胃癌患者外周血中单个核细胞及CD3^+CD8^(low)T细胞中ILT3的表达及意义

目的探讨免疫球蛋白样转录子3(ILT3)在胃癌患者外周血单个核细胞(PBMC)和CD3+CD8lowT淋巴细胞亚群中的表达,并分析其表达与胃癌生物学行为和临床病理学特征的关系。方法选择80例胃癌患者及40例健康人对照者为研究对象,用实时荧光定量PCR检测PBMC的ILT3 mRNA的表达,流式细胞术(FACS)检测外周血CD3+CD8lowT淋巴细胞亚群中ILT3的表达,并结合临床病理特征进行分析。结果胃癌患者与健康人对照者PBMC的ILT3 mRNA相对表达量分别为8.465±0.674和6.234±0.556,差异有统计学意义(t=2.160,P<0.05);低分化和中、高分化胃癌患者PBMC的ILT3 mRNA相对表达量分别为8.217±0.433(n=66)和3.656±0.195(n=14),差异有统计学意义(t=4.802,P<0.01);肿瘤未侵及深肌层与侵及深肌层比较,PBMC的ILT3 mRNA相对表达量分别为3.495±0.243(n=24)和7.316±0.404(n=56),差异有统计学意义(t=5.971,P<0.01)。胃癌患者和健康人对照者CD3+CD8lowT淋巴细胞亚群ILT3阳性率为(75.53±2.20)%和(12.83±0.94)%,差异有统计学意义(t=19.67,P<0.01)。结论胃癌患者PBMC的ILT3 mRNA表达与肿瘤细胞分化和浸润程度有关。ILT3在胃癌患者CD3+CD8lowT细胞亚群的表达增强,提示其可能参与肿瘤的免疫逃逸。

Changing roles of CD3^(+)CD8^(low) T cells in combating HIV-1 infection

Background:Cluster of differentiation 8(CD8 T)cells play critical roles in eradicating human immunodeficiency virus(HIV)-1 infection,but little is known about the effects of T cells expressing CD8 at low levels(CD8^(low))or high levels(CD8^(high))on HIV-1 replication inhibition after HIV-1 invasion into individual.Methods:Nineteen patients who had been acutely infected with HIV-1(AHI)and 20 patients with chronic infection(CHI)for≥2 years were enrolled in this study to investigate the dynamics of the quantity,activation,and immune responses of CD3^(+)CD8^(low) T cells and their counterpart CD3^(+)CD8^(high) T cells at different stages of HIV-1 infection.Results:Compared with healthy donors,CD3^(+)CD8^(low) T cells expanded in HIV-1-infected individuals at different stages of infection.As HIV-1 infection progressed,CD3^(+)CD8^(low) T cells gradually decreased.Simultaneously,CD3^(+)CD8^(high) T cells was significantly reduced in the first month of AHI and then increased gradually as HIV-1 infection progressed.The classical activation of CD3^(+)CD8^(low) T cells was highest in the first month of AHI and then reduced as HIV-1 infection progressed and entered the chronic stage.Meanwhile,activated CD38^(-)HLA-DR^(+)CD8^(low) T cells did not increase in the first month of AHI,and the number of these cells was inversely associated with viral load(r=-0.664,P=0.004)but positively associated with the CD4 T-cell count(r=0.586,P=0.014).Increased programmed cell death protein 1(PD-1)abundance on CD3^(+)CD8^(low) T cells was observed from the 1st month of AHI but did not continue to be enhanced,while a significant T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibition motif(ITIM)domains(TIGIT)abundance increase was observed in the 12th month of infection.Furthermore,increased PD-1 and TIGIT abundance on CD3^(+)CD8^(low) T cells was associated with a low CD4 T-cell count(PD-1:r=-0.456,P=0.043;TIGIT:r=-0.488,P=0.029)in CHI.Nonetheless,the nonincrease in PD-1 expression on classically activated CD3^(+)CD8^(low) T cells was inversely associated with HIV-1 viremia in the first month of AHI(r=-0.578,P=0.015).Notably,in the first month of AHI,few CD3^(+)CD8^(low) T cells,but comparable amounts of CD3^(+)CD8^(high) T cells,responded to Gag peptides.Then,weaker HIV-1-specific T-cell responses were induced in CD3^(+)CD8^(low) T cells than CD3^(+)CD8^(high) T cells at the 3rd and 12th months of AHI and in CHI.Conclusions:Our findings suggest that CD3^(+)CD8^(low) T cells play an anti-HIV role in the first month of infection due to their abundance but induce a weak HIV-1-specific immune response.Subsequently,CD3^(+)CD8^(low) T-cell number decreased gradually as infection persisted,and their anti-HIV functions were inferior to those of CD3^(+)CD8^(high) T cells.

