Effector T-cell trafficking between the leptomeninges and the cerebrospinal fluid.

In multiple sclerosis, brain-reactive T cells invade the central nervous system (CNS) and induce a self-destructive inflammatory process. T-cell infiltrates are not only found within the parenchyma and the meninges, but also in the cerebrospinal fluid (CSF) that bathes the entire CNS tissue. How the T cells reach the CSF, their functionality, and whether they traffic between the CSF and other CNS compartments remains hypothetical. Here we show that effector T cells enter the CSF from the leptomeninges during Lewis rat experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. While moving through the three-dimensional leptomeningeal network of collagen fibres in a random Brownian walk, T cells were flushed from the surface by the flow of the CSF. The detached cells displayed significantly lower activation levels compared to T cells from the leptomeninges and CNS parenchyma. However, they did not represent a specialized non-pathogenic cellular sub-fraction, as their gene expression profile strongly resembled that of tissue-derived T cells and they fully retained their encephalitogenic potential. T-cell detachment from the leptomeninges was counteracted by integrins VLA-4 and LFA-1 binding to their respective ligands produced by resident macrophages. Chemokine signalling via CCR5/CXCR3 and antigenic stimulation of T cells in contact with the leptomeningeal macrophages enforced their adhesiveness. T cells floating in the CSF were able to reattach to the leptomeninges through steps reminiscent of vascular adhesion in CNS blood vessels, and invade the parenchyma. The molecular/cellular conditions for T-cell reattachment were the same as the requirements for detachment from the leptomeningeal milieu. Our data indicate that the leptomeninges represent a checkpoint at which activated T cells are licensed to enter the CNS parenchyma and non-activated T cells are preferentially released into the CSF, from where they can reach areas of antigen availability and tissue damage.

The Role of Type III Interferons in Human Disease.

PURPOSE: This literature review summarizes the main immunological characteristics of type III interferons (IFN) and highlights the clinically relevant aspects and future therapeutic perspectives for these inflammatory molecules. SOURCE: Relevant articles in PubMed MEDLINE from the first publication (2003) until 2020. N=101 articles were included in this review. PRINCIPAL FINDINGS: Type III IFNs represent a relatively newly described inflammatory cytokine family. Although they induce substantially similar signalling to the well-known type I IFNs, significant functional differences make these molecules remarkable. Type III IFNs have extensive biological effects, contributing to the pathogenesis of several diseases and also offering new diagnostic and therapeutic approaches: 1) their potent anti-viral properties make them promising therapeutics against viral hepatitis and even against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is causing the current coronavirus disease 2019 (COVID-19) pandemic; 2) imbalances in the IFN-λs contribute to several forms of chronic inflammation (e.g., systemic and organ-specific autoimmune diseases) and potentially predict disease progression and therapeutic response to biologic therapies; and 3) the antitumor properties of the type III IFNs open up new therapeutic perspectives against malignant diseases. CONCLUSION: Over the last 18 years, researchers have gathered extensive information about the presence and role of these versatile inflammatory cytokines in human diseases, but further research is needed to clarify the mechanistic background of those observations. Better understanding of their biological activities will permit us to use type III IFNs more efficiently in new diagnostic approaches and individualized therapies, consequently improving patient care.

Toll-like receptor 2 activation induces C-C chemokine receptor 2-dependent natural killer cell recruitment to the peritoneum.

