Lymph Node Homing Receptor CCR7 Research Articles

The PDE4 Inhibitor Tanimilast Blunts Proinflammatory Dendritic Cell Activation by SARS-CoV-2 ssRNAs.

Phosphodiesterase 4 (PDE4) inhibitors are immunomodulatory drugs approved to treat diseases associated with chronic inflammatory conditions, such as COPD, psoriasis and atopic dermatitis. Tanimilast (international non-proprietary name of CHF6001) is a novel, potent and selective inhaled PDE4 inhibitor in advanced clinical development for the treatment of COPD. To begin testing its potential in limiting hyperinflammation and immune dysregulation associated to SARS-CoV-2 infection, we took advantage of an in vitro model of dendritic cell (DC) activation by SARS-CoV-2 genomic ssRNA (SCV2-RNA). In this context, Tanimilast decreased the release of pro-inflammatory cytokines (TNF-α and IL-6), chemokines (CCL3, CXCL9, and CXCL10) and of Th1-polarizing cytokines (IL-12, type I IFNs). In contrast to β-methasone, a reference steroid anti-inflammatory drug, Tanimilast did not impair the acquisition of the maturation markers CD83, CD86 and MHC-II, nor that of the lymph node homing receptor CCR7. Consistent with this, Tanimilast did not reduce the capability of SCV2-RNA-stimulated DCs to activate CD4+ T cells but skewed their polarization towards a Th2 phenotype. Both Tanimilast and β-methasone blocked the increase of MHC-I molecules in SCV2-RNA-activated DCs and restrained the proliferation and activation of cytotoxic CD8+ T cells. Our results indicate that Tanimilast can modulate the SCV2-RNA-induced pro-inflammatory and Th1-polarizing potential of DCs, crucial regulators of both the inflammatory and immune response. Given also the remarkable safety demonstrated by Tanimilast, up to now, in clinical studies, we propose this inhaled PDE4 inhibitor as a promising immunomodulatory drug in the scenario of COVID-19.

Reprogramming NK cells and macrophages via combined antibody and cytokine therapy primes tumors for elimination by checkpoint blockade.

SUMMARYTreatments aiming to augment immune checkpoint blockade (ICB) in cancer often focus on T cell immunity, but innate immune cells may have important roles to play. Here, we demonstrate a single-dose combination treatment (termed AIP) using a pan-tumor-targeting antibody surrogate, half-life-extended interleukin-2 (IL-2), and anti-programmed cell death 1 (PD-1), which primes tumors to respond to subsequent ICB and promotes rejection of large established tumors in mice. Natural killer (NK) cells and macrophages activated by AIP treatment underwent transcriptional reprogramming; rapidly killed cancer cells; governed the recruitment of cross-presenting dendritic cells (DCs) and other leukocytes; and induced normalization of the tumor vasculature, facilitating further immune infiltration. Thus, innate cell-activating therapies can initiate critical steps leading to a self-sustaining cycle of T cell priming driven by ICB.

The C-terminal peptide of CCL21 drastically augments CCL21 activity through the dendritic cell lymph node homing receptor CCR7 by interaction with the receptor N-terminus

The endogenous chemokines CCL19 and CCL21 signal via their common receptor CCR7. CCL21 is the main lymph node homing chemokine, but a weak chemo-attractant compared to CCL19. Here we show that the 41-amino acid positively charged peptide, released through C-terminal cleavage of CCL21, C21TP, boosts the immune cell recruiting activity of CCL21 by up to 25-fold and the signaling activity via CCR7 by ~ 100-fold. Such boosting is unprecedented. Despite the presence of multiple basic glycosaminoglycan (GAG) binding motifs, C21TP boosting of CCL21 signaling does not involve interference with GAG mediated cell-surface retention. Instead, boosting is directly dependent on O-glycosylations in the CCR7 N-terminus. As dictated by the two-step binding model, the initial chemokine binding involves interaction of the chemokine fold with the receptor N-terminus, followed by insertion of the chemokine N-terminus deep into the receptor binding pocket. Our data suggest that apart from a role in initial chemokine binding, the receptor N-terminus also partakes in a gating mechanism, which could give rise to a reduced ligand activity, presumably through affecting the ligand positioning. Based on experiments that support a direct interaction of C21TP with the glycosylated CCR7 N-terminus, we propose that electrostatic interactions between the positively charged peptide and sialylated O-glycans in CCR7 N-terminus may create a more accessible version of the receptor and thus guide chemokine docking to generate a more favorable chemokine-receptor interaction, giving rise to the peptide boosting effect.

