Memory CCR7 Research Articles

Altered memory CCR6+ Th17-polarised T-cell function and biology in people with HIV under successful antiretroviral therapy and HIV elite controllers

Despite successful antiretroviral therapy (ART), frequencies and immunological functions of memory CCR6+ Th17-polarised CD4+ T-cells are not fully restored in people with HIV (PWH). Moreover, long-lived Th17cells contribute to HIV persistence under ART. However, the molecular mechanisms underlying these observations remain understudied. mRNA-sequencing was performed using Illumina technology on freshly FACS-sorted memory CCR6+CD4+ T-cells from successfully ART-treated (ST), elite controllers (EC), and uninfected donors (HD). Gene expression validation was performed by RT-PCR, flow cytometry, and invitro functional assays. Decreased Th17cell frequencies in STs and ECs versus HDs coincided with reduced Th17-lineage cytokine production invitro. Accordingly, the RORγt/RORC2 repressor NR1D1 was upregulated, while the RORγt/RORC2 inducer Semaphorin 4D was decreased in memory CCR6+ T-cells of STs and ECs versus HDs. The presence of HIV-DNA in memory CCR6+ T-cells of ST and EC corresponded with the downregulation of HIV restriction factors (SERINC3, KLF3, and RNF125) and HIV inhibitors (tetraspanins), along with increased expression of the HIV-dependency factor MRE11, indicative of higher susceptibility/permissiveness to HIV-1 infection. Furthermore, markers of DNA damage/modification were elevated in memory CCR6+ T-cells of STs and ECs versus HDs, in line with their increased activation (CD38/HLA-DR), senescence/exhaustion phenotype (CTLA-4/PD-1/CD57) and their decreased expression of proliferation marker Ki-67. These results reveal new molecular mechanisms of Th17 cell deficit in ST and EC PWH despite a successful control of HIV-1 replication. This knowledge points to potential therapeutic interventions to limit HIV-1 infection and restore frequencies, effector functions, and senescence/exhaustion in Th17cells. This study was funded by the Canadian Institutes of Health Research (CIHR, operating grant MOP 142294, and the Canadian HIV Cure Enterprise [CanCURE 2.0] Team Grant HB2 164064), and in part, by the Réseau SIDA et maladies infectieuses du Fonds de recherche du Québec-Santé (FRQ-S).

Flow Cytometry Sorting of Memory CCR6+CD4+ T-Cells for HIV Reservoir Quantification.

Antiretroviral therapy (ART) has transformed the deadly human immunodeficiency virus type I (HIV-1) epidemic into a manageable chronic condition. Current ART is not curative and treatment interruption leads to viral rebound in people living with HIV-1 (PLWH). The main cause of viral rebound is the persistence of HIV reservoirs in long-lived memory CD4+ T cells. Accurate techniques to identify and quantify viral reservoirs are required to monitor therapeutic approaches designed to cure HIV infection. Th17-polarized CD4+ T cells are located at mucosal sites of HIV entry and are preferentially targeted for infection and viral reservoir persistence. They constitute an important reservoir in both blood and colon. In this chapter we describe a step-by-step flow cytometry-based protocol to isolate a fraction of Th17-enriched cells from PBMC based on their expression of the Th17 surface marker CCR6. The isolation of memory CCR6+CD4+ T cells allows subsequent PCR/RT-PCR-based HIV DNA/RNA quantifications, as well as their culture for quantitative viral outgrowth assays (QVOA). This method can be adapted for the isolation of CCR6+CD4+ T cells from peripheral tissues, such as the colon.

The heterogeneous human memory CCR6+ T helper-17 populations differ in T-bet and cytokine expression but all activate synovial fibroblasts in an IFN\u03b3-independent manner

