A Preliminary Research On The Mechanism And Role Of Vγ1 And Vγ4γδ T Cell Subpopulations In Anti-tumor Immune Response

γδT cells play a critical role in the protective immune response against tumor development.Mice lackingγδT cells(TCRδ^-/- mice) are more susceptible to MCA-induced tumor formation than wild-type mice.Moreover,it is shown thatγδT cells play a more important role in antitumor immune response thanαβT cells in carcinogenesis models.Vγ1 and Vγ4 are the two dominant subsets of peripheralγδT cells.More recently, it has been demonstrated that these two subsets have different roles in systemic listeriosis and asthma.However,the role of Vγ1 versus Vγ4γδT cells in tumor immune response is unclear.UnlikeαβT cells,most peripheralγδT cells exhibit a spontaneous activation phenotype,which is characterized by the surface expression of the activation marker (CD44^high) and by a fast turnover rate.Our previous studies also demonstrated that CD44^highγδT cells,but not CD44^low one,spontaneously express IFN-γand T-bet,and rapidly express IFN-γupon TCR triggering.This further supports the notion that they are indeed activated. However,the precise role of those CD44^highγδT cells in tumor immune response has not been clarified yet.Both the production of IFN-γand the ability to kill tumor cells were the critical physiologically relevant cellular effectors of immunosurveillance in eradicating developing tumors.IFN-γis a critical cytokine in antitumor immune responses.Endogenously produced IFN-γwas shown to inhibit the growth of transplanted tumors,in addition to cease the formation of primary chemically induced and spontaneous tumors.Our previous study also demonstrated thatγδT cells provided an early source of IFN-γwhich regulated theαβT cell effector function in the antitumor immune response.Early studies identified perforin as a critical cytolytic molecule in antitumor immune response.Compared with wild-type mice,the perforin^-/- mice developed tumors with greater frequency in response to the chemical carcinogen methylcholanthrene(MCA).However,the precise role of IFN-γand perforin in Vγ1 versus Vγ4γδT cells in tumor immune response is not fully understood.In Th1 cells,at least two distinct receptor-mediated pathways can induce IFN-γproduction.T cell receptor(TCR)-induced IFN-γproduction is antigen-specific as a part of the adaptive immune response.The differentiated Th1 cells can also produce IFN-γdirectly in response to IL-12 and IL-18 as part of the innate immune response.Furthermore,inαβT cells,the primary way to produce IFN-γis TCR-dependent pathways,whereas the primary way to induce IFN-γproduction in NK cells is based on cytokine pathway.As the bridge between the innate(NK and macrophages) and the adaptive immune response,whether theγδT cells can produce IFN-γunder the condition of cytokine stimulation is unclear. Moreover,it is remain unclear whether the expression of perforin can be regulated through the TCR and cytokine pathways in Vγ1 and Vγ4γδT cells.To answer these questions will not only shed light on the molecular mechanisms involved in tumor immune surveillance,but may also lead to the development of new strategies for tumor immunotherapy.Our main findings and conclusions of this study are summarized as follows:1.Vγ4γδT cells possess powerful capacity against tumor,while Vγ1γδT cells may be involved in regulation of Vγ4γδT cells resistance to tumor development.â‘ depletion experiment In order to define the roles of Vγ1 and Vγ4γδT cells in antitumor immune response,sex- and age-matched B6 mice were treated with Vγ1-depletion Ab(2.11) or Vγ4-depletion Ab(UC3) or the control antibody(n=15 per each group) at day -5 and -1,and on day 0,the mice were then inoculated subcutaneously with B16 F0 tumor cells(2×10⁵/mouse).Tumor growth was monitored and recorded daily.In our preliminary studies,injection of Vγ1 or Vγ4 antibody completely depleted Vγ1 and Vγ4γδT cells and the depletion lasted for about 3 weeks.Compared to Wt depleted mice,Vγ1 depleted mice showed delayed tumor development(p＜0.05) as well as smaller tumor size (p＜0.05);but it is not found significant difference between Wt depleted and Vγ4 depleted mice.