IGF2BP3 Enhances the Growth of Hepatocellular Carcinoma Tumors by Regulating the Properties of Macrophages and CD8^(+)T Cells in the Tumor Microenvironment

Background and Aims:Overexpression of IGF2BP3 is associated with the prognosis of hepatocellular carcinoma(HCC).However,its role in regulating tumor immune microenvironment(TME)is not well characterized.Here,we investigated the effects of IGF2BP3 on macrophages and CD8^(+)T cells within the TME of HCC.Methods:The relationship between IGF2BP3 and immune cell infiltration was analyzed using online bioinformatics tools.Knockout of IGF2BP3 in mouse hepatoma cell line Hepa1-6 was established using CRISPR/Cas9 technology.In vitro cell coculture and subcutaneously implanted hepatoma mice model were used to explore the effects of IGF2BP3 on immune cells.Expression of CCL50l transforming growth factor beta 1(TGF-β1)was detected with quantitative real-time polymerase chain reaction,western blotting,and enzyme-linked immunosorbent assay.The binding of IGF2BP3 and its target RNA was verified by trimolecular fluorescence complementation system and RNA immunoprecipitation followed by quantitative or semiquantitative polymerase chain reaction.Results:IGF2BP3 expression was elevated in HCC and was positively correlated with macrophage infiltration.Patients with higher IGF2BP3 expression and lower macrophage infiltration had a better survival rate.We found that IGF2BP3 could bind to the mRNA of CCL5 or TGF-β1,increasing their expression,and inducing macrophage infiltration and M2 polarization while inhibiting the activation of CD8^(+)T cells.Furthermore,inhibition of IGF2BP3 combined with anti-CD47 antibody treatment significantly suppressed the growth of hepatoma in Hepa1-6 xenograft tu-mor mice.Conclusions:IGF2BP3 promoted the infiltration and M2-polarization of macrophages and suppressed CD8^(+)T activation by enhancing CCL5 and TGF-β1 expression,which facilitated the progression of Hepa1-6 xenograft tumor.

Mettl3依赖的m^(6)A甲基化调控CD8^(+)T细胞效应分化和记忆形成

Efficient immune responses rely on the proper differentiation of CD8^(+)T cells into effector and memory cells.Here,we show a critical requirement of N^6-Methyladenosine(m^(6)A)methyltransferase Mettl3 during CD8^(+)T cell responses upon acute viral infection.Conditional deletion of Mettl3 in CD8^(+)T cells impairs effector expansion and terminal differentiation in an m^(6)A-dependent manner,subsequently affecting memory formation and the secondary response of CD8^(+)T cells.Our combined RNA-seq and m^(6)AmiCLIP-seq analyses reveal that Mettl3 deficiency broadly impacts the expression of cell cycle and transcriptional regulators.Remarkably,Mettl3 binds to the Tbx21 transcript and stabilizes it,promoting effector differentiation of CD8^(+)T cells.Moreover,ectopic expression of T-bet partially restores the defects in CD8^(+)T cell differentiation in the absence of Mettl3.Thus,our study highlights the role of Mettl3 in regulating multiple target genes in an m^(6)A-dependent manner and underscores the importance of m^(6)A modification during CD8^(+)T cell response.

