Interaction Between HRS And Tumor-infiltrating CD4~+T Cells In CHL

BackgroundLymphoma, also known as malignant lymphoma, can be mainly in the lymph nodes and extronodal lymphoid tissue.Because lymphocytes are the main components of the immune system,it is defined as a malignancy of the lymphatic system.In recent years,the incidence of lymphoma has a significant increasing trend.Therefore,many researchers have focused on it.Lymphoma is divided into Hodgkin lymphoma(HL) and Non-Hodgkin lymphoma (NHL). HL was first recognized and described its morphological characteriatics by Thomas Hodgkin in 1832.HL is categorised morphologically into classical HL(CHL) and nodular lymphocyte predominant HL(NLPHL).CHL is subclassified into nodular sclerosis HL(NSHL),mixed cellularity HL(MCHL), lymphocyte-rich HL(LRHL) and lymphocyte depleted HL(LDHL) depending on the architectural features and the cellular composition of the inflammatory background. NLPHL has been recognized as a separate B-cell lymphoma with a clinical course different from the other four forms of CHL.95% of all HL patients come down with CHL,the NSHL subtype dominating within the CHL.HL is characterized by the presence of relatively few giant neoplastic cell, Hodgkin and Reed-Sternberg(HRS) cells,embeded in a background of abundant reactive cells, including lymphocytes,plasma cells,eosinophils,mast cells and others.The typical R-S cell is "mirror image cell",mononuclear cell with the same characteristics as "mirror cell" is "Hodgkin cell",therefore,HRS cells are referred as HL tumor cells collectively. The functional role of these tumour-infiltrating lymphocytes(TIL) is controversially discussed.Generally,TIL are considered to represent a host immune response directed against neoplastic cells unsuccessfully,or may be due to need to rely on the surrounding microenvironment for its survival and proliferation.All the TIL and some extracelluar stroma constitute microenvironment. The interaction between tumour cells and microenvironment attracts more and more attention.At the same time,Organization-filed theory emerges.The theory points out that tumour cells and other components constitute a microecological system,the production of tumour cells is the resalt of imbalance in the microenvironment.So,in terms of research on tumor,we not only concern about the tumor cells,but also should pay attention to their environment.The theory also gets some support in the practice of cancer research.Research about epithelial tumor environment showed that the interaction between cancer epithelial cells and adjacent stromal cells may lead to changes in the tumor microenvironment,and thus promote the process of tumor.The actual origin of the cancer cells were composed of malignant epithelial cells and cancer-related eternal changes in fibroblast cells.When cancer-related fibroblasts cocultured with benign hyperplastic epithelium,it promoted tumorigenesis.Thus they extrapolated that the fibroblasts separated from primary tumor in the body had obtained the ability to promote tumor progression.Animal model of most tumor cell lines was achieved in nude mice.However,some lines can not achived by this means,but through the provison of microenvironment.For example,the experiment,non-tumorigenic prostate cancer cell line LNCaP with prostate stromal cell transplanting into nude mice, showed that it occurred with 50-63% tumor-formation rate when under three stomal cells-LNCaP cocultivation,but not independent LNCaP and with two stromal cells,which was isolated from clinical specimens. Kleinman et al holded that non-tumorigenic NIH-3T3 cells,when co-existing with the basement extraction of matrigel,would be shown to cause tumor,local invasion and strong angiogenesis.Additionally, in HL microenvironment research,Aldinucci et al made use of the coculture of fibroblasts and HRS cells,as a resalt,HRS can promote fibroblast cells secreting CCL5,while CCL5-CCR5 pathway can contribute to the maintence and proliferation of HRS. All this suggests the importance of CCL5 on HRS cells in the microenvironment.We have been trying to establish the animal model of CHL cell line L428. However, they were not achived in nude mice and Balb/C mice.The reason may be its unique histological characteristics.So environment evoke our enough attention in our research.First,we detected the phenotype in the background of pathological immune cells.It confirmed that most are T cells,especially CD4+ T cells in dominance.Therefore,follow-up experiments went on around the interaction between L428 and CD4+T cells.Using coculture methods,we focused on the effects of CD4+T cells on L428 biological activity.At the same time,detection of chemotactic effect of L428 on CD4+T was carried out.Two-way research was to demonstrate the interaction between both better and to provide certain theory in animal model construction.Contents1 HL clinical specimens T-related phenotype analysis of the background cells.IPP6.0 software was applied in the semi-quantitative analysis of the background celluar protein phenotype, indicators related to T and B markers,CD4 and CD8 analysis,GrB and TIA-1 analysis. 