 + T (CD45: 83.39 ± 2.56%, 82.74 ± 2.09%, 87.56 ± 2.11%; CD54: 3.80 ± 0.24%, 0.94 ± 0.40%, 3.41 ± 0.33% at postoperative days 3, 7, and 14, respectively, P < 0.05). The expressions of TNF-α, IL-1β, and iNOS in Group A were significantly lower than those in Groups B and C, and the values at postoperative day 7 were the lowest compared with those at postoperative days 3 and 14 (P < 0.05). No significant difference was found between Groups B and C.

CONCLUSIONSIntrathymic injection of MBP could suppress the immune reaction that might reduce the secondary immune injury of brain tissue induced by an inflammatory response.

Animals ; Anti-Inflammatory Agents ; pharmacology ; Antigens, Surface ; analysis ; Brain Injuries, Traumatic ; drug therapy ; CD4-CD8 Ratio ; Coculture Techniques ; Male ; Mice ; Mice, Inbred C57BL ; Microglia ; immunology ; Myelin Basic Protein ; administration & dosage ; pharmacology ; T-Lymphocytes ; immunology

A two-year-old spayed female pug presented with symmetrical hyperpigmented alopecic lesions on her axillary and inguinal regions. There were no remarkable findings in dermatologic examinations and hormonal assays. Histological examination of biopsied tissues revealed prominent lymphocytic perifolliculitis along with shrunk hair follicles. Immunohistochemistry for CD3, CD79a, CD4, and CD8 showed a positive stain for CD4 antigen around hair bulbs, suggesting CD4 positive T lymphocyte infiltration. This case suggests the possibility that CD4 T lymphocyte-mediated inflammatory reaction could be a main mechanism in canine alopecia areata. Additional studies are warranted to investigate the immunological mechanism in canine species.
Alopecia Areata* ; Alopecia* ; Antigens, CD4 ; Autoimmune Diseases ; Female ; Hair ; Hair Follicle ; Humans ; Immunohistochemistry ; Lymphocytes

Analysis of the T-cell receptor (TCR) repertoire of innate CD4+ T cells selected by major histocompatibility complex (MHC) class II-dependent thymocyte-thymocyte (T-T) interaction (T-T CD4+ T cells) is essential for predicting the characteristics of the antigens that bind to these T cells and for distinguishing T-T CD4+ T cells from other types of innate T cells. Using the TCRmini Tg mouse model, we show that the repertoire of TCRalpha chains in T-T CD4+ T cells was extremely diverse, in contrast to the repertoires previously described for other types of innate T cells. The TCRalpha chain sequences significantly overlapped between T-T CD4+ T cells and conventional CD4+ T cells in the thymus and spleen. However, the diversity of the TCRalpha repertoire of T-T CD4+ T cells seemed to be restricted compared with that of conventional CD4+ T cells. Interestingly, the frequency of the parental OT-II TCRalpha chains was significantly reduced in the process of T-T interaction. This diverse and shifted repertoire in T-T CD4+ T cells has biological relevance in terms of defense against diverse pathogens and a possible regulatory role during peripheral T-T interaction.
Amino Acid Sequence ; Animals ; Antigens, Surface/metabolism ; CD4-Positive T-Lymphocytes/cytology/*immunology/*metabolism ; Cell Communication ; Cell Differentiation/genetics/immunology ; Clonal Evolution ; Histocompatibility Antigens Class II/*immunology ; *Immunity, Innate ; Immunophenotyping ; Lymphocyte Count ; Mice ; Mice, Knockout ; Mice, Transgenic ; Peptide Fragments/chemistry ; Phenotype ; Receptors, Antigen, T-Cell/chemistry/*genetics/metabolism ; Receptors, Antigen, T-Cell, alpha-beta/chemistry/genetics ; Spleen/cytology ; Thymocytes/cytology/immunology/metabolism