Blockade of Tim-3 Pathway Ameliorates Interferon-γ Production from Hepatic CD8^+ T Cells in a Mouse Model of Hepatitis B Virus Infection

T cell immunoglobulin- and mucin-domain-containing molecule-3 （Tim-3） has been reported to participate in the pathogenesis of inflammatory diseases. However, whether Tim-3 is involved in hepatitis B virus （HBV） infection remains unknown. Here, we studied the expression and function of Tim-3 in a hydrodynamics-based mouse model of HBV infection. A significant increase of Tim-3 expression on hepatic T lymphocytes, especially on CD8^＋ T cells, was demonstrated in HBV model mice from day 7 to day 18. After Tim-3 knockdown by specific shRNAs, significantly increased IFN-γ production from hepatic CD8^＋ T cells in HBV model mice was observed. Very interestingly, we found Tim-3 expression on CD8^＋ T cells was higher in HBV model mice with higher serum anti-HBs production. Moreover, Tim-3 knockdown influenced anti-HBs production in vivo. Collectively, our data suggested that Tim-3 might act as a potent regulator of antiviral T-cell responses in HBV infection. Cellular ＆ Molecular Immunology.

Conversion of effector CD4^(+)T cells to a CD8^(+)MHC Ⅱ-recognizing lineage

CD4^(+)and CD8^(+)T cells are dichotomous lineages in adaptive immunity.While conventionally viewed as distinct fates that are fixed after thymic development,accumulating evidence indicates that these two populations can exhibit significant lineage plasticity,particularly upon TCR-mediated activation.We define a novel CD4^(-)CD8αβ^(+)MHC Ⅱ-recognizing population generated by lineage conversion from effector CD4^(+)T cells.CD4-CD8αβ^(+)effector T cells downregulated the expression of T helper cell-associated costimulatory molecules and inaeased the expression of cytotoxic T lymphocyte-associated cytotoxic molecules.This shift in functional potential corresponded with a CD8^(+)-lineage skewed transcriptional profile.TCRβ repertoire sequencing and in vivo genetic lineage tracing in acutely infected wild-type mice demonstrated that CD4^(-)CD8αβ^(+)effector T cells arise from fundamental lineage reprogramming of bona fide effector CD4^(+)T cells.Impairing autophagy via functional deletion of the initiating kinase Vps34 or the downstream enzyme Atg7 enhanced the generation of this cell population.These findings suggest that effector CD4^(+)T cells can exhibit a previously unreported degree of skewing towards the CD8^(+)T cell lineage,which may point towards a novel direction for HIV vaccine design.

Altered Carcinoembryonic Antigen-Related Cell Adhesion Molecule 1/T-Cell Immunoglobulin Mucin-3 Signaling Causes the Dysregulation of Decidual CD8+T Cells in the Third Trimester during Preeclamptic Pregnancies

Objective:To investigate the frequency and function of Tim-3^(+)CD8^(+)T cells in the third trimester of normal pregnancies(NPs)and preeclamptic(PE)pregnancies.Methods:T-cell immunoglobulin mucin-3(Tim-3)expression levels of CD8^(+)T cells in the decidua,peripheral blood,and umbilical cord blood obtained from women showing NPs and PE pregnancies were analyzed using flow cytometry.Decidual CD8^(+)T cells were cultured in the presence of recombinant human carcinoembryonic antigen-related cell adhesion molecule 1(CEACAM1)protein and/or Tim-3-specific neutralizing antibodies for analyzing CD107a and intracellular cytokine expression.The placental CEACAM1 protein expression was analyzed using immunohistochemistry.Results:Tim-3^(+)CD8^(+)T cells were more abundant in the decidua than in the peripheral blood.Tim-3 expression in the decidual CD8^(+)T cells was significantly lower in PE patients.Decidual Tim-3^(+)CD8^(+)T cells from PE patients expressed higher levels of CD107a and the Th1-type cytokine IFN-γ,but lower levels of the Th2-type cytokine IL-4.CEACAM1 altered the CD107a,IFN-γ,and IL-4 levels;this was reversed by anti-Tim-3 antibodies.The CEACAM1 protein levels were lower in the placental tissues of women with PE pregnancies than in those of women with NPs.Conclusions:Abnormal CEACAM1/Tim-3 regulation may participate in the development of PE,accompanied by disturbed Th2 cell predominance and higher cytotoxicity of decidual CD8^(+)T cells.

