Spatial dysregulation of T follicular helper cells impairs vaccine responses in aging.

The magnitude and quality of the germinal center (GC) response decline with age, resulting in poor vaccine-induced immunity in older individuals. A functional GC requires the co-ordination of multiple cell types across time and space, in particular across its two functionally distinct compartments: the light and dark zones. In aged mice, there is CXCR4-mediated mislocalization of T follicular helper (TFH) cells to the dark zone and a compressed network of follicular dendritic cells (FDCs) in the light zone. Here we show that TFH cell localization is critical for the quality of the antibody response and for the expansion of the FDC network upon immunization. The smaller GC and compressed FDC network in aged mice were corrected by provision of TFH cells that colocalize with FDCs using CXCR5. This demonstrates that the age-dependent defects in the GC response are reversible and shows that TFH cells support stromal cell responses to vaccines.

DUSP4-mediated accelerated T-cell senescence in idiopathic CD4 lymphopenia.

Idiopathic CD4 lymphopenia (ICL) is a rare heterogeneous immunological syndrome of unclear etiology. ICL predisposes patients to severe opportunistic infections and frequently leads to poor vaccination effectiveness. Chronic immune activation, expansion of memory T cells, and impaired T-cell receptor (TCR) signaling have been reported in ICL, but the mechanistic and causative links remain unclear. We show that late-differentiated T cells in 20 patients with ICL displayed defective TCR responses and aging markers similar to those found in T cells from elderly subjects. Intrinsic T-cell defects were caused by increased expression of dual-specific phosphatase 4 (DUSP4). Normalization of DUSP4 expression using a specific siRNA improved CD4(+) T-cell activity in ICL, as this restored TCR-induced extracellular signal-regulated kinase activation and increased the expression of the costimulatory molecules CD27 and CD40L. Conversely, repeated TCR stimulation led to defective signaling and DUSP4 overexpression in control CD4(+) T cells. This was associated with gradual acquisition of a memory phenotype and was curtailed by DUSP4 silencing. These findings identify a premature T-cell senescence in ICL that might be caused by chronic T-cell activation and a consequential DUSP4-dependent dampening of TCR signaling.

CCR1 inhibition ameliorates the progression of lupus nephritis in NZB/W mice.

Systemic lupus erythematosus is a chronic inflammatory autoimmune disease, the development of which is characterized by a progressive loss of renal function. Such dysfunction is associated with leukocyte infiltration in the glomerular and tubulointerstitial compartments in both human and experimental lupus nephritis. In this study, we investigated the role of the Ccr1 chemokine receptor in this infiltration process during the progression of nephritis in the lupus-prone New Zealand Black/New Zealand White (NZB/W) mouse model. We found that peripheral T cells, mononuclear phagocytes, and neutrophils, but not B cells, from nephritic NZB/W mice were more responsive to Ccr1 ligands than the leukocytes from younger prenephritic NZB/W mice. Short-term treatment of nephritic NZB/W mice with the orally available Ccr1 antagonist BL5923 decreased renal infiltration by T cells and macrophages. Longer Ccr1 blockade decreased kidney accumulation of effector/memory CD4(+) T cells, Ly6C(+) monocytes, and both M1 and M2 macrophages; reduced tubulointerstitial and glomerular injuries; delayed fatal proteinuria; and prolonged animal lifespan. In contrast, renal humoral immunity was unaffected in BL5923-treated mice, which reflected the unchanged numbers of infiltrated B cells in the kidneys. Altogether, these findings define a pivotal role for Ccr1 in the recruitment of T and mononuclear phagocyte cells to inflamed kidneys of NZB/W mice, which in turn contribute to the progression of renal injury.

Neutrophil extracellular traps downregulate lipopolysaccharide-induced activation of monocyte-derived dendritic cells.