Natural killer (NK) cells are innate effector cells with critical roles not only in tumor immunosurveillance and viral immunity, but also in bacterial and fungal infections. Toll-like receptor 2 (TLR2) can be important in the early and sustained immune responses to pathogens and tumors through the induction of cytokines and chemokines that recruit and activate immune effector cells. We investigated the role of TLR2 activation in NK cell recruitment with a view to informing approaches to induce or regulate peritoneal NK cell responses therapeutically. Peritoneal injection of TLR2 activators, including peptidoglycan and the lipopeptides FSL-1 and Pam3 CSK4 , resulted in NK cell recruitment after 16 h with increased NK cell numbers maintained for 48 h. TLR2 activators induced large amounts of CCR2 ligands, but much smaller amounts of CCR5 and CXCR3 ligands. Consistent with this observation, NK cell migration was abrogated in CCR2-deficient mice after peritoneal FSL-1 injection. Adoptive transfer of CCR2-deficient NK cells prior to peritoneal FSL-1 activation confirmed a cell-intrinsic component of CCR2-mediated NK cell migration. TLR2 activation did not induce an activated NK cell phenotype, but significant changes included an increase in the KLRG1+ subset and decreased NKG2D expression. Although not activated in vivo, peritoneal NK cells could be activated by interleukin (IL)-12 and IL-18 ex vivo to express CD69 and interferonγ. These data demonstrate that TLR2-mediated immune activation is a potent inducer of NK cell recruitment via a CCR2-dependent mechanism and that NK cells recruited by this mechanism can respond to additional signals to exert effector cell functions.

T-cell defects in patients with ARPC1B germline mutations account for combined immunodeficiency.

ARPC1B is a key factor for the assembly and maintenance of the ARP2/3 complex that is involved in actin branching from an existing filament. Germline biallelic mutations in ARPC1B have been recently described in 6 patients with clinical features of combined immunodeficiency (CID), whose neutrophils and platelets but not T lymphocytes were studied. We hypothesized that ARPC1B deficiency may also lead to cytoskeleton and functional defects in T cells. We have identified biallelic mutations in ARPC1B in 6 unrelated patients with early onset disease characterized by severe infections, autoimmune manifestations, and thrombocytopenia. Immunological features included T-cell lymphopenia, low numbers of naïve T cells, and hyper-immunoglobulin E. Alteration in ARPC1B protein structure led to absent/low expression by flow cytometry and confocal microscopy. This molecular defect was associated with the inability of patient-derived T cells to extend an actin-rich lamellipodia upon T-cell receptor (TCR) stimulation and to assemble an immunological synapse. ARPC1B-deficient T cells additionally displayed impaired TCR-mediated proliferation and SDF1-α-directed migration. Gene transfer of ARPC1B in patients' T cells using a lentiviral vector restored both ARPC1B expression and T-cell proliferation in vitro. In 2 of the patients, in vivo somatic reversion restored ARPC1B expression in a fraction of lymphocytes and was associated with a skewed TCR repertoire. In 1 revertant patient, memory CD8+ T cells expressing normal levels of ARPC1B displayed improved T-cell migration. Inherited ARPC1B deficiency therefore alters T-cell cytoskeletal dynamics and functions, contributing to the clinical features of CID.

ORAI1 mutations abolishing store-operated Ca2+ entry cause anhidrotic ectodermal dysplasia with immunodeficiency.

BACKGROUND: Store-operated Ca2+ entry (SOCE) through Ca2+ release-activated Ca2+ channels is an essential signaling pathway in many cell types. Ca2+ release-activated Ca2+ channels are formed by ORAI1, ORAI2, and ORAI3 proteins and activated by stromal interaction molecule (STIM) 1 and STIM2. Mutations in the ORAI1 and STIM1 genes that abolish SOCE cause a combined immunodeficiency (CID) syndrome that is accompanied by autoimmunity and nonimmunologic symptoms. OBJECTIVE: We performed molecular and immunologic analysis of patients with CID, anhidrosis, and ectodermal dysplasia of unknown etiology. METHODS: We performed DNA sequencing of the ORAI1 gene, modeling of mutations on ORAI1 crystal structure, analysis of ORAI1 mRNA and protein expression, SOCE measurements, immunologic analysis of peripheral blood lymphocyte populations by using flow cytometry, and histologic and ultrastructural analysis of patient tissues. RESULTS: We identified 3 novel autosomal recessive mutations in ORAI1 in unrelated kindreds with CID, autoimmunity, ectodermal dysplasia with anhidrosis, and muscular dysplasia. The patients were homozygous for p.V181SfsX8, p.L194P, and p.G98R mutations in the ORAI1 gene that suppressed ORAI1 protein expression and SOCE in the patients' lymphocytes and fibroblasts. In addition to impaired T-cell cytokine production, ORAI1 mutations were associated with strongly reduced numbers of invariant natural killer T and regulatory T (Treg) cells and altered composition of γδ T-cell and natural killer cell subsets. CONCLUSION: ORAI1 null mutations are associated with reduced numbers of invariant natural killer T and Treg cells that likely contribute to the patients' immunodeficiency and autoimmunity. ORAI1-deficient patients have dental enamel defects and anhidrosis, representing a new form of anhidrotic ectodermal dysplasia with immunodeficiency that is distinct from previously reported patients with anhidrotic ectodermal dysplasia with immunodeficiency caused by mutations in the nuclear factor κB signaling pathway (IKBKG and NFKBIA).

