技术资料

Chronic hepatitis C virus (HCV) infection causes generalized CD8+ T cell impairment,not limited to HCV-specific CD8+ T-cells. Liver-infiltrating monocyte-derived macrophages (MDMs) contribute to the local micro-environment and can interact with and influence cells routinely trafficking through the liver,including CD8+ T-cells. MDMs can be polarized into M1 (classically activated) and M2a,M2b,and M2c (alternatively activated) phenotypes that perform pro- and anti-inflammatory functions,respectively. The impact of chronic HCV infection on MDM subset functions is not known. Our results show that M1 cells generated from chronic HCV patients acquire M2 characteristics,such as increased CD86 expression and IL-10 secretion,compared to uninfected controls. In contrast,M2 subsets from HCV-infected individuals acquired M1-like features by secreting more IL-12 and IFN-gamma. The severity of liver disease was also associated with altered macrophage subset differentiation. In co-cultures with autologous CD8+ T-cells from controls,M1 macrophages alone significantly increased CD8+ T cell IFN-gamma expression in a cytokine-independent and cell-contact-dependent manner. However,M1 macrophages from HCV-infected individuals significantly decreased IFN-gamma expression in CD8+ T-cells. Therefore,altered M1 macrophage differentiation in chronic HCV infection may contribute to observed CD8+ T-cell dysfunction. Understanding the immunological perturbations in chronic HCV infection will lead to the identification of therapeutic targets to restore immune function in HCV+ individuals,and aid in the mitigation of associated negative clinical outcomes. View Publication

Cytokine responses from monocytes and macrophages exposed to bacteria are of particular importance in innate immunity. Focusing on the impact of the immunoregulatory cytokine interleukin (IL)-27 on control of innate immune system responses,we examined human immune responses to bacterial products and bacterial infection by E. coli and S. typhimurium. Since the effect of IL-27 treatment in human myeloid cells infected with bacteria is understudied,we treated human monocytes and macrophages with IL-27 and either LPS,flagellin,or bacteria,to investigate the effect on inflammatory signaling and cytokine responses. We determined that simultaneous stimulation with IL-27 and LPS derived from E. coli or S. typhimurium resulted in enhanced IL-12p40,TNF-$\alpha$,and IL-6 expression compared to that by LPS alone. To elucidate if IL-27 manipulated the cellular response to infection with bacteria,we infected IL-27 treated human macrophages with S. typhimurium. While IL-27 did not affect susceptibility to S. typhimurium infection or S. typhimurium-induced cell death,IL-27 significantly enhanced proinflammatory cytokine production in infected cells. Taken together,we highlight a role for IL-27 in modulating innate immune responses to bacterial infection. View Publication

Broadly neutralizing HIV-1 antibodies (bNAbs) isolated from infected subjects display protective potential in animal models. Their elicitation by immunization is thus highly desirable. The HIV-1 envelope glycoprotein (Env) is the sole viral target of bnAbs,but is also targeted by binding,non-neutralizing antibodies. Env-based immunogens tested so far in various animal species and humans have elicited binding and autologous neutralizing antibodies but not bNAbs (with a few notable exceptions). The underlying reasons for this are not well understood despite intensive efforts to characterize the binding specificities of the elicited antibodies; mostly by employing serologic methodologies and monoclonal antibody isolation and characterization. These approaches provide limited information on the ontogenies and clonal B cell lineages that expand following Env-immunization. Thus,our current understanding on how the expansion of particular B cell lineages by Env may be linked to the development of non-neutralizing antibodies is limited. Here,in addition to serological analysis,we employed high-throughput BCR sequence analysis from the periphery,lymph nodes and bone marrow,as well as B cell- and antibody-isolation and characterization methods,to compare in great detail the B cell and antibody responses elicited in non-human primates by two forms of the clade C HIV Env 426c: one representing the full length extracellular portion of Env while the other lacking the variable domains 1,2 and 3 and three conserved N-linked glycosylation sites. The two forms were equally immunogenic,but only the latter elicited neutralizing antibodies by stimulating a more restricted expansion of B cells to a narrower set of IGH/IGK/IGL-V genes that represented a small fraction (0.003-0.02{\%}) of total B cells. Our study provides new information on how Env antigenic differences drastically affect the expansion of particular B cell lineages and supports immunogen-design efforts aiming at stimulating the expansion of cells expressing particular B cell receptors. View Publication

Baboons naturally infected with simian T lymphotropic virus (STLV) are a potentially useful model system for the study of vaccination against human T lymphotropic virus (HTLV). Here we expanded the number of available full-length baboon STLV-1 sequences from one to three and related the T cell responses that recognize the immunodominant Tax protein to the tax sequences present in two individual baboons. Continuously growing T cell lines were established from two baboons,animals 12141 and 12752. Next-generation sequencing (NGS) of complete STLV genome sequences from these T cell lines revealed them to be closely related but distinct from each other and from the baboon STLV-1 sequence in the NCBI sequence database. Overlapping peptides corresponding to each unique Tax sequence and to the reference baboon Tax sequence were used to analyze recognition by T cells from each baboon using intracellular cytokine staining (ICS). Individual baboons expressed more gamma interferon and tumor necrosis factor alpha in response to Tax peptides corresponding to their own STLV-1 sequence than in response to Tax peptides corresponding to the reference baboon STLV-1 sequence. Thus,our analyses revealed distinct but closely related STLV-1 genome sequences in two baboons,extremely low heterogeneity of STLV sequences within each baboon,no evidence for superinfection within each baboon,and a ready ability of T cells in each baboon to recognize circulating Tax sequences. While amino acid substitutions that result in escape from CD8+ T cell recognition were not observed,premature stop codons were observed in 7{\%} and 56{\%} of tax sequences from peripheral blood mononuclear cells from animals 12141 and 12752,respectively.IMPORTANCE It has been estimated that approximately 100,000 people suffer serious morbidity and 10,000 people die each year from the consequences associated with human T lymphotropic virus (HTLV) infection. There are no antiviral drugs and no preventive vaccine. A preventive vaccine would significantly impact the global burden associated with HTLV infections. Here we provide fundamental information on the simian T lymphotropic virus (STLV) naturally transmitted in a colony of captive baboons. The limited viral sequence heterogeneity in individual baboons,the identity of the viral gene product that is the major target of cellular immune responses,the persistence of viral amino acid sequences that are the major targets of cellular immune responses,and the emergence in vivo of truncated variants in the major target of cellular immune responses all parallel what are seen with HTLV infection of humans. These results justify the use of STLV-infected baboons as a model system for vaccine development efforts. View Publication