BackgroundChronic synovial inflammation is an important hallmark of inflammatory arthritis, but the cells and mechanisms involved are incompletely understood. Previously, we have shown that CCR6+ memory T-helper (memTh) cells and synovial fibroblasts (SF) activate each other in a pro-inflammatory feedforward loop, which potentially drives persistent synovial inflammation in inflammatory arthritis. However, the CCR6+ memTh cells are a heterogeneous population, containing Th17/Th22 and Th17.1 cells. Currently, it is unclear which of these subpopulations drive SF activation and how they should be targeted. In this study, we examined the individual contribution of these CCR6+ memTh subpopulations to SF activation and examined ways to regulate their function.MethodsTh17/Th22 (CXCR3−CCR4+), Th17.1 (CXCR3+CCR4−), DP (CXCR3+CCR4+), and DN (CXCR3−CCR4−) CCR6+ memTh, cells sorted from PBMC of healthy donors or treatment-naïve early rheumatoid arthritis (RA) patients, were cocultured with SF from RA patients with or without anti-IL17A, anti-IFNγ, or 1,25(OH)2D3. Cultures were analyzed by RT-PCR, ELISA, or flow cytometry.ResultsTh17/Th22, Th17.1, DP, and DN cells equally express RORC but differ in production of TBX21 and cytokines like IL-17A and IFNγ. Despite these differences, all the individual CCR6+ memTh subpopulations, both from healthy individuals and RA patients, were more potent in activating SF than the classical Th1 cells. SF activation was partially inhibited by blocking IL-17A, but not by inhibiting IFNγ or TBX21. However, active vitamin D inhibited the pathogenicity of all subpopulations leading to suppression of SF activation.ConclusionsHuman CCR6+ memTh cells contain several subpopulations that equally express RORC but differ in TBX21, IFNγ, and IL-17A expression. All individual Th17 subpopulations are more potent in activating SF than classical Th1 cells in an IFNγ-independent manner. Furthermore, our data suggest that IL-17A is not dominant in this T cell-SF activation loop but that a multiple T cell cytokine inhibitor, such as 1,25(OH)2D3, is able to suppress CCR6+ memTh subpopulation-driven SF activation.

Human Memory Th17 Cell Populations Change Into Anti-inflammatory Cells With Regulatory Capacity Upon Exposure to Active Vitamin D.

Autoimmune diseases are characterized by an aberrantly activated immune system, resulting in tissue damage and functional disability in patients. An important therapeutic goal is to restore the deregulated immunological balance between pro- and anti-inflammatory T cells. This imbalance is illustrated by elevated levels and activity of memory Th17 cell populations, such as Th17, Th1/Th17, and Th17.1 cells, in various autoimmune diseases. These cells are characterized by the chemokine receptor CCR6, RORC expression and production of IL-17A, IFNγ, and TNFα. Using rheumatoid arthritis (RA) as a model of autoimmune disease, we here demonstrate that pro-inflammatory memory CCR6+ Th cells can switch into anti-inflammatory cells with regulatory capacity using the active vitamin D metabolite 1,25(OH)2D3. Memory CCR6+ Th cells, excluding Tregs, were sorted from healthy controls or treatment-naïve patients with early rheumatoid arthritis (RA) and cultured with or without 1,25(OH)2D3. Treatment with 1,25(OH)2D3 inhibited pro-inflammatory cytokines such as IL-17A, IL-17F, IL-22 and IFNγ in memory CCR6+ Th cells from both healthy controls and RA patients. This was accompanied by induction of anti-inflammatory factors, including IL-10 and CTLA4. Interestingly, these formerly pathogenic cells suppressed proliferation of autologous CD3+ T cells similar to classical Tregs. Importantly, the modulated memory cells still migrated toward inflammatory milieus in vitro, modeled by RA synovial fluid, and retained their suppressive capacity in this environment. These data show the potential to reset the pathogenic profile of human memory Th cells into non-pathogenic cells with regulatory capacity.

Human Treg Can Suppress Th17 Inflammatory Cytokine Secretion Independent of IL-2 Deprivation

Human Th17 cells are an inflammatory subset of CD4+ (CD4) T helper cells implicated in host of debilitating diseases and disorders such as multiple Sclerosis, rheumatoid arthritis, and psoriasis, as well as inhibitor formation in Hemophilia A. Although it is well established that regulatory CD4T cells (Tregs) can suppress other T helper (Th) effector cells (e.g. Th1, Th2, etc), their ability to suppress human Th17 cells is unresolved. This is attributed to several factors including plasticity between Treg and Th17 cells, the observation that Th17 cells express less IL-2 (and by extension are less dependent on IL-2) than other Th effector cells and consequently are less susceptible to Treg suppression via IL-2 deprivation. Using autologous naïve Tregs and effector memory CCR6+ CD4 T cells differentiated to Th17 cells from healthy human donor peripheral blood mononuclear cells, we performed a kinetic analysis of the IL-2 sensitivity via phosphorylated STAT5 expression. We show that Treg and Th17 cells have similar rates of pSTAT5 expression in vitro. We also show that both cell types have similar expression levels of the high affinity IL-2a receptor, CD25, following activation. Despite this, naïve Tregs successfully abrogated the secretion of IL17A by Th17 cells following 12hr co-culture. Furthermore, this abrogation is dependent on the strength of Treg activation and does not involve cytotoxic mechanisms, as viability of the Th17 cells remain high. These results suggest that human Tregs can suppress inflammatory IL-17A secretion from effector memory Th17 cells via mechanisms independent of IL-2 deprivation but dependent on strength of activation through Treg T-cell receptor. (Supported by NIH grant R01 HL126727) DisclosuresNo relevant conflicts of interest to declare.

