Heterogeneity of Human γδ T Cells and Their Role in Cancer Immunity

The γδ T cells are unconventional lymphocytes that function in both innate and adaptive immune responses against various intracellular and infectious stresses. The γδ T cells can be exploited as cancer-killing effector cells since γδ TCRs recognize MHC-like molecules and growth factor receptors that are upregulated in cancer cells, and γδ T cells can differentiate into cytotoxic effector cells. However, γδ T cells may also promote tumor progression by secreting IL-17 or other cytokines. Therefore, it is essential to understand how the differentiation and homeostasis of γδ T cells are regulated and whether distinct γδ T cell subsets have different functions. Human γδ T cells are classified into Vδ2 and non-Vδ2 γδ T cells. The majority of Vδ2 γδ T cells are Vγ9δ2 T cells that recognize pyrophosphorylated isoprenoids generated by the dysregulated mevalonate pathway. In contrast, Vδ1 T cells expand from initially diverse TCR repertoire in patients with infectious diseases and cancers. The ligands of Vδ1 T cells are diverse and include the growth factor receptors such as endothelial protein C receptor. Both Vδ1 and Vδ2 γδ T cells are implicated to have immunotherapeutic potentials for cancers, but the detailed elucidation of the distinct characteristics of 2 populations will be required to enhance the immunotherapeutic potential of γδ T cells. Here, we summarize recent progress regarding cancer immunology of human γδ T cells, including their development, heterogeneity, and plasticity, the putative mechanisms underlying ligand recognition and activation, and their dual effects on tumor progression in the tumor microenvironment.

Primary Peripheral Gamma Delta T-Cell Lymphoma of the Central Nervous System: Report of a Case Involving the Intramedullary Spinal Cord and Presenting with Myelopathy

Primary central nervous system lymphoma of T-cell origin (T-PCNSL) is rare, and its clinicopathological features remain unclear. Peripheral T-cell lymphoma of γδ T-cell origin is an aggressive lymphoma mainly involving extranodal sites. Here, we report a case of γδ T-PCNSL involving the intramedullary spinal cord and presenting with paraplegia. A 75-year-old Korean woman visited the hospital complaining of back pain and lower extremity weakness. Magnetic resonance imaging revealed multifocal enhancing intramedullary nodular lesions in the thoracic and lumbar spinal cord. An enhancing nodular lesion was observed in the periventricular white matter of the lateral ventricle in the brain. There were no other abnormalities in systemic organs or skin. Laminectomy and tumor removal were performed. The tumor consisted of monomorphic, medium-to-large atypical lymphocytes with pale-to-eosinophilic cytoplasm. Immunohistochemically, the tumor cells were CD3(+), TCRβF1(-), TCRγ(+), CD30(-), CD4(-), CD8(-), CD56(+), TIA1(+), granzyme B(+), and CD103(+). Epstein-Barr virus in situ was negative. This case represents a unique T-PCNSL of γδ T-cell origin involving the spinal cord.

Expression of PD1 and BTLA on the CD8+ T Cell and γδT Cell Subsets in Peripheral Blood of Non-Small Cell Lung Cancer Patients / 中国医学科学杂志(英文版)