â‘¡reconstitution experiment To further define the role of Vγ4γδT cells,sex- and age-matched B6 TCRδ^-/- mice were transferred with sorted Vγ1 or Vγ4γδT cells (1×10⁵/mouse)(n=6) and inoculated with the B16 F0 melanoma cell line(2×10⁵/mouse) on the following day,and the incidence of tumor growth was monitored.Similar to the depletion experiment above,mice reconstituted with Vγ4γδT cells,compared with Wt, were highly protective to tumor development(p＜0.05);but it is not found significant difference between Wt mice and Vγ1γδT cells reconstituted mice.2.Spontaneously activated,but not effector,Vγ1γδT cells may play a role in regulating otherγδT cells' anti-tumor activityâ‘ To access the production of IFN-γin Spontaneously Activated(CD44^high) Vγ1 or Vγ4γδT cells upon TCR stimulation,we sorted CD44^high Vγ1,CD44^high Vγ4,CD44^low Vγ1,CD44^low Vγ4γδT cells from naive B6 mice splenocytes and cultured them with anti-mouse CD3 antibody and IL-2 for 5 days,then the expanded cells were restimulated with anti-mouse CD3 antibody for 6 hours as described in material and method.Analysis of cytokine-producing cells revealed that just 18.94%Spontaneously Activated Vγ1γδT cells produced IFN-γ,which is much less than Spontaneously Activated Vγ4γδT cells(45.84%), naive Vγ1γδT cells(42.54%) and na(i|¨)ve Vγ4γδT cells(57.93%),indicating that Spontaneously Activated Vγ1γδT cells may have special function which is different from otherγδT cells.â‘¡Reconstitution experiment To define what different role of the spontaneously activated CD44^high and naive CD44^low Vγ1 or Vγ4γδT cells play in antitumor immune response,sex- and age-matched B6 TCRδ^-/- mice were reconstituted with the sorted activated(CD44^high) or na(i|¨)ve(CD44^low) Vγ1 and Vγ4γδT cells(1×10⁵/mouse) by i.v. injection and inoculated with B16 F0 melanoma cell line(2×10⁵/mouse).Starting on the following day,tumor growth was monitored and recorded daily.The mice reconstituted with activated Vγ4,na(i|¨)ve Vγ4 and na(i|¨)ve Vγ1γδT cells showed more protective tumor growth than those reconstituted with activated Vγ1γδT cells(p＜0.05),and no significant difference was observed in their tumor growth among mice reconstituted with activated Vγ4, na(i|¨)ve Vγ4 and na(i|¨)ve Vγ1γδT cells,indicating that,excepts the spontaneously activated Vγ1γδT cells population,otherγδT cells population have powerful effect in antitumor immune response. 3.Both IFN-γand perforin is required for spontaneously CD44^high Vγ4-mediated tumor protection;However,CD44^high Vγ1γδT cells do not show IFN-γ- or perforin-mediated antitumor effector function in vivoâ‘ tumor killing experiment in vivo To further confirm the role of the spontaneously activated Vγ1 and Vγ4γδT cells in tumor resistance,we expanded the spontaneously activated Vγ1 and Vγ4γδT cells in vitro,and then,the expanded CD44^high Vγ1 and Vγ4γδT cells populations were mixed with of B16 cells at ratio 1:4 respectively before coinjection into the skin of recipient B6 TCRδ^-/- mice.After injection,recipient mice were monitored for the presence of tumors daily.It can be seen that,compared to B6 TCRδ^-/- mice,CD44^high Vγ4γδT cells coinjection mice were more effective at controlling tumor formation and retarding tumor growth(P＜0.05),but CD44^high Vγ1γδT cells coinjection mice did not,indicating that the CD44^high Vγ4γδT cells have directly effector function to tumor development,but CD44^high Vγ1γδT cells do not.â‘¡IFN-γand perforin are two key factors for tumor immune surveillance;we next assessed the requirement of IFN-γand perforin for the role of CD44^high Vγ4γδT cell in antitumor immune response.The expanded Wt,IFN-γ^-/- or perforin^-/- CD44^high Vγ4γδT cells populations were prepared,and mixed with of B16 cells at a ratio of 1:4 respectively before coinjection into the skin of recipient B6 TCRδ^-/- mice(n=5 per each group).Tumor growth was monitored and recorded daily.In comparison to Wt CD44^high Vγ4 coinjection mice,both IFN-γ^-/-(P＜0.05) and perforin^-/-(P＜0.01) CD44^high Vγ4 coinjection mice were highly susceptible to tumor growth,therefore demonstrating that both IFN-γand perforin are required for the effector function of CD44^high Vγ4γδT cell in antitumor immune response.Notably,IFN-γ^-/- Vγ4γδT cells render B6 TCRδ^-/- mice more protective to tumor development than perforin^-/- Vγ4γδT cells(P＜0.05),indicating that perforin plays a more important role in Vγ4-mediated local tumor protection than IFN-γ.â‘¢In order to assessed the requirement of IFN-γand perforin for the role of CD44^high Vγ1γδT cell in antitumor immune response.The expanded Wt,IFN-γ^-/- or perforin^-/- CD44^high Vγ1γδT cells populations were prepared as described above,and mixed with of B16 cells at a ratio of 1:4 respectively before coinjection into the skin of recipient B6 TCRδ^-/- mice(n=5 per each group).Tumor growth was monitored and recorded daily as described above.No significant differences were detected between any groups of Vγ1γδT cells coinjection mice(Fig.3B),it was suggested that CD44^high Vγ1γδT cells might lack both IFN-γand perforin-mediated antitumor effector function.4.Spontaneously activated(CD44^high) Vγ4γδT cells produce a much higher level of IFN-γthan spontaneously activated(CD44^high) Vγ1γδT cells does through either TCR pathway or cytokine pathwayâ‘ CD3 stimulation ex vivo To access the production of IFN-γin spontaneously activated(CD44^high) Vγ1 or Vγ4γδT cells upon TCR stimulation,we firstly sorted the spontaneously activated(CD44^high) populations of Vγ1 and Vγ4γδT cells from naive B6 mice splenocytes and stimulated them immediately with anti-mouse CD3 antibody for 6 hours.Analysis of cytokine-producing cells revealed that the percentage of IFN-γ-producing Vγ1γδT cells(2.78%) were lower than that of IFN-γ-producing Vγ4γδT cells(4.27%).