PRAME peptide-specific CD8+ Tcells represent the predominant response against leukemia-associated antigens in healthy individuals.

Antigen-specific Tcells isolated from healthy individuals (HIs) have shown great therapeutic potential upon adoptive transfer for the treatment of viremia in immunosuppressed patients. The lack of comprehensive data on the prevalence and characteristics of leukemia-associated antigen (LAA)-specific Tcells in HIs still limits such an approach for tumor therapy. Therefore, we have investigated T-cell responses against prominent candidates comprising Wilms' tumor protein 1 (WT1), preferentially expressed antigen in melanoma (PRAME), Survivin, NY-ESO, and p53 by screening PBMCs from HIs using intracellular IFN-γ staining following provocation with LAA peptide mixes. Here, we found predominantly poly-functional effector/effector memory CCR7- /CD45RA+/- /CD8+ LAA peptide-specific Tcells with varying CD95 expression in 34 of 100 tested HIs, whereas CD4+ Tcells responses were restricted to 5. Most frequent LAA peptide-specific Tcell responses were directed against WT1 and PRAME peptides with a prevalence of 20 and 17%, respectively, showing the highest magnitude (0.16% ± 0.22% (mean ± SD)) for PRAME peptides. Cytotoxicity of PRAME peptide-specific Tcells was demonstrated by specific killing of PRAME peptide-pulsed T2 cells. Furthermore, the proliferative capacity of PRAME peptide-specific Tcells was confined to HIs responsive toward PRAME peptide challenge corroborating the accuracy of the screening results. In conclusion, we identified PRAME as a promising target antigen for adoptive leukemia therapy.

THU0506 Higher Proportions of Pathogenic Ccr6+ T Cell Subpopulations in Acpa+ Compared to ACPA- Patients with Early Rheumatoid Arthritis

BackgroundPresence of serum anti-citrullinated protein antibodies (ACPAs) in patients with rheumatoid arthritis (RA) predicts worse disease course and a more erosive disease. In the pathogenesis of RA, inflammatory T cells...

A1.76 Higher proportions of pathogenic CCR6+ T cell subpopulations in ACPA+ compared to ACPA- patients with early rheumatoid arthritis

Background and ObjectivesPresence of serum anti-citrullinated protein antibodies (ACPAs) in patients with rheumatoid arthritis (RA) predicts worse disease course and a more erosive disease. In the pathogenesis of RA, inflammatory...

Memory CCR6+CD4+ T Cells Are Preferential Targets for Productive HIV Type 1 Infection Regardless of Their Expression of Integrin β7

HIV type 1 infection is associated with a rapid depletion of Th17 cells from the GALT. The chemokine receptor CCR6 is a marker for Th17 lineage polarization and HIV permissiveness in memory CD4(+) T cells. CCR6(+) T cells have the potential to migrate into the GALT via the gut-homing integrin α(4)β(7), a newly identified HIV-gp120 binding receptor. In this study, we investigated whether memory T cells coexpressing CCR6 and integrin β(7) are selective HIV targets and whether retinoic acid (RA)-induced imprinting for gut-homing selectively increases CCR6(+) T cell permissiveness to infection. We demonstrated that β(7)(-)R6(+) and β(7)(+)R6(+) compared with β(7)(-)R6(-) and β(7)(+)R6(-) T cells were highly permissive to HIV, produced Th17 cytokines, and their frequency was decreased in the peripheral blood of HIV-infected subjects. RA upregulated integrin α(4) and β(7) coexpression in both CCR6(+) and CCR6(-) T cells, but increased HIV permissiveness selectively in CCR6(+) T cells via entry (CCR5 upregulation) and postentry mechanisms. In conclusion, these results demonstrate that CCR6, but not the integrin β(7), is a discriminative marker for memory T cells imprinted with a transcriptional program favorable to HIV replication. Nevertheless, given the ability of integrin β(7) to regulate cell migration into the GALT and bind HIV-gp120, CCR6(+) T cells coexpressing integrin β(7) and CCR5 might have an extraordinary ability to disseminate HIV from the portal sites of entry. Understanding the molecular mechanisms of memory CCR6(+) T cell differentiation is critical for the design of new therapeutic strategies that should interfere with viral permissiveness but not Th17 lineage commitment and gut-homing potential in CCR6(+) T cells.

