Effect Of Expression Level Of PD-1 On Tumor-associated Macrophage In Non-Hodgkin Lymphoma

Background:macrophage is one of the most important cell in innate immunity.The key biological feature of macrophage is the high plasticity when confront different microenvironmental effectors.Tumor always recruit the monocyte in peripheral circulation,and "educate" macrophage into tumor associated macrophage(TAM),which usually manifest as M2 type macrophage in terms of cell marker and functions.TAMs act as a tumor promotor in several ways,especially an important role in immoral evasion.Moreover,in NHL cases,TAMs are believed to be closely related to the lymphomagenesis.Another mechanism involved in immunity evasion is checkpoint immunity,and Programmed Death-1 is the most representative molecule in this family.PD-1 mainly expressed on activated T cell,but also detected on B cell?NK cell?Macrophage and other immune cells.The expression of PD-1 will inhibit the activity of T cell,but its expression on macrophage is not well understood.Through certain disease models,we discovered that PD-1 positive macrophage showed dysfunction of anti-inflammation activity,which lead to an immunity evasion.The percentage of PD-1 positive monocytes will cause an upregulation of IL-10 secretion,and dysfunction of monocyte-macrophage.Furthermore,the percentage of PD-1 positive macrophage may increase along with disease progression,also the phagocytosis is inhibited.Based on theories mentioned above.We attempt to detect the co-expression of TAMs marker and PD-1 on NHL patients' biopsy;and explore the effect of PD-1 expression on macrophage in NHL,including polarization,secretion and phagocytosis.Method:1.We collected 17 newly-diagnosed peripheral T cell lymphoma patients,and detected the markers for TAMs and PD01,using a multi-color Immunofluorescence staining technique.2.We structured the PD-1 overexpression THP-1 cell through Levi virus transfection.3.We incubated PD-1 over-expression THP-1 cell and mock THP-1 cell with Ml related cytokines,M2 related cytokines,and lymphoma cell strains(Jurkat cell and Raji cell),to examine the function of macrophage.We detected macrophage related markers(CD68 and CD 192 for M1 macrophage,while CD206 and CD 163 for M2 macrophage)through flow cytometry;we detected levels of IL-10 and IL-12 in cell culture supernatant through ELISA test;we also detected positivity rate of BSA-FITC through flow cytometry to evaluate the phagocytosis of macrophage.Result:1.Co-expression of TAMs marker and PD-1 in PTCL patients.(1)The dual positivity rate of CD68/PD-1 was 0.99%(range from 0.01?17.51%),and the dual positive cell number was 21.76/1000000pixels(range from 0.25?426.5/1 OOOOOOpixels);the dual positivity rate of CD206/PD-1 was 0.38%(range from 0-23.88%),and the cell number was 8.72/1000000pixels(range from 0?488.4/1 OOOOOOpixels).The co-expression of TAMs marker and PD-1 varies widely in PTCL patients.(2)The Ann Arbor staging system showed a significant relation with CD206/PD-1 co-expression rate,as well as dual positive cell number.No significant relation between other parameters.2.Exploration of effect of PD-1 expression on TAMs in NHL(1)After incubated with M1 related cytokines,the positivity rates of Ml related markers were significantly decreased when compared to mock cells(OE/MOCK group:CD68 23.5%vs 65.67%;CD192 28.1%vs 55.43%;CD68+/CD192+7.8%vs 40.07%,P<0.001).Also,the M2 related markers decreased significantly after incubation with M2 related cytokines,but in a smaller amplitude(OE/MOCK:CD206 58.38%vs 80.7%,P<0.001;CD163 7.37%vs 10.4%,P=0.049;CD206+/CD163+4.31%vs 8.68%,P=0.003).The phagocytosis was inhibited in PD-1 expression THP-1 cell in both circumstance when compared to MOCK cells(M1 group:OE/MOCK 13.3%vs 72.5%,P<0.001;M2 group:OE/MOCK 10.4%vs 64.17%).(2)After the stimulation of cytokines secreted by lymphoma cells(Jurkat or Ragi cells),the Ml related marker decreased significantly when compared to mock THP-1 cells(OE/MOCK group:CD68 18.7%vs 62.57%;CD192 22.37%vs 38.03%;CD68+/CD192+ 5.1%vs 25.87%,P<0.001),however,M2 related markers showed no significant change or a small drop(OE/MOCK:CD206 60.35%vs 75.93%,p<0.001;CD163 8.77%vs 7.19%,p=0.156;CD206+/CD163+5.91%vs 5.99%,p=0.924).And the phagocytosis was inhibited significantly when compared to mock cells(OE/MOCK:6.7%vs 41.1%,P<0.001)(3)The direct contact culture of lymphoma cells and THP-1 cells decreased the Ml related markers on macrophage significantly(OE/MOCK:CD68 19.83%vs 54.77%,p=0.002;CD192 55%vs 69.23%,p=0.04;CD68+/CD192+ 12.27%vs 40.07%,<0.001),however,the rate of M2 related marker showed no significant change or a small drop(OE/MOCK:CD206 71.73%vs 75.93%,p<0.001;CD1633.03%vs 7.19%,p=0.076;CD206+/CD163+ 2.3%vs 5.99%,p=0.024).The secretions of IL-12 were inhibited equally in both group(OE/MOCK:33.96pg/ml vs 30.72 pg/ml),but PD-1 expression THP-1 cell seemed to secret more IL-10(OE/MOCK:144.03pg/ml vs 32.33pg/ml).Also the phagocytosis was significantly inhibited when compared to mock cells(OE/MOCK:4.42%vs 40.7%,p<0.001).Conclusion:1.The co-expression of TAMs marker and PD-1 varied widely in PTCL patients,but the co-expression of CD206 and PD-1 showed significant relation with Ann Arbor staging,implicating the potential value in prognostic classification.2.The PD-1 expression on macrophage may inhibit the polarization of macrophage into Ml type,however its polarization into M2 macrophage seems to be preserved.In the circumstance with lymphoma,PD-1 expression macrophage trended to secret more IL-10,while the IL-12 secretion was equally inhibited.Also,the expression of PD-1 on macrophage inhibited its phagocytosis.