Severe, persistent, and fatal T-cell immunodeficiency following therapy for infantile leukemia.

We describe five cases of children who completed chemotherapy for infantile acute lymphoblastic leukemia (ALL) and soon after were diagnosed with severe T-cell, non-HIV immunodeficiency, with varying B-cell and NK-cell depletion. There was near absence of CD3(+) , CD4(+) , and CD8(+) cells. All patients developed multiple, primarily opportunistic infections. Unfortunately, four patients died, although one was successfully treated by hematopoietic stem cell transplantation. These immunodeficiencies appeared to be secondary to intensive infant ALL chemotherapy. Our report highlights the importance of the early consideration of this life-threatening immune complication in patients who received chemotherapy for infantile ALL.

CCR4 and CXCR3 play different roles in the migration of T cells to inflammation in skin, arthritic joints, and lymph nodes.

CCR4 and CXCR3 are expressed on several T-cell subsets in inflamed tissues, yet their role in tissue-specific recruitment is unclear. We examined the contributions of CCR4 and CXCR3 to T-cell recruitment into inflamed joints in collagen-induced arthritis, antigen-draining lymph nodes (LNs) and dermal inflammatory sites (poly I:C, LPS, concanavalin A, and delayed type hypersensitivity), using labeled activated T cells from CXCR3(-/-), CCR4(-/-), and WT mice. Both CXCR3 and CCR4 deficiency reduced the development of arthritis, but did not affect Th1-cell recruitment to the inflamed joints. Accumulation in inflamed LNs was highly CXCR3 dependent. In contrast, CCR4-deficient Th1 cells had an increased accumulation in these LNs. Migration to all four dermal inflammatory sites by activated Th1 and T cytotoxic cells and memory CD4(+) T cells was partially CXCR3-dependent, but Treg-cell migration was independent of CXCR3. The subset of cells expressing CCR4 has skin-migrating properties, but CCR4 itself is not required for the migration. Thus, migration into these inflamed tissues is CCR4-independent, and partially dependent on CXCR3, except for Treg cells, which require neither receptor. CCR4 may therefore affect retention of T cells in different tissues rather than trafficking out of the blood.

Mast cells protect against Pseudomonas aeruginosa-induced lung injury.

Pseudomonas aeruginosa, an opportunistic pathogen, is the leading cause of morbidity and mortality in immune-compromised individuals. Maintaining the integrity of the respiratory epithelium is critical for an effective host response to P. aeruginosa. Given the close spatial relationship between mast cells and the respiratory epithelium, and the importance of tightly regulated epithelial permeability during lung infections, we examined whether mast cells influence airway epithelial integrity during P. aeruginosa lung infection in a mouse model. We found that mast cell-deficient Kit(W-sh)/Kit(W-sh) mice displayed greatly increased epithelial permeability, bacterial dissemination, and neutrophil accumulation compared with wild-type animals after P. aeruginosa infection; these defects were corrected on reconstitution with mast cells. An in vitro Transwell co-culture model further demonstrated that a secreted mast cell factor decreased epithelial cell apoptosis and tumor necrosis factor production after P. aeruginosa infection. Together, our data demonstrate a previously unrecognized role for mast cells in the maintenance of epithelial integrity during P. aeruginosa infection, through a mechanism that likely involves prevention of epithelial apoptosis and tumor necrosis factor production. Our understanding of mechanisms of the host response to P. aeruginosa will open new avenues for the development of successful preventative and treatment strategies.