Polymorphonuclear neutrophils (PMN) play a central role in inflammation and participate in its control, notably by modulating dendritic cell (DC) functions via soluble mediators or cell-cell contacts. Neutrophil extracellular traps (NETs) released by PMN could play a role in this context. To evaluate NET effects on DC maturation, we developed a model based on monocyte-derived DC (moDC) and calibrated NETs isolated from fresh human PMN. We found that isolated NETs alone had no discernable effect on moDC. In contrast, they downregulated LPS-induced moDC maturation, as shown by decreased surface expression of HLA-DR, CD80, CD83, and CD86, and by downregulated cytokine production (TNF-α, IL-6, IL-12, IL-23), with no increase in the expression of tolerogenic DC genes. Moreover, the presence of NETs during moDC maturation diminished the capacity of these moDC to induce T lymphocyte proliferation in both autologous and allogeneic conditions, and modulated CD4(+) T lymphocyte polarization by promoting the production of Th2 cytokines (IL-5 and IL-13) and reducing that of Th1 and Th17 cytokines (IFN-γ and IL-17). Interestingly, the expression and activities of the lymphoid chemokine receptors CCR7 and CXCR4 on moDC were not altered when moDC matured in the presence of NETs. Together, these findings reveal a new role for NETs in adaptive immune responses, modulating some moDC functions and thereby participating in the control of inflammation.

CXCR4 dysfunction in non-alcoholic steatohepatitis in mice and patients.

Homing of inflammatory cells to the liver is key in the progression of non-alcoholic steatohepatitis (NASH). An abnormal response of CD4+ T-cells from obese mice to the chemotactic effect of CXCL12 has been reported but the mechanism involved in this process and relevance in patients are unknown. We aimed to explore the mechanism involved in the abnormal chemotaxis of CXC chemokine ligand 12 (CXCL12) in several mouse models of NASH and the relevance in the context of human non-alcoholic fatty liver disease (NAFLD). We assessed chemotactic responsiveness of CD4+ T-cells to CXCL12, the effect of AMD3100, a CXC chemokine receptor 4 (CXCR4) antagonist, in mice and lymphocytes from patients with NAFLD, and the affinity of CXCL12 for CXCR4. CXCL12-promoted migration of CD4+ T-cells from three different mouse models of NASH was increased and dependent of CXCR4. CD4+ T-cells from patients with NASH, but not from patients with pure steatosis, responded more strongly to the chemotactic effect of CXCL12, and this response was inhibited by AMD3100. Treatment with AMD3100 decreased the number of CD4+ T-cells to the liver in ob/ob mice. CXCL12 expression in the liver, CXCR4 and CXCR7 expression in CD4+ T-cells were not increased in three different mouse models of NASH. However, the affinity of CXCL12 for CXCR4 was increased in CD4+ T-cells of ob/ob mice. In conclusion, the CXCL12/CXCR4 pathway contributes in both mice and patients to the enhanced recruitment of CD4+ T-cells in NASH. An increased affinity of CXCL12 to CXCR4 rather than a higher expression of the chemokine or its receptors is involved in this process.

Complementary methods provide evidence for the expression of CXCR7 on human B cells.

PTMs of extracellular domains of membrane proteins can influence antibody binding and give rise to ambivalent results. Best proof of protein expression is the use of complementary methods to provide unequivocal evidence. CXCR7, a member of the atypical chemokine receptor family, mainly functions as scavenger for the chemokines CXCL12 and CXCL11. The expression of CXCR7 on nonhematopoietic cells and neoplasms is widely accepted, however, its expression on leukocytes was recently challenged. To solve the dissent, we thoroughly analyzed the expression of CXCR7 on human B cells. We validated the efficiency of different epitope-specific monoclonal antibodies to detect CXCR7 on transfected cells and primary human B cells. The specificity of the used antibodies was further confirmed by an experimentally independent double labeling approach. Examination of CXCR7-dependent scavenging of fluorescent-labeled CXCL12 revealed functional expression of the receptor on human B cells. Moreover, real-time PCR analysis of CXCR7 mRNA showed the presence of transcripts in human leukocytes. Finally, two CXCR7-specific peptides were identified by MS in immunoprecipitates from primary human B cells. Thus, we present a strategy based on combined proteomic and functional approaches that can be used to solve dissents on protein expression, i.e. demonstrating the expression of CXCR7 on human leukocytes.

Expression of CXCL12 receptors in B cells from Mexican Mestizos patients with systemic Lupus erythematosus.