The PDE4 inhibitor CHF6001 modulates pro-inflammatory cytokines, chemokines and Th1- and Th17-polarizing cytokines in human dendritic cells

Phosphodiesterase 4 (PDE4) inhibitors are used to treat autoimmune and inflammatory diseases, such as psoriasis and chronic obstructive pulmonary disease (COPD). CHF6001 is a novel, potent and selective inhaled PDE4 inhibitor in development for the treatment of COPD. When tested in vitro on human dendritic cells (DCs), CHF6001 decreased the release of pro-inflammatory cytokines (TNF-α and IL-6), chemokines (CXCL8, CCL3, CXCL10 and CCL19) and of Th1- and Th17-polarizing cytokines (IL-12, IL-23 and IL-1β). In contrast to β-methasone, a reference steroid anti-inflammatory drug, CHF6001 increased the secretion of CCL22, a Th2 recruiting chemokine, and the expression of the lymph node homing receptor CCR7. Accordingly, the migration of DCs to CCR7 ligands was increased, while migration to pro-inflammatory chemokines was decreased. Of note, the action of CHF6001 was apparently mediated by a promoter-specific decrease in NF-κB p65 recruitment, independent of perturbation of LPS signalling or NF-κB nuclear translocation. Our results indicate that CHF6001 can modulate DC pro-inflammatory Th1/Th17 polarizing potential by fine tuning the transcriptional activity of the master inflammatory transcription factor NF-κB. Therefore, CHF6001 may prove useful to control Th1/Th17-polarized inflammatory diseases such as COPD.

A high migratory capacity of donor T-cells in response to the lymph node homing receptor CCR7 increases the incidence and severity of GvHD.

The pathogenesis of GvHD involves migration of donor T-cells into the secondary lymphoid organs in the recipient, which is steered by two homing molecules, CD62L and CCR7. Therefore, we investigated whether the migratory capacity of donor T-cells is associated with GvHD. This single center prospective study included 85 donor-recipient pairs. In vitro chemotaxis assays of the lymphocytes of the apheresis product were performed in parallel to the analysis of CD62L and CCR7 by flow cytometry. The migratory index to the CCR7 ligands, CCL19 and CCL21, was higher in T-cells from donors whose recipients will develop GvHD. Similarly, the acute GvHD (aGvHD) group received higher percentage of CD4+CCR7+ T-cells, whereas chronic GvHD (cGvHD) patients were transplanted with higher percentages of CD8+CCR7+ T-cells compared with the non-GvHD group. These results were confirmed when patients were subdivided according to degrees of severity. Further, multivariate analysis confirmed that the proportions of CCR7+ CD4+ and CCR7+ CD8+ T-cells are risk factors for the development and severity of aGvHD and cGvHD, respectively. Functional experiments demonstrated that CCR7+ T-cells exhibited higher potential for activation than CCR7- T-cells did. We therefore propose that the selective depletion of CCR7-expressing T-cells may be an effective preventive therapy for GvHD.

Human γδ T cells augment antigen presentation in Listeria Monocytogenes infection.