Effector T cell interactions with meningeal vascular structures in nascent autoimmune CNS lesions.

The tissues of the central nervous system are effectively shielded from the blood circulation by specialized vessels that are impermeable not only to cells, but also to most macromolecules circulating in the blood. Despite this seemingly absolute seclusion, central nervous system tissues are subject to immune surveillance and are vulnerable to autoimmune attacks. Using intravital two-photon imaging in a Lewis rat model of experimental autoimmune encephalomyelitis, here we present in real-time the interactive processes between effector T cells and cerebral structures from their first arrival to manifest autoimmune disease. We observed that incoming effector T cells successively scanned three planes. The T cells got arrested to leptomeningeal vessels and immediately monitored the luminal surface, crawling preferentially against the blood flow. After diapedesis, the cells continued their scan on the abluminal vascular surface and the underlying leptomeningeal (pial) membrane. There, the T cells encountered phagocytes that effectively present antigens, foreign as well as myelin proteins. These contacts stimulated the effector T cells to produce pro-inflammatory mediators, and provided a trigger to tissue invasion and the formation of inflammatory infiltrations.

Fatal pneumococcal meningitis in a 7-year-old girl with interleukin-1 receptor activated kinase deficiency (IRAK-4) despite prophylactic antibiotic and IgG responses to Streptococcus pneumoniae vaccines.

UNLABELLED: IRAK-4 deficiency causes IL-1R and TLR signaling failure, resulting in minimal clinical features despite invasive bacterial infection. We report the course of a 7-year-old IRAK-4-deficient girl presenting in the first year with multiple occult Staphylococcus aureus lymphadenitis. She was managed with antibiotic prophylaxis (sulfa/trimethoprim/PenV, then - due to neutropenia - Cefprozil), pneumococcal vaccination (PCV-7, Pneumovax23, PCV-13) and vigilance. Pneumococcal-specific IgG levels were monitored. No bacterial infections occurred on prophylaxis for 6 years after initial presentation. IgG response to pneumococcal polysaccharide was satisfactory but short-lived, requiring frequent boosting. At age 7, patient developed a morning headache and vomited once. Cefprozil was administered and re-dosed. Over 12 h, she was fatigued without other symptoms. Low fever accompanied another emesis. A few hours later she was confused, and purpuric rash appeared. Emergency physicians diagnosed sepsis/meningitis and started vancomycin-ceftriaxone. Respiratory failure and cerebellar herniation occurred <24 h after first symptoms. Blood and CSF grew Streptococcus pneumoniae type 6C resistant to second-generation cephalosporins. The patient's latest PCV-13 vaccination was 6 weeks before death, which included serotype 6A. Immunoglobulins were normal except IgG4 was increased (3.4 g/L). IgG response to vaccine antigens was satisfactory. IgG to 6A is reported to cross-react with 6C, but this was not the case here. CONCLUSION: Despite antibiotic prophylaxis and repeated vaccination, even older IRAK-4-deficient patients are at high risk of rapidly fatal infection due to emergence of antibiotic resistance. These patients need early assessment at any age, bacterial culturing, alternative empiric antibiotic therapy and close observation when even vaguely unwell. Based on increasingly recognized immunological and/or clinical impairments in B cell function, and possibly other defects, long-term IgG prophylaxis in addition to antibiotics is recommended.

Early fibroblast progenitor cell migration to the AngII-exposed myocardium is not CXCL12 or CCL2 dependent as previously thought.