BACKGROUND: Systemic Lupus Erythematosus (SLE) is a chronic autoimmune disease characterized by B-cell hyper-reactivity and the production of pathogenic anti-nuclear-directed auto-antibodies (Abs). B-cell ontogeny is partly dependent on the CXCL12/CXCR4 axis for which the contribution to SLE pathogenesis remains unclear. CXCR7, the novel receptor for CXCL12, is differentially expressed among memory B-cell subsets. However, its biological role in SLE remains to be explored. METHODS: Relative CXCR4 and CXCR7 expression levels were compared by quantitative PCR in leukocytes from blood samples of 41 Mexican Mestizos patients with SLE and 45 ethnicity-matched healthy subjects. Intracellular and membrane expression of both receptors was analyzed by flow cytometry in naive and Ab-secreting B cells. B-cell responsiveness to CXCL12 was investigated using Transwell-based chemotaxis assays. Data were analyzed using the Kruskal-Wallis test for comparisons of values amongst healthy controls and patients with inactive or active SLE, and non-parametrically using the Mann-Whitney U-test for multiple comparisons and unpaired samples. Correlations were determined by Spearman's ranking. RESULT: SLE leukocytes displayed reduced levels of CXCR4 and CXCR7 transcripts. In SLE patients, a significant defect in CXCR4 expression was detected at the surface of naive and Ab-secreting B cells, associated with an abnormal intracellular localization of the receptor. CXCR7 predominantly localized in cytosolic compartments of B cells from healthy and SLE individuals. Disease activity did not impact on these expression patterns. Altered receptor compartmentalization correlated with an impaired CXCL12-promoted migration of SLE B cells. CONCLUSIONS: Our data highlight a down-regulation of CXCL12 receptors on circulating B cells from SLE patients that likely influences their migratory behavior and distribution.

[CXCR4, a therapeutic target in rare immunodeficiencies?]. / CXCR4, une cible thérapeutique dans certains déficits immunitaires rares?

Currently, more than 200 primary immunodeficiency diseases have been discovered. In most cases, genetic defects affect the expression or the function of proteins involved in immune development and homeostasis. Some orphan immuno-hematological disorders are characterized by an abnormal leukocyte trafficking, a notion predictive of an anomaly of the chemokine/chemokine receptor system. In this review, we focus on recent advances in the characterization of dysfunctions of the CXCL12 (SDF-1)/CXCR4 signaling axis in two rare human immunodeficiencies, one associated with a loss of CXCR4 function, the Idiopathic CD4(+) T-cell Lymphocytopenia, and the other with a gain of CXCR4 function, the WHIM syndrome.

Ageing promotes early T follicular helper cell differentiation by modulating expression of RBPJ.

Ageing profoundly changes our immune system and is thought to be a driving factor in the morbidity and mortality associated with infectious disease in older people. We have previously shown that the impaired immunity to vaccination that occurs in aged individuals is partly attributed to the effect of age on T follicular helper (Tfh) cell formation. In this study, we examined how age intrinsically affects Tfh cell formation in both mice and humans. We show increased formation of Tfh precursors (pre-Tfh) but no associated increase in germinal centre (GC)-Tfh cells in aged mice, suggesting age-driven promotion of only early Tfh cell differentiation. Mechanistically, we show that ageing alters TCR signalling which drives expression of the Notch-associated transcription factor, RBPJ. Genetic or chemical modulation of RBPJ or Notch rescues this age-associated early Tfh cell differentiation, and increased intrinsic Notch activity recapitulates this phenomenon in younger mice. Our data offer mechanistic insight into the age-induced changes in T-cell activation that affects the differentiation and ultimately the function of effector T cells.

Rejuvenating conventional dendritic cells and T follicular helper cell formation after vaccination.

Germinal centres (GCs) are T follicular helper cell (Tfh)-dependent structures that form in response to vaccination, producing long-lived antibody secreting plasma cells and memory B cells that protect against subsequent infection. With advancing age the GC and Tfh cell response declines, resulting in impaired humoral immunity. We sought to discover what underpins the poor Tfh cell response in ageing and whether it is possible to correct it. Here, we demonstrate that older people and aged mice have impaired Tfh cell differentiation upon vaccination. This deficit is preceded by poor activation of conventional dendritic cells type 2 (cDC2) due to reduced type 1 interferon signalling. Importantly, the Tfh and cDC2 cell response can be boosted in aged mice by treatment with a TLR7 agonist. This demonstrates that age-associated defects in the cDC2 and Tfh cell response are not irreversible and can be enhanced to improve vaccine responses in older individuals.

Escherichia coli Heat-Labile Enterotoxin B Limits T Cells Activation by Promoting Immature Dendritic Cells and Enhancing Regulatory T Cell Function.