â‘¡CD3 stimulation in vitro Secondly,we expanded CD44^high Vγ1 and Vγ4γδT cells in vitro.The expanded cells were restimulated with anti-mouse CD3 antibody for 6 hours.Analysis of cytokine-producing cells revealed that the percentage of IFN-γ-producing Vγ1γδT cells(18.94%) were much fewer than that of IFN-γ-producing Vγ4γδT cells(45.84%),but the percentage of both IL-4-producing Vγ1γδT cells (0.47%)and IL-4-producing Vγ4γδT cells(0.10%) are less than 0.50%.â‘¢IL12+IL18 stimulation ex vivo We further assessed the production of IFN-γin spontaneously activated(CD44^high) Vγ1 or Vγ4γδT cells upon cytokine stimulation.The sorted activated(CD44^high) populations of Vγ1 and Vγ4γδT cells were cultured with IL12 and IL18 for 6 hours for cytokine analysis.The results showed that the percentage of IFN-γ-producing Vγ4γδT cells(20.24%) were almost two fold of that of IFN-γ-producing Vγ1γδT cells(12.5%).â‘£IL12+IL18 stimulation in vitro The expanded CD44^high Vγ1 and Vγ4γδT cells in vitro were restimulated with IL12 and IL18 for cytokine analysis.The results show that the percentage of IFN-γ-producing Vγ1γδT cells(29.16%) were much fewer than that of IFN-γ-producing Vγ4γδT cells(60.24%).⑤Both T-bet and Eomes have been defined as the transcription factors that contribute to IFN-γproduction in CD8⁺ T cells.To assess the transcription of T-bet and Eomes in CD44^high Vγ1γδT Cells and CD44^high Vγ4γδT Cells,CD44^high Vγ1γδT Cells and CD44^high Vγ4γδT Cells sorted from B6 mice splenocytes were used for real-time PCR analysis.The transcription of T-bet in CD44^high Vγ4γδT Cells was approximately two fold below that in CD44^high Vγ1γδT Cells.Strikingly,The transcription of Eomes in CD44^high Vγ1γδT Cells was approximately twenty fold below that in CD44^high Vγ4γδT Cells, indicating that Eomes play more important role in function of CD44^high Vγ4γδT Cells.5.Spontaneously activated(CD44^high) Vγ4γδT cells have more CTL ability than spontaneously activated(CD44^high) Vγ1γδT cells does through producing more perforinâ‘ CTL assay To assess the cytotoxicity of Vγ4γδT cells to tumor cell in vitro, sorted CD44^high Vγ4 and Vγ1γδT cells were expanded,and the CTL ability of these expanded cells was analyzed using the JAM test.YAC-1 cells were used as the target cells. Vγ4γδT cells showed significantly increased CTL activity at higher E:T ratios(10:1,20:1 and 40:1)(P＜0.05),suggesting that CD44^high Vγ4γδT cells have higher cytotoxicity to tumor cells than CD44^high Vγ1γδT cell in vitro.â‘¡CD3 stimulation ex vivo Given the information that CD44^high Vγ4γδT cells produce more IFN-γ,we wanted to determine whether there was any difference of the production of perforin between in CD44^high Vγ4 and Vγ1γδT cells.To do this, spontaneously activated(CD44^high) populations of Vγ1 and Vγ4γδT cells were sorted from B6 mice splenocytes,and stimulated with anti-mouse CD3 antibody for 6 hours,and used for intracellular cytokine staining.Analysis of cytokine-producing cells revealed that the percentage of perforin-producing Vγ1γδT cells(2.67%) were much fewer than that of perforin-producing Vγ4γδT cells(8.47%).â‘¢IL12+IL18 stimulation ex vivo Sorted spontaneously activated(CD44^high) populations of Vγ1 and Vγ4γδT cells were then stimulated with IL 12 and IL 18 for 6 hours. The results shown that the percentage of perforin-producing Vγ4γδT cells(2.56%) were ahnost two fold of that of perforin-producing Vγ1γδT cells(20.27%).The results demonstrate that CD44^high Vγ4γδT cells produce higher levels of perforin compared to Vγ1γδT cells upon TCR and cytokine stimulation.â‘£Infiltrating Vγ1 and Vγ4γδT cells produce perforin in situ To test whether does the infiltrating Vγ1 and Vγ4γδT cells produce perforin in situ,B6 mice were injected with B16 melanoma cells,the tissue at the tumor injection site was then digested,and infiltrating cells were stained directly for anti-Vγ1 or anti-Vγ4 followed by intracellular perforin staining.The results also demonstrate that there are much more infiltrating perforin-producing Vγ4γδT cells(1.09%) than the infiltrating perforin-producing Vγ1γδT cells(0.07%).