Circulating γδ T cells in healthy individuals rapidly respond to bacterial and viral pathogens. Many studies have demonstrated that γδ T cells are activated and expanded by Listeria monocytogenes (L.monocytogenes), a foodborne bacterial pathogen with high fatality rates. However, the roles of γδ T cells during L.monocytogenes infection are not clear. In the present study, we characterized the morphological characteristics of phagocytosis in γδ T cells after L.monocytogenes infection using transmission electron microscopy. Results show activation markers including HLA-DR and lymph node-homing receptor CCR7 on γδ T cells were upregulated after stimulation via L.monocytogenes. Significant proliferation and differentiation of primary αβ T cells was also observed after co-culture of peripheral blood mononuclear cells with γδ T cells anteriorly stimulated by L.monocytogenes. L.monocytogenes infection decreased the percentage of γδ T cells in mouse IELs and increased MHC-II expression on the surface of γδ T cells in vivo. Our findings shed light on antigen presentation of γδ T cells during L.monocytogenes infection.

Acquistion of Effector and Memory Responses is Reciprocally Regulated by IL-12 and IFN-α/β in Human CD8+ T Cells (96.8)

Abstract The innate cytokines IL-12 and IFN-α/β act in concert to facilitate clearance of intracellular pathogens. However their precise role in promoting human T cell responses is still unclear. In this study, we demonstrate that these cytokines regulate distinct aspects of effector and memory human CD8+ T cell differentiation. We found that IL-12 programs the development of TEM cells which secrete high levels of IFN-γand TNF-α and attain strong cytolytic activity. Programming of TEM cells by IL-12 correlated to the induction of the IL-12 receptor β 2 (IL-2Rβ2) and was associated with cells which had undergone rapid division. Alternatively, IFN- α/β enhanced a population of TCM cells which expressed high levels of the lymph node homing receptor CCR7. This population did not display immediate effector activity however, upon secondary activation, were capable of rapid proliferation and acquisition of both memory and effector phenotypes. TCM development was regulated by IFN-αand proceeded by slowing the progression of cell division in a subpopulation of cells that selectively expressed elevated IFN- α/βreceptor-2 (IFNAR2). We further examined the effects of the strength of TCR signal on TEM and TCM development. In the presence of both IL-12 and IFN- α/β, these cytokine signals were amplified as the strength of the TCR signal was increased, promoting the simultaneous development of both TCM and TEM. Together, our results support a novel model in which IL-12 and IFN- α/βact in a non-redundant manner to regulate the co-linear generation of both effector and memory cells. This study is supported by a grant from the National Institutes of Health/National Institute of Allergy and Infectious Disease (AI56222) and by a predoctoral fellowship from the National Instititue of Health/Nation Institute of Allergy and Infectious Disease (AI68622).

A high-mobility, low-cost phenotype defines human effector-memory CD8+ T cells

AbstractT cells move randomly (“random-walk”), a characteristic thought to be integral to their function. Using migration assays and time-lapse microscopy, we found that CD8+ T cells lacking the lymph node homing receptors CCR7 and CD62L migrate more efficiently in transwell assays, and that these same cells are characterized by a high frequency of cells exhibiting random crawling activity under culture conditions mimicking the interstitial/extravascular milieu, but not when examined on endothelial cells. To assess the energy efficiency of cells crawling at a high frequency, we measured mRNA expression of genes key to mitochondrial energy metabolism (peroxisome proliferator–activated receptor γ coactivator 1β [PGC-1β], estrogen-related receptor α [ERRα], cytochrome C, ATP synthase, and the uncoupling proteins [UCPs] UCP-2 and -3), quantified ATP contents, and performed calorimetric analyses. Together these assays indicated a high energy efficiency of the high crawling frequency CD8+ T-cell population, and identified differentially regulated heat production among nonlymphoid versus lymphoid homing CD8+ T cells.