Fibroblast progenitor cells (fibrocytes) are important to the development of myocardial fibrosis and are suggested to migrate to the heart via CXCL12 and chemokine ligand (CCL) 2. We hypothesized that if these chemokines are recruiting fibrocytes, disrupting their signaling will reduce early (3-day) fibrocyte infiltration and, consequently, fibrosis in the myocardium. C57/Bl6 and CCR2(-/-) mice were infused with saline or angiotensin (Ang) II, with or without CXC receptor 4 blockade (AMD3100). Hearts were assessed for chemokine up-regulation, immunofluorescence, and histological features. AngII caused early myocardial up-regulation of CXCL12 and CCL2, which corresponded to significant myocardial infiltration and fibrosis compared with controls. Animals receiving AMD3100 and/or with the genotype CCR2(-/-) failed to demonstrate reductions in infiltrate or fibrosis after 3 days of AngII, and AngII + AMD3100 animals showed exacerbated fibrocyte infiltration and fibrosis compared with AngII alone. CCR2(-/-) mice demonstrated significant reductions in myocardial fibrosis relative to wild type, but this was after 28 days of AngII infusion and was the result of reduced infiltrating cell proliferation. An alternative CCR2 ligand, CCL12, was found to be increasing infiltrating cell proliferation in the heart after AngII infusion, which we confirmed in vitro. In conclusion, early fibrocyte recruitment cannot be inhibited through modulating CXCL12 or CCL2, as previously thought. Ablating CCR2 signaling did confer myocardial fibrosis reductions, but these benefits were not observed until much later and were likely the result of modulated proliferation through ablating the CCL12-CCR2 interaction.

Differing requirements for CCR4, E-selectin, and α4ß1 for the migration of memory CD4 and activated T cells to dermal inflammation.

CCR4 on T cells is suggested to mediate skin homing in mice. Our objective was to determine the interaction of CCR4, E-selectin ligand (ESL), and α(4)ß(1) on memory and activated T cells in recruitment to dermal inflammation. mAbs to rat CCR4 were developed. CCR4 was on 5-21% of memory CD4 cells, and 20% were also ESL(+). Anti-TCR-activated CD4 and CD8 cells were 40-55% CCR4(+), and ∼75% of both CCR4(+) and CCR4(-) cells were ESL(+). CCR4(+) memory CD4 cells migrated 4- to 7-fold more to dermal inflammation induced by IFN-γ, TNF, TLR agonists, and delayed-type hypersensitivity than CCR4(-) cells. CCR4(+) activated CD4 cells migrated only 5-50% more than CCR4(-) cells to these sites. E-selectin blockade inhibited ∼60% of CCR4(+) activated CD4 cell migration but was less effective on memory cells where α(4)ß(1) was more important. Anti-α(4)ß(1) also inhibited CCR4(-) activated CD4 cells more than CCR4(+) cells. Anti-E-selectin reduced activated CD8 more than CD4 cell migration. These findings modify our understanding of CCR4, ESL, α(4)ß(1), and dermal tropism. There is no strict relationship between CCR4 and ESL for skin homing of CD4 cells, because the activation state and inflammatory stimulus are critical determinants. Dermal homing memory CD4 cells express CCR4 and depend more on α(4)ß(1) than ESL. Activated CD4 cells do not require CCR4, but CCR4(+) cells are more dependent on ESL than on α(4)ß(1), and CCR4(-) cells preferentially use α(4)ß(1). The differentiation from activated to memory CD4 cells increases the dependence on CCR4 for skin homing and decreases the requirement for ESL.

Differences in IDO1+ dendritic cells and soluble CTLA-4 are associated with differential clinical responses to methotrexate treatment in rheumatoid arthritis.