IL-17+ Regulatory T Cells in the Microenvironments of Chronic Inflammation and Cancer

Foxp3(+)CD4(+) regulatory T (Treg) cells inhibit immune responses and temper inflammation. IL-17(+)CD4(+) T (Th17) cells mediate inflammation of autoimmune diseases. A small population of IL-17(+)Foxp3(+)CD4(+) T cells has been observed in peripheral blood in healthy human beings. However, the biology of IL-17(+)Foxp3(+)CD4(+) T cells remains poorly understood in humans. We investigated their phenotype, cytokine profile, generation, and pathological relevance in patients with ulcerative colitis. We observed that high levels of IL-17(+)Foxp3(+)CD4(+) T cells were selectively accumulated in the colitic microenvironment and associated colon carcinoma. The phenotype and cytokine profile of IL-17(+)Foxp3(+)CD4(+) T cells was overlapping with Th17 and Treg cells. Myeloid APCs, IL-2, and TGF-β are essential for their induction from memory CCR6(+) T cells or Treg cells. IL-17(+)Foxp3(+)CD4(+) T cells functionally suppressed T cell activation and stimulated inflammatory cytokine production in the colitic tissues. Our data indicate that IL-17(+)Foxp3(+) cells may be "inflammatory" Treg cells in the pathological microenvironments. These cells may contribute to the pathogenesis of ulcerative colitis through inducing inflammatory cytokines and inhibiting local T cell immunity, and in turn may mechanistically link human chronic inflammation to tumor development. Our data therefore challenge commonly held beliefs of the anti-inflammatory role of Treg cells and suggest a more complex Treg cell biology, at least in the context of human chronic inflammation and associated carcinoma.

IL-23 inhibits IL-22 and AhR signalling and with IL-1β regulates the human peripheral Th17/Th22 balance

Background and objectiveHuman peripheral memory (CD45RO+) Th17 cells express the chemokine receptor CCR6 and these cells have been found in elevated numbers in chronic inflammatory diseases including rheumatoid arthritis (RA)...

Identification of a novel subset of human circulating memory CD4+ T cells that produce both IL-17A and IL-4

IL-17A has been suggested to play a pathogenic role in bronchial asthma and other allergic disorders. Study of the relationship between human IL-17A-producing CD4(+) T(H) cells (T(H)17) and IL-4-producing CD4(+) T(H) (T(H)2) cells. T-cell clones generated from the CCR6(+)CD161(+) fraction of human circulating CD4(+) T cells, which contains virtually all T(H)17 cells, as well as circulating CD4(+) T cells from both healthy subjects and patients with asthma, were assessed by flow cytometry for their cytokine production profile. A small proportion of CCR6(+)CD161(+)CD4(+) T-cell clones showed the ability to produce both IL-17A and IL-4 (T(H)17/T(H)2). T(H)17/T(H)2 clones also produced IL-5, IL-8, IL-9, IL-13, IL-21, and IL-22 and displayed the ability to induce the in vitro secretion of IgE. A very few T(H)17/T(H)2 cells were found among circulating CD4(+) T cells from normal subjects, but their proportions were significantly increased in the circulation of patients with chronic asthma. T(H)17/T(H)2 cells could not be derived from naive umbilical cord blood CD4(+) T cells under any experimental condition. However, when circulating memory CCR6(+)CD161(+)CD4(+) T cells were cloned under appropriate polarizing conditions, T(H)17/T(H)2 clones originated in the presence of IL-4, suggesting that an IL-4-rich microenvironment may induce the shifting of memory T(H)17 cells into T(H)17/T(H)2 cells. Because of its peculiar functional properties and the increased numbers in the circulation of patients with bronchial asthma, this previously unknown population of T(H)17/T(H)2 cells may play some role in the pathogenesis of this disease.

Regulatory T Cells Sequentially Migrate from Inflamed Tissues to Draining Lymph Nodes to Suppress the Alloimmune Response

To determine the site and mechanism of suppression by regulatory T (Treg) cells, we investigated their migration and function in an islet allograft model. Treg cells first migrated from blood to the inflamed allograft where they were essential for the suppression of alloimmunity. This process was dependent on the chemokine receptors CCR2, CCR4, and CCR5 and P- and E-selectin ligands. In the allograft, Treg cells were activated and subsequently migrated to the draining lymph nodes (dLNs) in a CCR2, CCR5, and CCR7 fashion; this movement was essential for optimal suppression. Treg cells inhibited dendritic cell migration in a TGF-beta and IL-10 dependent fashion and suppressed antigen-specific T effector cell migration, accumulation, and proliferation in dLNs and allografts. These results showed that sequential migration from blood to the target tissue and to dLNs is required for Treg cells to differentiate and execute fully their suppressive function.