Brain antigens in functionally distinct antigen-presenting cell populations in cervical lymph nodes in MS and EAE

Drainage of central nervous system (CNS) antigens to the brain-draining cervical lymph nodes (CLN) is likely crucial in the initiation and control of autoimmune responses during multiple sclerosis (MS). We demonstrate neuronal antigens within CLN of MS patients. In monkeys and mice with experimental autoimmune encephalomyelitis (EAE) and in mouse models with non-inflammatory CNS damage, the type and extent of CNS damage was associated with the frequencies of CNS antigens within the cervical lymph nodes. In addition, CNS antigens drained to the spinal-cord-draining lumbar lymph nodes. In human MS CLN, neuronal antigens were present in pro-inflammatory antigen-presenting cells (APC), whereas the majority of myelin-containing cells were anti-inflammatory. This may reflect a different origin of the cells or different drainage mechanisms. Indeed, neuronal antigen-containing cells in human CLN did not express the lymph node homing receptor CCR7, whereas myelin antigen-containing cells in situ and in vitro did. Nevertheless, CLN from EAE-affected CCR7-deficient mice contained equal amounts of myelin and neuronal antigens as wild-type mice. We conclude that the type and frequencies of CNS antigens within the CLN are determined by the type and extent of CNS damage. Furthermore, the presence of myelin and neuronal antigens in functionally distinct APC populations within MS CLN suggests that differential immune responses can be evoked.

The Adjuvants Aluminum Hydroxide and MF59 Induce Monocyte and Granulocyte Chemoattractants and Enhance Monocyte Differentiation toward Dendritic Cells

Aluminum hydroxide (alum) and the oil-in-water emulsion MF59 are widely used, safe and effective adjuvants, yet their mechanism of action is poorly understood. We assessed the effects of alum and MF59 on human immune cells and found that both induce secretion of chemokines, such as CCL2 (MCP-1), CCL3 (MIP-1alpha), CCL4 (MIP-1beta), and CXCL8 (IL-8), all involved in cell recruitment from blood into peripheral tissue. Alum appears to act mainly on macrophages and monocytes, whereas MF59 additionally targets granulocytes. Accordingly, monocytes and granulocytes migrate toward MF59-conditioned culture supernatants. In monocytes, both adjuvants lead to increased endocytosis, enhanced surface expression of MHC class II and CD86, and down-regulation of the monocyte marker CD14, which are all phenotypic changes consistent with a differentiation toward dendritic cells (DCs). When monocyte differentiation into DCs is induced by addition of cytokines, these adjuvants enhanced the acquisition of a mature DC phenotype and lead to an earlier and higher expression of MHC class II and CD86. In addition, MF59 induces further up-regulation of the maturation marker CD83 and the lymph node-homing receptor CCR7 on differentiating monocytes. Alum induces a similar but not identical pattern that clearly differs from the response to LPS. This model suggests a common adjuvant mechanism that is distinct from that mediated by danger signals. We conclude that during vaccination, adjuvants such as MF59 may increase recruitment of immune cells into the injection site, accelerate and enhance monocyte differentiation into DCs, augment Ag uptake, and facilitate migration of DCs into tissue-draining lymph nodes to prime adaptive immune responses.

IL‐12 and IFNα/β regulate non‐redundant functions in human CD8+ T cells

During viral infection, dendritic cells secrete the innate cytokines IL‐12 and IFNα/β to facilitate viral clearance. These cytokines have been linked to CD8+ T cell and are widely thought to act in a redundant manner to facilitate cytokine secretion and cytolytic activity. We examined the ability of IL‐12 and IFNα/β to drive these responses in human CD8+CD45RA+ T cells. We found that IL‐12 was sufficient for the generation of CD8+ effector cell activity, marked by IFNγ and TNFα secretion and strong cytolytic activity. In contrast, IFNα/β alone did not promote IFNγ or TNFα. Although, IFNα/β markedly induced perforin and granzyme B expression as a whole, these cells displayed functionally defective lytic activity in the absence of IL‐12 signaling. Thus, IL‐12 but not IFNα/β regulates primary effector functions in human CD8+ T cells. Interestingly, IFNα/β enhanced the expression of lymph node homing receptor CCR7 on a subset of T cells. This subpopulation displayed various characteristics of a memory phenotype, such as low CXCR3 and perforin expression while retaining granzyme B expression. Further, development of this CCR7 hi sub population was regulated solely by IFNα/β and was independent of the effects of IL‐12. These results demonstrate that IL‐12 and IFNα/β are not functionally redundant and in fact, impart unique phenotypes on human CD8+ T cells.This study is supported by a grant from the National Institutes of Health/National Institute of Allergy and Infectious Disease (AI56222) and by a predoctoral fellowship from the National Instititue of Health/Nation Institute of Allergy and Infectious Disease (AI68622).