Objective To investigate the expression and regulation of programmed cell death protein 1 (PD1), B lymphocyte and T lymphocyte attenuator (BTLA) in peripheral blood of patients with non-small cell lung cancer (NSCLC); to examine the correlation of the mRNA levels between PD and BTLA in NSCLC. Methods Flow cytometry was used to detect the expression of PD1 and BTLA on the surfaces of CD8+ T cells and γδ+ T cells in the peripheral blood samples collected from 32 in-patients with stage IV NSCLC and 30 healthy individuals. We compared the expression of PD1 and BTLA on the surfaces of γδ+ T cells in the NSCLC patients with bone metastasis before and after the treatment of zoledronic acid. The correlations of PD1 and BTLA, as well as their ligands were analyzed using Pearson correlation analysis with the cBioPortal data platform. Results The frequency of PD1 on the surfaces of CD8+ T cells was significantly higher than that of the γδT cells in both healthy controls (t=2.324, P=0.024) and NSCLC patients（t=2.498, P=0.015). The frequency of PD1 on CD8+ T cells, rather than on γδ+ T cells, was significantly upregulated in advanced NSCLC patients compared with that in healthy controls (t=4.829, P<0.001). The PD1+ BTLA+γδT cells of the healthy controls were significantly lower than that of the NSCLC patients (t=2.422, P=0.0185). No differences in percentage of PD1+γδ+ and BTLA+γδ+ T cells were observed in 7 NSCLC patients with bone metastasis before and after zoledronic acid treatment. PD1 was positively correlated with BTLA in both lung adenocarcinoma (r=0.54; P<0.05) and lung squamous cell carcinoma (r=0.78; P<0.05). Conclusions The upregulation of co-inhibitory molecules occurs on the surfaces of both CD8+ T cells and γδT cells in advanced NSCLC, suggesting that these molecules were involved in regulating the inactivation of CD8+ T cells and γδ+ T cells, immune escape and tumor invasion.

Phenotype analysis of γδ T cell and their subsets in acute HIV-infected patients / 中国免疫学杂志

Objective:To analyze the phenotypes of γδ T cell in acute HIV-infected patients,and to clarify the role of γδ T cell in acute HIV infection(AHI). Methods:The expressions of CD38,programmed cell death protein-1(PD-1),CD160,CD95 and tumor necrosis factor-related apoptosis inducing ligand-death receptor 5 ( TRAIL-DR5 ) were detected by using flow cytometry. Results:Compared with healthy controls,the frequencies of Vδ1 T cells were significantly increased and the frequencies of Vδ2 T cells were de-creased,however,the frequencies of total γδ T cells were not significantly changed in acute HIV-infected patients. The frequencies of CD38+γδ T cells,CD38+Vδ1 T cells and CD38+Vδ2 T cells were significantly increased compared with healthy controls. Moreover, compared with healthy controls,the frequencies of PD-1+γδ T cells and DR5+γδ T cells were increased,but the expression of PD-1 and DR5 on Vδ1 and Vδ2 T cells were not significantly changed. However,the frequencies of CD38+Vδ1 T cells,CD160+Vδ1 T cells,PD-1+Vδ1 T cells,CD95+Vδ1 T cells and DR5+Vδ1 T cells were significantly increased compared with Vδ2 T cells in acute HIV-infected patients. Conclusion:Activation,inhibitor and apoptosis markers are highly expressed on γδ T cells,especially on Vδ1 T cells in acute HIV-infected patients,suggest that Vδ1 T cells play more important roles in acute HIV infection and disease progression.

Optimization of in vitroamplification of peripheral blood γδ T cells with cytokines / 基础医学与临床

Objective To optimize in vitro amplification of human γδ T cells with cytokines for tumor adoptive immunotherapy.Methods On the basis of the immobilized anti-TCR γδ antibody plus IL-2 system, other γ chain receptor family cytokines, including IL-7, IL-15 and IL-21, were tested to amplify human peripheral blood γδ T cells either alone or in diversity combination.The percentage of γδ T cells was measured by flow cytometry, and the proliferation efficiency of γδ T cells was calculated.The expression of proliferation-or cytotoxicity-related molecules on γδ T cells was examined by flow cytometry in order to explore the relevant mechanisms.The cytotoxicity of γδ T cells to Daudi cells was detected by lactate dehydrogenase.Results IL-15 alone but not IL-7 or IL-21 increases the γδ T cell purity, amplification efficiency and cytotoxicity to reach comparable levels to those of IL-2.IL-2 plus IL-15 up-regulates the expression of CD69 on γδ T cells and significantly increases their amplificationefficiency (P<0.05).IL-2 plus IL-21 enhanced the cytotoxicity of γδ T cells against Daudi cells by increasing the expression of granzyme A (P<0.001).The combination of IL-2, IL-15 and IL-21 significantly improves cytotoxicity of γδ T cells but reduces their amplification efficiency.In addition, when IL-21 was applied for a short time, it also enhanced the cytotoxicity of γδ T cells (P<0.05).Conclusions The combination of IL-2 and IL-15 as well as a short time addition of IL-21 is the best cytokine recipe to amplify human peripheral blood γδ T cells in vitro with immobilized anti-TCR γδ antibody, which can increase both the proliferation efficiency and the cytotoxicity to tumor cells of γδ T cells.