Objective: Antigen-presenting dendritic cells (DCs) and monocytes play an essential role in rheumatoid arthritis (RA) pathogenesis, however, their tolerogenic potential remains unclear. Herein, the tolerogenic profiles of DCs are characterized in treatment-naïve RA patients to determine their role to inflammatory arthritis management. Methods: Thirty-six treatment-naïve RA patients were enrolled, of which 62% were non-responders to methotrexate (MTX) monotherapy based on disease activity score (DAS) after 6-months of therapy. DC and monocyte subset frequencies, activation (CD40, CD86, CD209 expression), and tolerogenic profile (intracellular indoleamine-2,3-dioxygenase [IDO1] and cytotoxic T lymphocyte antigen 4 [CTLA-4] expression) were examined in the baseline peripheral blood by multicolor flow-cytometry. Soluble CTLA-4 (sCTLA-4) levels in plasma were measured. Results: DC subsets were decreased in RA compared to healthy controls (HC), and the frequency of conventional DCs (cDC) inversely correlated with inflammatory markers and improvement in disease activity. CD141+ cDC1s were the major IDO1-expressing cells. IDO1+cDC1s were reduced in RA patients compared to HC. The baseline frequency of IDO1+cDC1s inversely correlated with improvement in disease activity. CTLA-4 expression in CD1c+ cDC2s and monocytes was lower in RA patients compared to HC. Moreover, MTX-responders had a significantly lower frequency of IDO1+cDC1 cells and higher level of sCTLA-4 in the plasma compared to MTX non-responders. There was a strong predictive association of low IDO1+cDC1 cells, low sCTLA-4 and non-response to MTX. Conclusions: Our findings reveal altered DC and monocytes immunophenotypes that are associated with RA pathology and treatment response. The frequencies of tolerogenic IDO1+cDC1s and the low level of sCTLA-4 are strongly associated with MTX non-responsiveness and therapeutic outcome. These results suggest that investigation of the association IDO1+cDC1 and sCTLA-4 with response to treatment may be more generalizable to other autoimmune diseases.

Virus stimulation of human mast cells results in the recruitment of CD56⁺ T cells by a mechanism dependent on CCR5 ligands.

The trafficking of effector cells to sites of infection is crucial for antiviral responses. However, the mechanisms of recruitment of the interferon-γ-producing and cytotoxic CD56(+) T cells are poorly understood. Human mast cells are sentinel cells found in the skin and airway and produce selected proinflammatory mediators in response to multiple pathogen-associated signals. The role of human mast cell-derived chemokines in T-cell recruitment to virus infection was examined. Supernatants from primary human cord blood-derived mast cells (CBMCs) infected with mammalian reovirus were examined for chemokine production and utilized in chemotaxis assays. Virus-infected CBMCs produced several chemokines, including CCL3, CCL4, and CCL5. Supernatants from reovirus-infected CBMCs selectively induced the chemotaxis of CD8(+) T cells (10±1%) and CD3(+)CD56(+) T cells (19±5%). CD56(+) T-cell migration was inhibited by pertussis toxin (65±9%) and met-RANTES (56±7%), a CCR1/CCR5 antagonist. CD56(+) T cells expressed CCR5, but little CCR1. The depletion of CCL3, CCL4, and CCL5 from reovirus-infected CBMC supernatants significantly (41±10%) inhibited CD56(+) T-cell chemotaxis. This study demonstrates a novel role for mast cells and CCR5 in CD56(+) T-cell trafficking and suggests that human mast cells enhance immunity to viruses through the selective recruitment of cytotoxic effector cells to virus infection sites. These findings could be exploited to enhance local T-cell responses in chronic viral infection and malignancies at mast cell-rich sites.

Differentially Expressed Inflammation-Regulating MicroRNAs in Oligoarticular Juvenile Idiopathic Arthritis.

OBJECTIVE: To evaluate microRNA expression in synovial fluid (SF), plasma, and leukocytes from patients with juvenile idiopathic arthritis (JIA). METHODS: MicroRNA expression in pooled JIA plasma and SF was assessed by absolute quantitative droplet digital PCR array. The results were validated in individual patient samples. MicroRNA content in leukocytes and extracellular vesicles was evaluated by real-time PCR in JIA blood and SF. Blood microRNA expression was compared with healthy controls (HCs). Principal component analysis was used to profile JIA plasma and SF microRNAs, and the potential biological consequences of microRNA dysregulation were investigated by pathway analysis. RESULTS: MiR-15a-5p and miR-409-3p levels were higher in JIA plasma than in HC plasma. JIA SF contained elevated levels of miR-21-5p, miR-27a-3p, miR-146b-5p, miR-155-5p, and miR-423-5p, and decreased miR-192-5p and miR-451a, compared to JIA plasma. Extracellular vesicle analysis demonstrated variable encapsulation among selected microRNAs, with only miR-155-5p being represented substantially in extracellular vesicles. SF leukocytes also had higher expression of miR-21-5p, miR-27a-3p, miR-146b-5p, and miR-155-5p, and lower expression of miR-409-3p and miR-451a, relative to blood. No differences were observed between JIA and HC blood leukocytes. Clusters of microRNAs were commonly altered in JIA joint fluid and leukocytes compared to JIA blood samples. In silico analysis predicted that differentially expressed microRNAs in JIA target the transforming growth factor (TGF)-ß pathway. CONCLUSION: The expression of multiple microRNAs is dysregulated in JIA both locally and systemically, which may inhibit the TGF-ß pathway. These findings advance our knowledge of JIA immunopathogenesis and may lead to the development of targeted therapies.