Function-Associated Surface Molecules on Airway Dendritic Cells in Cigarette Smokers

Airway dendritic cells (DCs) control pulmonary immune responses to inhaled particles. However, the profile of function-associated surface molecules on airway DCs in smokers is unknown. In this study, function-associated surface molecules were analyzed using four-color flow cytometry on myeloid DCs (mDCs) in bronchoalveolar lavage fluid (BALF) of cigarette smokers and never-smokers. Furthermore, the lung function was assessed directly before bronchoscopy in all participants. There was a 7-fold increase in total cell numbers in BALF of smokers, as compared with never-smokers. The percentage of mDCs among BALF cells and the expression of the maturation marker CD83 on mDCs did not differ between smokers and never-smokers. However, there was a strong increase in the expression of Langerin and CD1a (markers of Langerhans cells) on mDCs of smokers. Furthermore, mDCs of smokers were characterized by an increased expression of antigen presentation markers such as CD80 and CD86. By contrast, mDCs of smokers displayed a decreased expression of the lymph node homing receptor CCR7, as compared with mDCs of never-smokers. Decreased expression of CCR7 on mDCs, but not any of the other surface molecules studied, was specifically associated with airway obstruction and pulmonary hyperinflation in smokers. In conclusion, our data suggest that smoking affects the expression profile of function-associated surface molecules on airway mDCs. We provide the first evidence that a reduced CCR7 expression on airway mDCs is associated with airflow limitation in smokers.

Only the CD45RA+ subpopulation of CD4+CD25high T cells gives rise to homogeneous regulatory T-cell lines upon in vitro expansion

AbstractThymus-derived CD4+CD25+ regulatory T cells suppress autoreactive CD4+ and CD8+ T cells and thereby protect from autoimmunity. In animal models, adoptive transfer of CD4+CD25+ regulatory T cells has been shown to prevent and even cure autoimmune diseases as well as pathogenic alloresponses after solid organ and stem-cell transplantations. We recently described methods for the efficient in vitro expansion of human regulatory T cells for clinical applications. We now demonstrate that only CCR7- and L-selectin (CD62L)–coexpressing cells within expanded CD4+CD25high T cells maintain phenotypic and functional characteristics of regulatory T cells. Further analysis revealed that these cells originate from CD45RA+ naive cells within the CD4+CD25high T-cell compartment, as only this subpopulation homogeneously expressed CD62L, CCR7, cytotoxic T lymphocyte–associated antigen-4 (CTLA-4), and forkhead box P3 (FOXP3), produced no inflammatory cytokines and maintained robust suppressive activity after expansion. In contrast, cell lines derived from CD45RA– memory-type CD4+CD25high T cells lost expression of lymph node homing receptors CCR7 and CD62L, contained interleukin-2 (IL-2) and interferon-γ (IFN-γ) as well as IL-10–secreting cells, showed only moderate suppression and, most importantly, did not maintain FOXP3 expression. Based on these unexpected findings, we suggest that isolation and expansion of CD45RA+ naive CD4+ CD25high T cells is the best strategy for adoptive regulatory T (Treg)–cell therapies.