A novel cyclic peptide targeting LAG-3 for cancer immunotherapy by activating antigenspecific CD8^+ T cell responses

PD-1 and CTLA-4 antibodies offer great hope for cancer immunotherapy.However,many patients are incapable of responding to PD-1 and CTLA-4 blockade and show low response rates due to insufficient immune activation.The combination of checkpoint blockers has been proposed to increase the response rates.Besides,antibody drugs have disadvantages such as inclined to cause immune-related adverse events and infiltration problems.In this study,we developed a cyclic peptide C25 by using Ph.D.-C7C phage display technology targeting LAG-3.As a result,C25 showed a relative high affinity with human LAG-3 protein and could effectively interfere the binding between LAG-3 and HLA-DR(MHC-II).Additionally,C25 could significantly stimulate CD8^+T cell activation in human PBMCs.The results also demonstrated that C25 could inhibit tumor growth of CT26,B16 and B 16-OVA bearing mice,and the infiltration of CD8^+T cells was significantly increased while FOXP3^+Tregs significantly decreased in the tumor site.Furthermore,the secretion of IFN-γby CD8^+T cells in spleen,draining lymph nodes and especially in the tumors was promoted.Simultaneously,we exploited T cells depletion models to study the anti-tumor mechanisms for C25 peptide,and the results combined with MTT assay confirmed that C25 exerted anti-tumor effects via CD8+T cells but not direct killing.In conclusion,cyclic peptide C25 provides a rationale for targeting the immune checkpoint,by blockade of LAG-3/HLA-DR interaction in order to enhance anti-tumor immunity,and C25 may provide an alternative for cancer immunotherapy besides antibody drugs.

An altered CD8^(+) T cell epitope of insulin prevents type 1 diabetes in humanized NOD mice

Autoreactive CD8^(+)T cells,which play an indispensable role inβcell destruction,represent an emerging target for the prevention of type 1 diabetes(T1D).Altered peptide ligands(APLs)can efficiently induce antigen-specific T cells anergy,apoptosis or shifts in the immune response.Here,we found that HLA-A*0201-restricted CD8^(+)T cell responses against a primaryβ-cell autoantigen insulin epitope InsB15–14 were present in both NOD.β2m null.HHD NOD mice and T1D patients.We generated several APL candidates for InsB15–14 by residue substitution at the p6 position.Only H6F exhibited an inhibitory effect on mInsB1_(5–14)-specific CD8^(+)T cell responses in vitro.H6F treatment significantly reduced the T1D incidence,which was accompanied by diminished autoreactive CD8^(+)T cell responses to mInsB15-14,inhibited infiltration of CD8^(+)and CD4^(+)T cells in the pancreas and reduced pro-inflammatory cytokine production in pancreatic and splenic T cells in NOD.β2m^(null).HHD mice.Mechanistically,H6F treatment significantly augmented a tiny portion of CD8^(+)CD25^(+)Foxp3^(+)T cells in the spleen and especially in the pancreas.This subset exhibited typical Treg phenotypes and required peptide-specific restimulation to exert immunosuppressive activity.Therefore,this APL H6F may be a promising candidate with potential clinical application value for antigen-specific prevention of T1D.

Enhancement of antitumor immunity by low-dose total body irradiation is associated with selectively decreasing the proportion and number of T regulatory cells

Low-dose total body irradiation （LTBI） is used in the treatment of some cancers mainly for immune enhancement rather than cell killing. However, the mechanism underlying LTBI remains unknown. In this study, by analyzing the immune patterns of lymphocytes, we found that the percentage and absolute number of CD4^＋CD25^＋Foxp3^＋ regulatory T cells are markedly decreased in naive mice following treatment with LTBI. On the contrary, the CD4^＋CD44^＋/CD8^＋CD44^＋ effector-memory T cells are greatly increased. Importantly, naive mice treated with dendritic cell-gp100 tumor vaccines under LTBI induced an enhancement of antigen-specific proliferation and cytotoxicity as well as interferon-γ, （IFN-γ） secretion against FIO melanoma tumor challenge, compared to treatment with either the tumor vaccine or LTBI alone. Consequently, the treatment resulted in a reduced tumor burden and prolonged mouse survival. Our data demonstrate that LTBI＇s enhancement of antitumor immunity was mainly associated with selectively decreasing the proportion and number of T regulatory cells, implying the potential application of the combination of LTBI and a tumor vaccine in antitumor therapy.