2 basic identification on L428 cell line.The basic authentication on L428 was carried out from morphological characteristics and related immune markers.3 effects of CD4+T cell line Jurkat on L428We focused on the coculture of T lymphocyte line Jurkat on L428 by direct and indirect coculture.The detection of L428 proliferation was integrated used of Notch1 quantitative detection,the growth cycle and MTT method.At the same time, immortalized B cells coculture with L428 as a control.4 effects of mixed cells including CD4+T on L428To better explain the effect of CD4+T cell cocultivation on L428,first, lymph node with predilection sites was used,by its variety of cells;also with PBMC(peripheral blood mononuclear cell) of T cells in the majority.The same test was used on the L428 growth and proliferation.5 chemotatic effects of L428-related medium on CD4+ T cellsL428 supernatants, human CCL5 cell factor and its antibody in different combinations as the conditioned medium (conditioned media, CM) to detect their chemotactic effects on CD4+ T cells.Methods1 immunohistochemistry and computer image software analysis.Major steps:Paraffin sections,conventional dewaxing to water,0.01mol/L sodium citrate buffer (PH=6.0) microwave antigen retrieval,dropping endogenous peroxidase blocking agent,anti-complexⅠ,Ⅱ, DAB staining hematoxylin,dehydrated, transparent and neutral gum sealing.Each case with the positive film coming with the experiment as positive control.PBS buffer instead of primary antibody as negative control.At the same time,computer image analysis software IPP6.0,measuring positive area in each view and mean optical density value. Immunohistochemical staining index(Positive Index,PI)=positive areaxpositive signal average optical density value,calculating the corresponding ratio between the two PI.2 Morphological observation and immune markersMorphological observation on L428 cell line was obtained from inverted microscope,centrifugal slice HE and slice HE of wax block.The latter was also used to mark CD30,CD15,Mum-1 and so on.3 CocultivationThree groups were as follows:the first group was pure L428;the second group was indirect cocultivation group;the third group was direct cocultivation group.Three holes in each group,37℃,5% CO2 cell incubator for 72h.3.1 Indirect cocultivationMain cell L428 1000ul on the 24 holes, effected cell 200ul placed on the top room, the ratio of 1:10 between the cells with cell density ratio of 1:2,namely,the main cell L428 was 2×105/ml and the effected cell was 105/ml.The medium was 10% FBS concentration.When add cell suspension to the top room,make sure that the action be appropriate to avoid air bubbles,otherwise affect the membrane permeability.3.2 Direct cocultivationThe effected cell for conventional cell culture incubator 72h,collected supernants when the cell density was 2×105/ml,diluted to 1:lwith serum;833ul 2×105/ml of L428 was placed in 24 well plate plus 167ul supernatant of the above diluted,normal culture incubator conditions for 72h.4 Detection of biological activity related indicatorsQuantitative detection of Notch1,which is the indicator of cell growth and proliferation in mRNA leval;cell cycle and immunophenotyping by flow cytometry;proliferation on the number of living cells by MTT.5 Preparation of PBMCPreparation of 30ml fresh anticoagulant blood,mixed uniformly with equal volume of PBS,each time a mixture of 20ml peripheral blood was added to the same volume of liquid surface.This operation must be careful,to keep the interface clear.300g centrifugation for 15 minutes,then divided into four layers,from top to bottom as follows:serum leval,white coating(purpose PBMC),separated liquid layer,red blood cell layer.Siphoning serum layers to 5mm thickness left,carefully aspirate white film to a new centrifuge tube,added PBS and centrifuged,repeated this operation twice.If the precipitation mixed with red blood cell,lysis buffer can be added,after washing then suctioned to the culture flask.After 2h of monocyte adhesion,siphoning the cell suspension to the new flask for culture.6 Preparation of lymph node cell suspensionCollect clinically normal lymph nodes, cuted into pieces in the small bottle of penicillin,200 mesh filter with a filter,assisting with the syringe piston grinding to achieve the purpose of single-cell,after filtration washing single cell with PBS,if mixed with red blood cells, lysis buffer was necessary. After washing,suctioed to the culture flask.After 4h of monocyte adhesion,siphoned the cell suspension to the new flask for culture.7 CD4 cell chemotaxis assayMACS immunomagnetic separation CD4 cells from PBMC was for the use of detecting chemotactic effect of different conditioned media on CD4 cells.8 Statistical analysisThe experimental results were processed and analyzed by SPSS 13.0 statistical software.Measurement data was represented by mean±standard deviation(x±s).Different group of cell proliferation capacity in vitro by MTT assay were analyzed by factorial design analysis of variance and One-way ANOVA.The corresponding indicator inensity ratio by IPP6.0, quantitative analysis of Notchl,the data of cell cycle and chemotaxis assay were processed by one-way ANOVA.Results1 The cell phenotype expression intensity of the background cells of clinical samplesCollected for a comprehensive study of the HL cases-37 cases,of which NLPHL 4 cases,CHL 33 cases.Various subtypes of CHL were MCHL 6 cases,NSHL 14 cases,LRHL 13 cases respectively.Another 10 cases only did for T/B ratio analysis,including NLPHL 1 case,NSHL 4 cases,LRHL 5 cases.Statistical analysis of T/B ratio:on the whole,CHL was higher than NLPHL, approximate t test showed the difference was significant (t=-10.771,P=0.000).In HL the ratio was LRHL>NSHL>MCHL>NLPHL,resultes showed that Welch ANOVA showed that subtypes of HL were different significantly (F=49.274, P=0.000).DunnettT3 multiple comparison showed there was no significant difference between NLPHL and MCHL (P=0.168),while NLPHL and NSHL, LRHL were significantly different(P=0.000).But there was no significant difference between subtypes of CHL(P>0.05).Statistical analysis of CD4/CD8 ratio:on the whole, NLPHL was higher than CHL,Independent t test showed the difference was not significant (t=0.567, P=0.574); In CHL the ratio was MCHL>NSHL> LRHL.Fisher ANOVA showed that subtypes of CHL were different significantly (F=4.511,P=0.019).Further LSD multiple comparison showed there was significant difference between LRHL and MCHL, NSHL (P=0.011,P=0.030)Statistical analysis of GrB/TIA-1 ratio:on the whole,CHL was higher than NLPHL,independent t test showed the difference was not significant (t=-0.168, P=0.868);In CHL, the ratio was MCHL>NSHL>LRHL.One-way analysis of variance showed that subtypes of CHL was variance heterogeneity (F=6.754, P=0.004).Welch ANOVA showed that there was no significant difference between subtypes of CHL (F=4.584,P=0.075).2 Basic idenfication of L428Morphological verification of the L428 was in line with its size and diversity of the characteristics of nuclear morphology;Phenotype is also consistent with its unique identity.3 Cocultivation with JurkatRealtime PCR:expression of Notchl in L428 cocultured with Jurkat before and after,Levene ANOVO test showed that variance was equal(F=3.410,P=0.103),based on this,Fisher analysis showed they were different significantly (F=254.849,P=0.000),multiple comparison of LSD showed the difference was significant(P=0.000).Cell cycle:We focused on the growth fraction(S phase+G2/M phase),large values indicated faster proliferation.The overall values relationship was direct group>indirect group>simple goroup. Levene ANOVO test showed that variance was equal(F=0.417,P=0.677), based on this,Fisher analysis showed they were different significantly (F=177.817,P=0.000),multiple comparison of LSD showed the difference was significant(P=0.000).MTT:results showed that the overall relationship was direct group>indirect group>simple group, the cross interaction was significant (F=58.678,P=0.000);The main effects of time were different significantly(F=9047.268,P=0.000);The main effects of group were different significantly(F=420.007,P=0.000)4 Cocultivation with mixed cells including T lymphocytesRealtime PCR:expression of Notchl in L428 cocultured with LN before and after,Levene ANOVO test showed that variance was equal(F=0.768,P=0.505),based on this,Fisher analysis showed they were different significantly (F=24.094, P=0.002),multiple comparison of LSD showed the difference was significant (P<0.05); expression of Notchl in L428 cocultured with PBMC before and after,Levene ANOVO test showed that variance was equal(F=0.186,P=0.835),based on this,Fisher analysis showed they were different significantly (F=91.474, P=0.000), multiple comparison of LSD showed the difference was significant (P<0.05).Cell cycle:We focused on the growth fraction(S phase+G2/M phase),large values indicated faster proliferation.The overall values relationship was direct group>indirect group>simple goroup.Cocultivation with LN:Levene ANOVO test showed that variance is equal(F=0.975,P=0.430), based on this,Fisher analysis showed they were different significantly (F=40.235,P=0.000),multiple comparison of LSD showed the difference was significant(P<0.05); Cocultivation with PBMC:Levene ANOVO test showed that variance was equal (F=2.218,P=0.190), based on this,Fisher analysis showed they were different significantly (F=37.272,P=0.000),multiple comparison of LSD showed the difference was significant(P<0.05);MTT:results showed that the overall relationship was direct group>indirect group>simple group.Cocultivation with LN:the cross interaction was significant (F=13.57,P=0.000);The main effects of time were different significantly (F=3598.391,P=0.000);The main effects of group were different significantly (F=145.723,P=0.000). Cocultivation with PBMC:the cross interaction was significant (F=12.187,P=0.000);The main effects of time were different significantly (F=2876.748,P=0.000);The main effects of group were different significantly (F=124.226,P=0.000).5 Chemotaxis comparison under different conditioned medium. The isolated CD4+ T cells was for the chemotaxis assay.The results showed that when normal medium as control only a small number of cells be across,when the chemjotatic factors were CM and CM-CCL5,increased in turn,while the factor was anti-CCL5 with CM,decresed sharply.There was a significant difference among them(F=108.591,P=0.000),multiple comparison showed there was significant difference between them(P<0.05).Conclusion1 CHL and NLPHL are T cells and B cells with the dominance respectively. In terms of T subtypes distribution,both are TIA-1 and CD4 positive in majority;2 Proliferation is enhanced significantly of L428 cocultured with CD4+T cell line Jurkat;3 Mixed cells of LN and PBMC,including CD4+T lymphocytes,have a significant stimulation on L428,but weaker than Jurkat;4 The overall coculture effect is direct better than indirect;5 CCL5 cytokine has a significant chemotatic effect on CD4+T lymphocytes.In short,mutual influence and mutual choice among HRS and the background,eventually are associated with favorable survival of HRS cells.Innovation1 Semi-quantitative analysis of T-related protein expression in clinical samples.2 The simulation of microenvironment on cell line level.