Construction of lung cancer cell model overexpressing human MutS homologue 2(hMSH2) / 基础医学与临床

Objective To construct human lung cancer cell model with human MutS homologous protein 2 (hMSH2) overexpression for exploring the effect of hMSH2 molecule in the cytotoxicity of γδ T cell against lung cancer cells.Methods hMSH2 coding sequence was cloned by PCR for construction of recombinant vector which over expressed hMSH2-EGFP fusion protein using homologous recombination.The recombinant vector was transfected to lung cancer cell line NCI-H520 to construct human lung cancer cell model overexpressing hMSH2 molecule.The expression of hMSH2 molecule in NCI-H520 was detected by Western blot.The expression of hMSH2 on the cell membrane was measured by flow cytometry.Cytotoxicity of expanded γδ T cells from peripheral blood mononuclear cells against NCI-H520 cells was detected by LDH release assay in vitro.Results hMSH2 coding sequence (2805 bp) was cloned and the result of restriction endonuclease digestion of Fugw-hMSH2 recombinant vector was accordance with the anticipated objective strip size.Exogenous hMSH2-EGFP fusion protein was expressed in NCIH520 cells.The level of hMSH2 molecule on the surface of NCI-H520 cells with overexpression of hMSH2 was significantly increased (P＜0.001).Cytotoxicity of γδ T cells against NCI-H520 cells with overexpression of hMSH2 was significantly increased compared to the wild type NCI-H520 cells (P＜0.05).Conclusions Lung cancer cell model that overexpresses hMSH2 molecule is successfully constructed,hMSH2 molecule on the cell membrane is increased and the cytotoxicity of γδ T cells against lung cancer cells is enhanced.

Cytotoxic role of γδT cells to latency cells in patients with early human immunodeficiency virus-1 infection / 基础医学与临床

Objective To investigate the cytotoxicity of γδ T cells to HIV-1 latency cells in patients with early HIV-1 infection.Methods Sixteen early HIV-1-infected patients were enrolled in this study.Peripheral blood mononuclear cells (PBMCs) of patients were isolated and γδ T cells were expanded using zoledronate (5 μmol/L) and interleukin (IL)-2 (1 000 IU/mL) ex vivo.Lactic dehydrogenase (LDH) was used to detect the cytotoxic role of γδ T cells to HIV-1 latency cells(J-Lat Full Length Clonel0.6).The phenotype of γδ T cells before and after expansion and the intensity of GFP in HIV-1 latency cells were detected by flow cytometry.Results Zoledronate plus IL-2 stimulated rapid and large γδ T cells proliferation ex vivo (P＜0.001).γδ T cells showed high cytotoxici ty to latency cells,and the intensity of GFP in latency cells was decreased significantly (P＜0.05).Moreover,expanded γδ T cells displayed cytotoxic NK-like phenotype,the frequency of CD56+ Vδ2 T cells in patients with early HIV-1 infection was significantly higher than that of healthy controls.Conclusions γδ T cell has an ability to eradicate HIV-1 latency,and γδ T cell-based autologous or xenogenous adoptive immunotherapy will have promise prospects to cure HIV-1 infection.

