技术资料

CD4(+) T cells develop distinct and often contrasting helper,regulatory,or cytotoxic activities. Typically a property of CD8(+) T cells,granzyme-mediated cytotoxic T cell (CTL) potential is also exerted by CD4(+) T cells. However,the conditions that induce CD4(+) CTLs are not entirely understood. Using single-cell transcriptional profiling,we uncover a unique signature of Granzyme B (GzmB)(+) CD4(+) CTLs,which distinguishes them from other CD4(+) T helper (Th) cells,including Th1 cells,and strongly contrasts with the follicular helper T (Tfh) cell signature. The balance between CD4(+) CTL and Tfh differentiation heavily depends on the class of infecting virus and is jointly regulated by the Tfh-related transcription factors Bcl6 and Tcf7 (encoding TCF-1) and by the expression of the inhibitory receptors PD-1 and LAG3. This unique profile of CD4(+) CTLs offers targets for their study,and its antagonism by the Tfh program separates CD4(+) T cells with either helper or killer functions. View Publication

The AP-1 factor basic leucine zipper transcription factor,ATF-like (BATF) is important for CD4(+) Th17,Th9,and follicular Th cell development. However,its precise role in Th2 differentiation and function remains unclear,and the requirement for BATF in nonallergic settings of type-2 immunity has not been explored. In this article,we show that,in response to parasitic helminths,Batf(-/-) mice are unable to generate follicular Th and Th2 cells. As a consequence,they fail to establish productive type-2 immunity during primary and secondary infection. Batf(-/-) CD4(+) T cells do not achieve type-2 cytokine competency,which implies that BATF plays a key role in the regulation of IL-4 and IL-13. In contrast to Th17 and Th9 cell subsets in which BATF binds directly to promoter and enhancer regions to regulate cytokine expression,our results show that BATF is significantly enriched at Rad50 hypersensitivity site (RHS)6 and RHS7 of the locus control region relative to AP-1 sites surrounding type-2 cytokine loci in Th2 cells. Indeed,Batf(-/-) CD4(+) T cells do not obtain permissive epigenetic modifications within the Th2 locus,which were linked to RHS6 and RHS7 function. In sum,these findings reveal BATF as a central modulator of peripheral and humoral hallmarks of type-2 immunity and begin to elucidate a novel mechanism by which it regulates type-2 cytokine production through its modification of the Th2 locus control region. View Publication

New genetic tools are needed to understand the functional interactions between HIV and human host factors in primary cells. We recently developed a method to edit the genome of primary CD4(+) T cells by electroporation of CRISPR/Cas9 ribonucleoproteins (RNPs). Here,we adapted this methodology to a high-throughput platform for the efficient,arrayed editing of candidate host factors. CXCR4 or CCR5 knockout cells generated with this method are resistant to HIV infection in a tropism-dependent manner,whereas knockout of LEDGF or TNPO3 results in a tropism-independent reduction in infection. CRISPR/Cas9 RNPs can furthermore edit multiple genes simultaneously,enabling studies of interactions among multiple host and viral factors. Finally,in an arrayed screen of 45 genes associated with HIV integrase,we identified several candidate dependency/restriction factors,demonstrating the power of this approach as a discovery platform. This technology should accelerate target validation for pharmaceutical and cell-based therapies to cure HIV infection. View Publication

Spatial organization of the genome plays a central role in gene expression,DNA replication,and repair. But current epigenomic approaches largely map DNA regulatory elements outside of the native context of the nucleus. Here we report assay of transposase-accessible chromatin with visualization (ATAC-see),a transposase-mediated imaging technology that employs direct imaging of the accessible genome in situ,cell sorting,and deep sequencing to reveal the identity of the imaged elements. ATAC-see revealed the cell-type-specific spatial organization of the accessible genome and the coordinated process of neutrophil chromatin extrusion,termed NETosis. Integration of ATAC-see with flow cytometry enables automated quantitation and prospective cell isolation as a function of chromatin accessibility,and it reveals a cell-cycle dependence of chromatin accessibility that is especially dynamic in G1 phase. The integration of imaging and epigenomics provides a general and scalable approach for deciphering the spatiotemporal architecture of gene control. View Publication