Coexpression of chemokine receptors CCR5, CXCR3, and CCR4 and ligands for P- and E-selectin on T lymphocytes of patients with juvenile idiopathic arthritis.

OBJECTIVE: To investigate P- and E-selectin ligand coexpression with chemokine receptors (CKRs) on T cells in the synovial fluid (SF) and blood of children with juvenile idiopathic arthritis (JIA). METHODS: Sixteen patients with polyarticular or persistent oligoarticular JIA (ages 5.3-15.1 years) were studied. SF and venous blood were collected, and immunostaining for the expression of CCR4, CCR5, CXCR3, and P- or E-selectin ligands was performed. RESULTS: Compared to blood, SF was greatly enriched for CD4+ T cells bearing CCR5, CCR4, CXCR3, and both P- and E-selectin ligand. Twenty-five percent of the CD4+ T cells in SF expressed both CCR5 and CCR4, some also coexpressing CXCR3. Such cells were rare in blood. Half of the few CCR5+ T cells in blood coexpressed P- or E-selectin ligand, a phenotype that was enriched up to 50-fold in SF. A minority of CCR4+ and CXCR3+ cells in blood (∼25%) coexpressed selectin ligand; these were enriched 4-8-fold in SF. Most CCR4-expressing CD4+ T cells expressed both E-selectin ligand and cutaneous lymphocyte antigen. CONCLUSION: CCR4-, CCR5-, CXCR3-, and selectin ligand-expressing CD4+ T cells preferentially accumulate in the joints of children with JIA. The marked enrichment of CCR5+ T cells coexpressing P-selectin and/or E-selectin ligand in CD4+ SF T cells suggests that the few such cells in blood selectively migrate to inflamed joints via endothelial P- and E-selectin- and CCR5-activating chemokines. The predominance of CCR4-expressing CD4+ T cells coexpressing E-selectin ligand suggests that such cells migrate not only to areas of cutaneous inflammation, as previously reported, but also to the joints in JIA. Combined targeting of CCR5- and E-selectin-dependent mechanisms may be a relevant treatment strategy.

Prolidase deficiency, a rare inborn error of immunity, clinical phenotypes, immunological features, and proposed treatments in twins.

BACKGROUND: Prolidase deficiency (PD) is an autosomal recessive inborn multisystemic disease caused by mutations in the PEPD gene encoding the enzyme prolidase D, leading to defects in turnover of proline-containing proteins, such as collagen. PD is categorized as a metabolic disease, but also as an inborn error of immunity. PD presents with a range of findings including dysmorphic features, intellectual disabilities, recurrent infections, intractable skin ulceration, autoimmunity, and splenomegaly. Despite symptoms of immune dysregulation, only very limited immunologic assessments have been reported and standard therapies for PD have not been described. We report twin females with PD, including comprehensive immunologic profiles and treatment modalities used. CASE PRESENTATION: Patient 1 had recurrent infections in childhood. At age 13, she presented with telangiectasia, followed by painful, refractory skin ulcerations on her lower limbs, where skin biopsy excluded vasculitis. She had typical dysmorphic features of PD. Next-generation sequencing revealed pathogenic compound heterozygous mutations (premature stop codons) in the PEPD gene. Patient 2 had the same mutations, typical PD facial features, atopy, and telangiectasias, but no skin ulceration. Both patients had imidodipeptiduria. Lymphocyte subset analysis revealed low-normal frequency of Treg cells and decreased frequency of expression of the checkpoint molecule CTLA-4 in CD4+ TEM cells. Analysis of Th1, Th2, and Th17 profiles revealed increased inflammatory IL-17+ CD8+ TEM cells in both patients and overexpression of the activation marker HLA-DR on CD4+ TEM cells, reflecting a highly activated proinflammatory state. Neither PD patient had specific antibody deficiencies despite low CD4+CXCR5+ Tfh cells and low class-switched memory B cells. Plasma IL-18 levels were exceptionally high. CONCLUSIONS: Immunologic abnormalities including skewed frequencies of activated inflammatory CD4+ and CD8+ TEM cells, decreased CTLA-4 expression, and defects in memory B cells may be a feature of immune dysregulation associated with PD; however, a larger sample size is required to validate these findings. The high IL-18 plasma levels suggest underlying autoinflammatory processes.