Nodal peripheral T‐cell lymphomas correspond to distinct mature T‐cell populations

Peripheral T-cell lymphomas (PTCL) have not been successfully correlated with specific developmental stages of reactive T-cells. Mature T-cells pass through distinct stages upon antigen encounter. Naïve T-cells are CD45RA(+)/CD45R0(-)/CD27(+)/CCR7(+). After antigen contact they replace CD45RA expression with CD45R0. The mature T-cells differentiate to central memory cells, which retain CD27 and CCR7, or to effector memory cells, which lose expression of both molecules depending on the strength of the antigen interaction. In this study, we evaluated lymph node biopsies from eight PTCL-not otherwise specified (PTCL-NOS), seven angioimmunoblastic T-cell lymphomas (AILT), and 15 anaplastic large cell lymphomas (ALCL). Detection of tumour cells with antibodies that recognize specific rearranged T-cell receptor Vbeta segments allowed us to investigate the expression of various differentiation-associated molecules. Results were analysed by hierarchical cluster analysis. All AILT and ALCL showed a homogeneous effector cell phenotype (CD45RA(-)/CD45R0(+)/CD27(-)), but differed in the cytotoxic and activation markers expressed. Several (5/8) PTCL-NOS clustered together; these cases all exhibited a CD4(+) central memory cell phenotype (CD45RA(-)/CD45R0(+)/CD27(+)) and four expressed the lymph node homing receptor CCR7. In conclusion, AILT and ALCL tumour cells correspond to different subsets of effector cells, while a subset of PTCL-NOS correlates with a non-effector T-cell population.

Chemokine receptor expression identifies Pre-T helper (Th)1, Pre-Th2, and nonpolarized cells among human CD4+ central memory T cells.

We previously reported that central–memory T cells (TCM cells), which express lymph node homing receptors CCR7 and CD62L, are largely devoid of effector functions but acquire characteristics of effector–memory T cells (TEM cells) (i.e., CCR7− T helper [Th]1 or Th2 cells) after stimulation with T cell receptor agonists or homeostatic cytokines. Here we show that three chemokine receptors identify functional subsets within the human CD4+ TCM cell pool. TCM cells expressing CXCR3 secreted low amounts of interferon γ, whereas CCR4+ TCM cells produced some interleukin (IL)-4, but not IL-5. In response to IL-7 and IL-15, CXCR3+ TCM and CCR4+ TCM cells invariably generated fully differentiated CCR7− Th1 and Th2 cells, respectively, suggesting that they represent pre-Th1 and pre-Th2 cells. Conversely, CXCR5+ TCM cells lacking CXCR3 and CCR4 remained nonpolarized and retained CCR7 and CD62L expression upon cytokine-driven expansion. Unlike naive cells, all memory subsets had a low T cell receptor rearrangement excision circle content, spontaneously incorporated bromodeoxyuridine ex vivo, and contained cells specific for tetanus toxoid. Conversely, recall responses to cytomegalovirus and vaccinia virus were largely restricted to CXCR3+ TCM and TEM cells. We conclude that antigen-specific memory T cells are distributed between TEM cells and different subsets of TCM cells. Our results also explain how the quality of primary T cell responses could be maintained by TCM cells in the absence of antigen.

A pool of central memory-like CD4 T cells contains effector memory precursors.

The L51S mutation in the D10.G4.1 TCR alpha-chain reduces the affinity of the TCR to its ligand by affecting the interactions among the TCR, the beta-chain of I-A(k), and the bound peptide. We show that this mutation drives the generation of a pool of memory CD44(high)CD62L(neg)CD45RB(neg) CD4 TCR transgenic T cells. Their activation threshold is low, such that they proliferate in response to lower concentrations of agonist peptides than naive L51S CD4 T cells. Unlike effector memory CD4 T cells, however, they lack immediate effector function in response to TCR stimulation. These cells express IL-2R alpha only after culture with specific peptide. Although they can be recovered from lymph nodes, the majority lack the expression of the lymph node homing receptor CCR7. When these cells receive a second TCR stimulation in vitro, they differentiate into potent Th2-like effector cells, producing high levels of IL-4 at doses of agonist peptide too low to stimulate cytokine release from similarly differentiated naive L51S CD4 T cells. Having these properties, the L51S TCR transgenic memory CD4 T cells cannot be classified as either strict central memory or effector memory, but, rather, as a pool of memory T cells containing effector memory precursors.