A Study Of The Effect And Mechanism Of IgG4 On Human CD8~+T Cells

Background and ObjectivesCytotoxic T cells（CD8⁺T cells）play a major role in the cellular immune response of the immune system,and could specifically kill infected,damaged or dysfunctional cells including tumor cells.However,in tumor microenvironment（TME）,antigens constantly exist,some CD8⁺T cells eventually differentiate into Exhausted T cells（TEX）,decrease secretion of pro-inflammatory cytokines and increase of expression inhibitory receptors on CD8⁺T cells,leading to local immune inhibition.It has been reported that immunoglobulin G（IgG）could inhibit the proliferation of cytotoxic T cells,but the subclasses of IgG have not been investigated.IgG4 was highly expressed in esophageal cancer tumor tissue,mainly in a subtype of B lymphocytes,and could inhibit tumor immunity by competitively binding to IgG1 via Fc-Fc reaction while the IgG1 was specifically bound to tumor antigen.Thus,IgG4might have directly or indirectly effect on CD8⁺T cells,and this has not been reported so far.In this study,we focused on the effects of IgG4 and other IgG subclasses on proliferation,cytokine secretion and inhibitory receptor expression of CD8⁺T cell,and attempted to reveal the possible effects and mechanism of IgG4 and other IgG subclasses on CD8⁺T cell proliferation and function.Materials and MethodsIn this study,peripheral blood mononuclear cells（PBMC）were extracted from healthy adults.CD8⁺T cells were isolated by CD8⁺T cell negative selection kit then cultured in 10%Fetal Bovine Serum（FBS）,150 U/m L recombinant human Interleukin-2（rh IL-2）,5 ng/m L recombinant human Interleukin-15（rh IL-15）and X-VIVO 15 Serum-free hematopoietic cell medium in a humidified atmosphere of 5%CO₂ at 37°C.Cells were stimulated with 10μg/m L anti-CD3 antibody（OKT3）and 2μg/m L anti-CD28 antibody.IgG1/IgG2/IgG3/IgG4/IVIG protein were added respectively.CD8⁺T cell proliferation activities were detected through chemiluminescence with Cell Counting Kit.The secretions of Granzyme B,Interferonγand Tumor Necrosis Factorα（TNFα）cellcular supernatant were detected with Enzyme Linked Immunosorbent Assay（ELISA）.RNA Expression of inhibitory receptor on CD8⁺T cells including programmed cell death protein 1（PD-1）,Cytotoxic T Lymphocyte Antigen 4（CTLA-4）or Leukocyte-associated Immunoglobulin-like Receptor 1（LAIR-1）were detected with Quantitative real-time PCR（RT-q PCR）.The binding effect of FITC-conjugated IgG4 to CD8⁺T cells and the binding effect of IgG4 to CD8⁺T cells blocked by Fc receptor blocker were detected with cellular immunofluorescence on cytospin.Results and DiscussionThe results showed that 0.05 mg/m L and 0.5 mg/m L IgG4 significantly inhibited the proliferation of CD8⁺T cells through Cell Counting Kit,while other proteins did not affect cell proliferation.IgG4（0.5 mg/m L）significantly inhibited the secretion of cytokines TNFα,IFNγand Granzyme B by CD8+T cells.IgG1（0.5 mg/m L）and IVIG（0.5 mg/m L）reduced the production of IFNγby CD8⁺T cells.IgG1（0.5 mg/m L）and IgG3（0.5 mg/m L）reduced the generation of Granzyme B by CD8⁺T cells similarly.Among these effects the inhibition by IgG4 were the most significant.IgG4（0.5 mg/m L）could marked increase RNA expression of inhibitory receptor PD-1/LAIR/CTLA-4 on CD8⁺T cells.Through cellular immunofluorescence,we found that FITC-conjugated IgG4 could bind to activated CD8⁺T cells,and this binding could be blocked by Fc receptor blocker.RNA-seq data based on the cancer genome atlas（TCGA）and Genotype-Tissue Expression（GTEx）showed that expression of gene IGHG4 in tumor tissue was much higher than that in normal tissue in most cancers and this was contributed mainly by an increase of IgG4 positive B lymphocytes.ConclusionIgG4 was highly expressed in most tumor tissues,and could bind to CD8⁺T cells through Fcγreceptor.It inhibited CD8⁺T cell proliferation and the secretion of proinflammatory cytokines,and promoted the m RNA expression of inhibitory receptors.IgG4 could inhibit the function of CD8⁺T cell and turn it into the exhaustion state,contributing to tumor immune evasion.