CD46 is a glycoprotein with important functions in innate and adaptive immune responses. Functionally different isoforms are generated by alternative splicing at exons 7-9 (BC and C isoforms) and exon 13 (CYT-1 and CYT-2 isoforms) giving rise to BC1,BC2,C1 and C2. We developed a novel real-time PCR assay that allows quantitative comparisons between these isoforms. Their relative frequency in CD4(+) T cells from 100 donors revealed a distribution with high interpersonally variability. Importantly,the distribution between the isoforms was not random and although splicing favoured inclusion of exon 8 (BC isoforms),exclusion of exon 8 (C isoforms) was significantly linked to exclusion of exon 13 (CYT-2 isoforms). Despite inter-individual differences,CD4(+) and CD8(+) T cells,B cells,NK cells and monocytes expressed similar isoform profiles intra-individually. However,memory/effector CD4(+) T cells had a significantly higher frequency of CYT-2 when compared with naïve CD4(+) T cells. Likewise,in vitro activation of naïve and total CD4(+) T cells increased the expression of CYT-2. This indicates that although splicing factors determine a certain expression profile in an individual,the profile can be modulated by external stimuli. This suggests a mechanism by which alterations in CD46 isoforms may temporarily regulate the immune response. View Publication

Leukocyte microvilli are flexible projections enriched with adhesion molecules. The role of these cellular projections in the ability of T cells to probe antigen-presenting cells has been elusive. In this study,we probe the spatial relation of microvilli and T-cell receptors (TCRs),the major molecules responsible for antigen recognition on the T-cell membrane. To this end,an effective and robust methodology for mapping membrane protein distribution in relation to the 3D surface structure of cells is introduced,based on two complementary superresolution microscopies. Strikingly,TCRs are found to be highly localized on microvilli,in both peripheral blood human T cells and differentiated effector T cells,and are barely found on the cell body. This is a decisive demonstration that different types of T cells universally localize their TCRs to microvilli,immediately pointing to these surface projections as effective sensors for antigenic moieties. This finding also suggests how previously reported membrane clusters might form,with microvilli serving as anchors for specific T-cell surface molecules. View Publication

The persistence of latent HIV proviruses in long-lived CD4(+) T cells despite antiretroviral therapy (ART) is a major obstacle to viral eradication. Because current candidate latency-reversing agents (LRAs) induce HIV transcription,but fail to clear these cellular reservoirs,new approaches for killing these reactivated latent HIV reservoir cells are urgently needed. HIV latency depends upon the transcriptional quiescence of the integrated provirus and the circumvention of immune defense mechanisms. These defenses include cell-intrinsic innate responses that use pattern-recognition receptors (PRRs) to detect viral pathogens,and that subsequently induce apoptosis of the infected cell. Retinoic acid (RA)-inducible gene I (RIG-I,encoded by DDX58) forms one class of PRRs that mediates apoptosis and the elimination of infected cells after recognition of viral RNA. Here we show that acitretin,an RA derivative approved by the US Food and Drug Administration (FDA),enhances RIG-I signaling ex vivo,increases HIV transcription,and induces preferential apoptosis of HIV-infected cells. These effects are abrogated by DDX58 knockdown. Acitretin also decreases proviral DNA levels in CD4(+) T cells from HIV-positive subjects on suppressive ART,an effect that is amplified when combined with suberoylanilide hydroxamic acid (SAHA),a histone deacetylase inhibitor. Pharmacological enhancement of an innate cellular-defense network could provide a means by which to eliminate reactivated cells in the latent HIV reservoir. View Publication

Immune recognition by T cells relies on the presentation of pathogen-derived peptides by infected cells,but the persistence of chronic infections calls for new approaches to modulate immune recognition. Ag cross-presentation,the process by which pathogen Ags are internalized,degraded,and presented by MHC class I,is crucial to prime CD8 T cell responses. The original degradation of Ags is performed by pH-dependent endolysosomal cathepsins. In this article,we show that HIV protease inhibitors (PIs) prescribed to HIV-infected persons variably modulate cathepsin activities in human APCs,dendritic cells and macrophages,and CD4 T cells,three cell subsets infected by HIV. Two HIV PIs acted in two complementary ways on cathepsin hydrolytic activities: directly on cathepsins and indirectly on their regulators by inhibiting Akt kinase activities,reducing NADPH oxidase 2 activation,and lowering phagolysosomal reactive oxygen species production and pH,which led to enhanced cathepsin activities. HIV PIs modified endolysosomal degradation and epitope production of proteins from HIV and other pathogens in a sequence-dependent manner. They altered cross-presentation of Ags by dendritic cells to epitope-specific T cells and T cell-mediated killing. HIV PI-induced modulation of Ag processing partly changed the MHC self-peptidome displayed by primary human cells. This first identification,to our knowledge,of prescription drugs modifying the regulation of cathepsin activities and the MHC-peptidome may provide an alternate therapeutic approach to modulate immune recognition in immune disease beyond HIV. View Publication

