Lymphoma Endothelium Preferentially Expresses Tim-3 And Facilitates The Progression Of Lymphoma By Mediating Immune Evasion

[Background and objective] Angiogenesis is increasingly being recognized as animportant prognosticator associated with the progression of lymphoma,and as an attractivetarget for next-generation modalities.However,our understanding of lymphomaangiogenesis is still in its infancy.In present study,we use global gene expressionmicroarray analysis to identify tumor-specific molecular aberration of lymphoma.[Methods] The homogeneous endothelial cells were obtained from frozen lymphoma andreactive lymph nodes sections by using LCM.The small amount of total RNA was extractedusing the RNAqueous micro kit and the purified RNA was linearly amplified in vitro.TheAffymetrix GeneChip array HG U133 Plus 2.0 were then hybridized against the cDNAprobe mixture with high through input and efficiency and the fluorescent signals werescanned.The obtained data were analyzed using Feature Extraction software.[Results] Thirteen transcripts were found to be at least 1-fold more abundant in reactivelymph node-derived endothelium than in lymphoma-derived endothelium.Fourteentranscripts,on the other hand,were found to be at least 1-fold more abundant inlymphoma-derived endothelium than in reactive lymph node-derived endothelium.[Conclusion] The specific molecular aberration of endothelial cells were primarilyscreened through cDNA microarray analysis of the microvascular endothelial cells fromlymphoma harvested by laser capture microdissection,and it will provide new targetmolecules for anti-angiogenesis therapy. [Objective] To identify the expression levels of Tim-3 in endohelial cells of lymphoma andreactive lymph modes and to further confirm that Tim-3 is preferentially expressed inlymphoma-derived endothelial cells.[Methods] Tim-3 expression in endothelial cells of lymphoma and reactive lymph nodeswere detected by in situ hybridization,immunocytochemistry.Immunofluorescence stainingwas used to detect the expression and location of Tim-3 in endothelial cells isolated fromfresh lymphoma and reactive lymph nodes.To address whether lymph node EC producedtwo isofonns of Tim-3 transcripts,mRNA from freshly purified lymph node EC wassubjected to PCR analysis.[Results] In only two out of ten non-neoplastic lymph nodes,weakly stained Tim-3 mRNAcould be detected in endothelium.In contrast,strong staining of Tim-3 mRNA was detectedin endothelium in eight out of ten lymphoma specimens.Furthermore,positive endotheliumstaining for Tim-3 protein was detected in only 10.34% (3/29) of reactive lymph nodes.Incontrast,positive staining of Tim-3 protein was detected in approximately 52.38% (55/105)of the lymphoma specimens examined.EC from seven out of ten lymphoma tissues wasstained strongly for Tim-3 protein on the cell membrane.Conversely,EC from two reactivelymph nodes examined was negative for Tim-3.A full-length Tim-3 transcript was detectedin EC from eight out of ten lymphoma samples,the shorter Tim-3 transcript was notdetected in any of the ten samples.And EC from two reactive lymph nodes was negativefor Tim-3 either as a full-length or a shorter transcript.[Conclusion] Tim-3 is preferentially expressed in lymphoma-derived endothelial cells. [Objective] To investigate the correlation of Tim-3 expression in endothelium with localimmune profiles and advanced clinical stages ofNon-hodgkin's lymphoma.[Methods] Immunohistochemistry was applied to detect the expressions of Tim-3 andCD34 in 85 cases of non-hodgkin's lymphoma,and CD34 was used as an indicator ofmicrovessel density.12 cases of endothelial Tim-3-positive or Tim-3-negative diffuse largeB cell lymphomas(DLBCL) were randomly selected to analyse the percentage of CD4,CD8 and CDla in serial sections.[Results] Endothelial Tim-3 protein levels were found to significantly correlate with MVDof lymphoma (r = 0.446,P＜0.001).Tim-3-positive DLBCL had a significantly lowerpercentage of CD4+ T cells (4.41%±6.87% versus 11.30%±7.51%,P = 0.030) There wasno significant difference,in the percentage of CD8 and CDla expression betweenTim-3-positive and negative DLBCL(P＞0.05).high Tim-3 expression was statisticallycorrelated with higher lymphoma stages (P = 0.043),B symptoms (fever,night sweats andweight loss;P = 0.037),and higher international prognosis index (IPI) scores (P = 0.002).[Conclusion] Tim-3 expression in endothelium correlated with local immune profiles andadvanced clinical stages of lymphoma,mayde participate in immune evasion andangiogenesis ofNon-Hodgkin's lymphoma. [Objective] It is necessary to provide a favorable cell model in vitro for studyinginteractions between Tim-3-expressing endothelial cells and autologous T lymphocytes.Our aims is to validate the costimulating cell model of human umbilical veinendothelial cells(UVEC) and autologous cord blood T lymphocytes.