Double negative T cells,a potential biomarker for systemic lupus erythematosus

Systemic lupus erythematosus(SLE)is an autoimmune disease that is a challenge to diagnose and treat.There is an urgent need for biomarkers to help define organ involvement,and more effective therapies.A unique population of T cells,the CD3^(+)CD4^(−)CD8^(−)(DNeg)cells,is significantly increased in lupus patients.Twentyseven cases(53%)of pediatric SLE patients had elevated DNeg cells in their peripheral blood,which correlated with kidney function(R^(2)=0.54).Significant infiltration of DNeg cells was observed in both adult and pediatric lupus kidneys by immunofluorescence.For the first time,this study provides direct evidence that DNeg cells facilitate kidney injury in preclinical 8-week-old MRL/lpr lupus mice.In lupus mice,the increase in DNeg cells tracked with worsening disease and correlated with kidney function(R^(2)=0.85).Our results show that DNeg cells per se can cause kidney dysfunction,increase in number with increase in disease pathology,and could serve as a potential biomarker.

High baseline tumor burden-associated macrophages promote an immunosuppressive microenvironment and reduce the efficacy of immune checkpoint inhibitors through the IGFBP2-STAT3-PD-L1 pathway

Background:Several clinical studies have uncovered a negative correlation between baseline tumor burden and the efficacy of immune checkpoint inhibitor(ICI)treatment.This study aimed to uncover the specific mechanisms underlying the difference in sensitivity to ICI treatment between tumors with high(HTB)and low(LTB)tumor burden.Methods:For in vivo studies,several mouse models of subcutaneous tumors were established,and transcriptome sequencing,immunohistochemistry,and flow cytometry assays were used to detect the immune status in these subcutaneous tumors.For in vitro experiments,co-culture models,cytokine antibody arrays,western blotting,flow cytometry,and enzyme-linked immunosorbent assays were used to explore the underlying molecular mechanisms Results:We found that MC38 or B16 subcutaneous tumors from the HTB group did not show any response to anti-programmed cell death protein-1(PD-1)therapy.Through flow cytometry assays,we found that the infiltration with CD8^(+)T cellswas significantly decreasedwhereasM2-like macrophageswere enriched in subcutaneous tumors of HTB groups compared with those of LTB group.These changes were not affected by the initial number of injected tumor cells or tumor age,nor could they be reversed by surgical tumor reduction.Intraperitoneal colony-stimulating factor 1 receptor(CSF-1R)inhibitor PLX3397 injection at different time points of tumor growth only had an effect when administered in the early tumor stage to maintain the“heat”of the tumor microenvironment during the process of tumor growth,thereby achieving a response to ICI treatment when the tumor grew to a large size.Mechanistically,we found that insulin-like growth factor binding protein 2(IGFBP2)expression levelswere significantly elevated in HTB tumor tissues.IGFBP2 promoted the programmed death-ligand 1(PD-L1)expression in M2-like macrophages by activating signal transducer and activator of transcription 3(STAT3),and PD-L1^(+)M2-likemacrophages exerted an immunosuppressive effect by inhibiting the proliferation and activation of CD8^(+)T cells in a PD-L1-dependent fashion.Conclusions:This study suggested that the low efficacy of ICI treatment in HTB tumors is mainly attributed to the intratumoral accumulation of PD-L1^(+)M2-like macrophages via the IGFBP2-STAT3-PD-L1 signaling pathway and their substantial inhibitory effects on T cell proliferation and activation.