Celastrol increases osteosarcoma cells line HOS lysis by γδ T cells through TRAIL way / 中国免疫学杂志

Objective:To investigate whether celastrol can increase the cytotoxic activity of γδ T cells against osteosarcoma (OS) cells line HOS through TRAIL way. Methods:The death receptors 4/5 (DR4/5) protein levels in the OS cell lines HOS was in-vestigated by Western blot analysis. Zoledronate ( ZOL) was used to induceγδT cells from PBMCs of healthy volunteers.γδT cell cy-totoxicity against HOS cells was investigated by a standard lactate dehydrogenase release assay ( LDH ) . Results:Celastrol increased DR4/5 protein expression in HOS ( P<0. 05 ) .γδ T cells from PBMCs of healthy volunteers showed cytotoxicity against HOS ( P<0. 05). Following co-culture with HOS pre-treated with celastrol (1 μmol/L) for 24 h,γδ T cells showed significantly higher cytotoxicity against HOS (P<0. 05). The induction of DR4/5 molecules increased lysis of HOS by γδ T cells which was abolished by addition of a blocking TRAIL antibody. Conclusion:Celastrol can enhance γδ T cells'cytotoxic activity against OS cells line HOS through TRAIL way.

Υδ T cells play an important role in the development of RSV-influenced allergic airway inflammation / 中华微生物学和免疫学杂志

ObjectiveTo investigate whether γδ T cells act as a regulatory factor during respiratory syncytial virus (RSV) infections was responsible for the subsequent changes in asthmatic-type inflammation in allergic mice.MethodsMice were sensitized and challenged with OVA,and infected intranasaly with RSV before or after OVA sensitization.Lung sections were stained with HE for determination of inflammatory reaction.Real-time RT-PCR was used to analyze the expression of cytokine mRNA of γδ T cells in the lung and spleen of tested mice.The number of γδ T cells in the spleen and lung of BALB/c mice was determined by flow cytometry.Adoptive transfer of γδ T cells was performed to identify the role of γδ T cells in allergic asthma.ResultsOVA-sensitized and challenged mice exhibited significantly peribronchial inflammation with larger number of mononuclear cells and granulocytes in the lung tissue sections.RSV infection before OVA-sensitization diminished the grade of inflammatory responses induced by OVA treatment.The expression of IFN-γ mRNA was increased siguificantly in RSV-infected,OVA-sensitized mice.In contrast,the level of IL-4 mRNA was diminished.The number of total γδ T cells as well as activated γδ T cells was decreased in the spleen and lung of OVA-sensitized mice by prior RSV infection.Adoptive transfer of γδ T cells obtained from OVA-sensitized and challenged mice induced a slight inflammation in the lung of normal mice,and enhanced inflammatory responses in RSV-infected OVA-sensitized mice.Conclusionγδ T cells may play an important role in the development of allergen-induced allergic airway inflammation.

CD45 MOLECULE AND GENERATION OF EPIDERMAL γδ T CELLS FROM MICE / 免疫学杂志

To investigate the role of CD45 protein tyrosine phosphatase in γδ T cell development,we exa-mined whether Vγ3 dendritic epidermal T cells (DETC),a subset of γδ T cells uniquely reside in the murine epidermis were altered in the CD45-gene -deficient mice.In situ immunolabelling on epidermal sheets demonstrsted that the CD45-deficient mice had a normal density and immunophenotype of Vγ3 DETC in comparison to the wild-type control mice.RT-PCR revealed that similar levels of Vγ3 TCR mRNA were present in the epidermis of both CD45-deficient mice and wild-type controls.FCM showed no significant difference in the proportion of Vγ3 T cells in the epidermal cells between the two genotypes.In addition, the frequency of Vγ2 T cells, another subset of γδT cells in lymph nodes was normal in CD45-deficient mice.These results indicate that althouh CD45 is crucial for the development of αβΤ cells,it might be not necessary for the thymic maturation of γδ T cells including Vγ3 DETC and Vγ2 T cells.