[Methods] following pretreatment with IFN-γ,MHC and costimulatory molecules in themembrane of UVEC were determined by Flow cytometry.We construct an adenoviral DNAvaccine named ADV-TT which contained the N-terminal domain of fragment C of tetanustoxin and a portion of the second domain which can induce strong cytotoxic T cell (CTL)responses,and ADV-TT as a surrogate lymphoma antigen was transfected into UVECs.Theproliferation capacity of T lymphocytes after coculture with transfected UVECs wasmeasured by CFSE-flow cytometry.The effect of ADV-TT infection on UVEC proliferationand survival were analyzed.[Results] UVEC was found to constitutively express classⅠMHC and CD58 molecules.Following pretreatment with IFN-γ,MHC classⅡbut not CD86 molecules were detectedon UVEC.ADV-TT-infected UVEC stimulated autologous lymphocytes to proliferate in anADV-TT dose-dependent fashion.Not only CD4+ but also CD8+ T lymphocytes wereactivated.ADV-TT infection did not lead to an increase in UVEC cell numbers whencompared with the UVEC/PBS control.[Conclusion] surrogate antigen TT-expressed UVEC could activate both CD4+ and CD8+T lymphocytes,and thus could be used as a cell model to investigate the direct effects ofEC expressing Tim-3 on T lymphocytes. [Objective] To explore the influence of Tim-3-expressing endothelial cells on the immuneresponse of antigen-specific T lymphoeytes and initially to illuminate the effect ofTim-3-expressing endothelial cells on lymphoma immune tolerance.[Methods] CFSE-Flow cytometry was used to analyze the proliferation of thelymphocytes in response to UVEC transfected different adenoviral mutants;Biomed-2multiplex T cell receptor PCR was exploited to analyze the T cell repertoire of all treatedgroups;T lymphocytes were incubated with ADV-TT-infected dendritic cells (DC_TT).Theresulting CTL were added to ADV-TT-infected UVEC in the presence or absence ofADV-Tim-3.The culture supematant was then determined by a LDH release assay forspecific lysis;The lymphocyte subsets of all treated groups were analyzed by flowcytometry.The production of T_H1/T_H2 type cytokines of all treated groups were determinedby cytometric bead array.[Results]The lymphocyte proliferation in response to ADV-TT infected UVEC wassignificantly suppressed by ADV-Tim-3 in a dose-dependent fashion;The TCRγclonalexpansion against ADV-TT was completely inhibited by ADV-Tim-3;The killing efficacyof activated autologous T lymphocytes was significantly inhibited by ADV-Tim-3 but notby ADV-GFP(P＜0.05);The proliferation of CD4+ and CD8+ T cells was significantlyinhibited by ADV-Tim-3,the CD4+/CD28^high T lymphocyte decreased significantlyfollowing exposure to ADV-Tim-3,the CD8+/CD28^high and Treg cell populations were notaltered by ADV-Tim-3,ADV-Tim-3 suppressed activation of CD4+/IFN-γ+ but not CD8+/IFN-γ+;ADV-Tim-3 appeared to promote production of T_H2 type cytokines (IL-10;ADV-Tim-3 versus ADV-GFP,P = 0.005) and inhibit production of T_H1 type cytokines (IFN-γ,TNF-α;ADV-Tim-3 versus ADV-GFP).[Conclusion] Tim-3-expressing endothelial cells suppressed antigen-induced activation ofCD4+ T cells and provided protective immunity,and this may a new mechanism of tumorimmune evasion. [Objective] To study the effect of Tim-3-expressing endothelial cells on the progressionand regulaion anti-tumor immune response of lymphoma.[Methods] Mouse model of transplantable B lymphoma was established in TA2 mice,Thetumorigenesis probability and tumor volume of all groups was determined,The percentageof CD4+ and CD8+ lymphocytes in peripheral blood and spleens was analyzed by Flowcytometry.The serum,spleen cells,and lymphoma tissue for basal levels of T_H1 (IFN-γ,TNF-α,and IL-2) - and T_H2 (IL-5 and IL-4)-type cytokines of all treated groups were alsotested.CD4+ and CD8+ T cells population of local tumor was determined byimmunofluorescence.[Results] Tim-3-expressing EC significantly accelerated the onset and the growth oflymphoma when compared with ADV-GFP- or PBS-treated EC.The percentage of CD4+ Tlymphocytes from ADV-Tim-3-treated mice in peripheral blood and spleens wassignificantly lower than that of PBS- or ADV-GFP-treated mice.In the ADV-Tim-3 group,basal levels of T_H1-type cytokines (TNF-αin serum,TNF-αand IFN-γin spleen cells andlymphoma tissue) were significantly lower than those in the ADV-GFP or PBS group.TheCD4+ T cell subset that had infiltrated the tumor was significantly lower in theADV-Tim-3 group.[Conclusion] Tim-3-expressing endothelial cells facilitated the progression of lymphoma invivo,invovled in immune evasion of lymphoma.