HIV-infected slow progressors (SP) represent a heterogeneous group of subjects who spontaneously control HIV infection without treatment for several years while showing moderate signs of disease progression. Under conditions that remain poorly understood,a subgroup of these subjects experience failure of spontaneous immunological and virological control. Here we determined the frequency of SP subjects who showed loss of HIV control within our Canadian Cohort of HIV(+) Slow Progressors and identified the proinflammatory cytokine IL-32 as a robust biomarker for control failure. Plasmatic levels of the proinflammatory isoforms of IL-32 (mainly β and γ) at earlier clinic visits positively correlated with the decline of CD4 T-cell counts,increased viral load,lower CD4/CD8 ratio and levels of inflammatory markers (sCD14 and IL-6) at later clinic visits. We present here a proof-of-concept for the use of IL-32 as a predictive biomarker for disease progression in SP subjects and identify IL-32 as a potential therapeutic target. View Publication

Immune tolerance is executed partly by Foxp3(+)regulatory T (Treg) cells,which suppress autoreactive T cells. In autoimmune type 1 diabetes (T1D) impaired tolerance promotes destruction of insulin-producing β-cells. The development of autoantigen-specific vaccination strategies for Foxp3(+)Treg-induction and prevention of islet autoimmunity in patients is still in its infancy. Here,using human haematopoietic stem cell-engrafted NSG-HLA-DQ8 transgenic mice,we provide direct evidence for human autoantigen-specific Foxp3(+)Treg-induction in vivo. We identify HLA-DQ8-restricted insulin-specific CD4(+)T cells and demonstrate efficient human insulin-specific Foxp3(+)Treg-induction upon subimmunogenic vaccination with strong agonistic insulin mimetopes in vivo. Induced human Tregs are stable,show increased expression of Treg signature genes such as Foxp3,CTLA4,IL-2Rα and TIGIT and can efficiently suppress effector T cells. Such Foxp3(+)Treg-induction does not trigger any effector T cells. These T1D vaccine candidates could therefore represent an expedient improvement in the challenge to induce human Foxp3(+)Tregs and to develop novel precision medicines for prevention of islet autoimmunity in children at risk of T1D. View Publication

T-bet is essential for natural regulatory T cells (nTreg) to regulate Th1 inflammation,but whether T-bet controls other Treg functions after entering the inflammatory site is unknown. In an islet allograft model,T-bet(-/-) nTreg,but not induced Treg,failed to prolong graft survival as effectively as wild-type Treg. T-bet(-/-) nTreg had no functional deficiency in vitro but failed to home from the graft to draining lymph nodes (dLN) as efficiently as wild type. T-bet regulated expression of adhesion- and migration-related molecules,influencing nTreg distribution in tissues,so that T-bet(-/-) nTreg remained in the grafts rather than migrating to lymphatics and dLN. In contrast,both wild-type and T-bet(-/-) CD4(+) conventional T cells and induced Treg migrated normally toward afferent lymphatics. T-bet(-/-) nTreg displayed instability in the graft,failing to suppress Ag-specific CD4(+) T cells and prevent their infiltration into the graft and dLN. Thus,T-bet regulates nTreg migration into afferent lymphatics and dLN and consequently their suppressive stability in vivo. View Publication

Myasthenia gravis (MG) is a prototypical autoimmune disease that is among the few for which the target Ag and the pathogenic autoantibodies are clearly defined. The pathology of the disease is affected by autoantibodies directed toward the acetylcholine receptor (AChR). Mature,Ag-experienced B cells rely on the action of Th cells to produce these pathogenic Abs. The phenotype of the MG Ag-reactive T cell compartment is not well defined; thus,we sought to determine whether such cells exhibit both a proinflammatory and a pathogenic phenotype. A novel T cell library assay that affords multiparameter interrogation of rare Ag-reactive CD4(+) T cells was applied. Proliferation and cytokine production in response to both AChR and control Ags were measured from 3120 T cell libraries derived from 11 MG patients and paired healthy control subjects. The frequency of CCR6(+) memory T cells from MG patients proliferating in response to AChR-derived peptides was significantly higher than that of healthy control subjects. Production of both IFN-γ and IL-17,in response to AChR,was also restricted to the CCR6(+) memory T cell compartment in the MG cohort,indicating a proinflammatory phenotype. These T cells also included an elevated expression of GM-CSF and absence of IL-10 expression,indicating a proinflammatory and pathogenic phenotype. This component of the autoimmune response in MG is of particular importance when considering the durability of MG treatment strategies that eliminate B cells,because the autoreactive T cells could renew autoimmunity in the reconstituted B cell compartment with ensuing clinical manifestations. View Publication