Treatments to limit T cell activation are essential for managing autoimmune and inflammatory disorders. The B subunit of Escherichia coli heat-labile enterotoxin (EtxB) is known to ameliorate inflammatory disease in vivo but the mechanism by which this is mediated is not well understood. Here, we show that following intranasal administration, EtxB acts on two key cellular regulators of T cell activation: regulatory T cells and dendritic cells (DCs). EtxB enhances the proliferation of lung regulatory T cells and doubles their suppressive function, likely through an increase in expression of the Treg effector molecule CTLA-4. EtxB supports the generation of interleukin-10-producing DCs that are unable to activate T cells. These data show, for the first time, that mucosal EtxB treatment limits T cells activation by acting jointly on two distinct types of immune cells.

Follicular regulatory T cells can be specific for the immunizing antigen and derive from naive T cells.

T follicular regulatory (Tfr) cells are a subset of Foxp3(+) regulatory T (Treg) cells that form in response to immunization or infection, which localize to the germinal centre where they control the magnitude of the response. Despite an increased interest in the role of Tfr cells in humoral immunity, many fundamental aspects of their biology remain unknown, including whether they recognize self- or foreign antigen. Here we show that Tfr cells can be specific for the immunizing antigen, irrespective of whether it is a self- or foreign antigen. We show that, in addition to developing from thymic derived Treg cells, Tfr cells can also arise from Foxp3(-) precursors in a PD-L1-dependent manner, if the adjuvant used is one that supports T-cell plasticity. These findings have important implications for Tfr cell biology and for improving vaccine efficacy by formulating vaccines that modify the Tfr:Tfh cell ratio.

Altered chemotactic response to CXCL12 in patients carrying GATA2 mutations.

GATA2 deficiency-formerly described as MonoMAC syndrome; dendritic cells, monocytes, B cells, and natural killer cell deficiency; familial myelodysplastic syndrome/acute myeloid leukemia; or Emberger syndrome-encompasses a range of hematologic and nonhematologic anomalies, mainly characterized by monocytopenia, B lymphopenia, natural killer cell cytopenia, neutropenia, immunodeficiency, and a high risk of developing acute myeloid leukemia. Herein, we present 7 patients with GATA2 deficiency recruited into the French Severe Chronic Neutropenia Registry, which enrolls patients with all kinds of congenital neutropenia. We performed extended immunophenotyping of their whole blood lymphocyte populations, together with the analysis of their chemotactic responses. Lymphopenia was recorded for B and CD4(+) T cells in 6 patients. Although only 3 patients displayed natural killer cell cytopenia, the CD56(bright) natural killer subpopulation was nearly absent in all 7 patients. Natural killer cells from 6 patients showed decreased CXCL12/CXCR4-dependent chemotaxis, whereas other lymphocytes, and most significantly B lymphocytes, displayed enhanced CXCL12-induced chemotaxis compared with healthy volunteers. Surface expression of CXCR4 was significantly diminished in the patients' natural killer cells, although the total expression of the receptor was found to be equivalent to that of natural killer cells from healthy individual controls. Together, these data reveal that GATA2 deficiency is associated with impaired membrane expression and chemotactic dysfunctions of CXCR4. These dysfunctions may contribute to the physiopathology of this deficiency by affecting the normal distribution of lymphocytes and thus potentially affecting the susceptibility of patients to associated infections.

[Dysfunctions of the CXCL12 (SDF-1)/CXCR4 signaling axis in the WHIM syndrome and the idiopathic CD4(+) T-cell lymphocytopenia]. / Anomalies de l'axe de signalisation CXCL12 (SDF-1)/CXCR4 dans le syndrome WHIM et la lymphopénie T CD4(+) idiopathique.

Chemokines are small cytokine-like secreted proteins that govern migration of leukocytes to their specific niches in lymphoid organs and to inflammatory sites. They mediate their functions by binding to and activating chemokine receptors, which belong to the heptahelical G protein-coupled receptor family. The CXC chemokine Stromal cell Derived Factor-1 (SDF-1/CXCL12) is the sole natural ligand for the broadly expressed CXCR4 receptor and acts as a chemoattractant for many leukocyte subsets. The CXCL12/CXCR4 axis exerts critical activities in homeostatic processes such as organogenesis, hematopoiesis and leukocyte trafficking. Dysregulations of CXCR4 signaling and/or expression are associated with several infectious, inflammatory, autoimmune and malignant conditions. In light of recent data, we review here CXCR4 dysfunctions unveiled in two rare human immunodeficiency disorders, one characterized by a gain of CXCR4 function, the WHIM syndrome, and the other by a loss of CXCR4 function, the idiopathic CD4(+) T-cell lymphocytopenia.