CXCR3 blockade inhibits T-cell migration into the CNS during EAE and prevents development of adoptively transferred, but not actively induced, disease.

Autoreactive T-cell infiltration into the CNS is critical in MS and EAE. The chemokine receptor CXCR3 and its ligands are implicated in MS and mouse EAE, but the contribution of CXCR3 to T-cell migration into the inflamed CNS remains controversial. During active disease in a rat EAE model, blood T-cell, spleen T-cell and T lymphoblast migration into the CNS was inhibited by a CXCR3 blocking mAb by, 30-70%, ∼75% and 50-80%, respectively. However, CXCR3 blockade after active immunization did not inhibit EAE, did not alter total T-cell accumulation in the CNS and did not affect Treg accumulation or the presence of cells producing IFN-γ or IL-17. Conversely, CXCR3 blockade during EAE induced by adoptive transfer of myelin basic protein-activated T cells delayed disease onset, shortened its duration and reduced disease severity. Moreover, CXCR3 blockade inhibited leukocyte infiltration of the CNS>95%, virtually abolishing infiltration of transferred T cells. Thus, CXCR3 plays a major role in T-cell migration to the CNS and can be critical for encephalitogenic T-cell migration into the CNS to induce disease, but CXCR3-independent recruitment can also produce EAE.

High Dose Intravenous IgG Therapy Modulates Multiple NK Cell and T Cell Functions in Patients With Immune Dysregulation.

Intravenous immunoglobulin (IVIG) is an effective immunomodulatory treatment for immune dysregulation diseases. However, the mechanisms by which it reduces systemic inflammation are not well understood. NK cell cytotoxicity is decreased by IVIG in women with reduced fertility, but IVIG effects on NK cells in immune dysregulation are less clear. We hypothesized that IVIG modulation of lymphocyte function, especially in NK cells, is important for resolution of inflammation. Our aim was to identify IVIG-induced changes in a cohort of patients with Kawasaki disease (KD) and those that occur broadly in pediatric patients with various immune dysregulatory diseases. Peripheral blood mononuclear cells (PBMCs) of patients with KD or autoimmune/inflammatory diseases were phenotyped pre and post high dose IVIG treatment by flow cytometry. In KD patients, after IVIG infusion Treg cell frequency and the proportion of activated CD25+ immunoregulatory CD56bright NK cells was increased, and multiple lymphocyte subsets showed increased expression of the lymphoid tissue homing receptor CD62L. Importantly, IVIG treatment decreased the frequency of cells expressing the degranulation marker CD107a among cytotoxic CD56dim NK cells, which was reflected in a significant reduction in target cell killing and in decreased production of multiple pro-inflammatory mediators. Interestingly, the activating receptor CD336 was expressed on a higher proportion of CD56bright NK cells after IVIG in both KD and autoimmune/inflammatory patients while other NK receptors were increased differentially in each cohort. In autoimmune/inflammatory patients IVIG induced the proliferation marker CD71 on a higher percentage of CD56dim NK cells, and in contrast to KD patients, CD107a+ cells were increased in this subset. Furthermore, when PBMCs were stimulated ex vivo with IL-2 or Candida antigen in autologous plasma, more of the CD4+ T cells of KD patients expressed CD25 after IVIG therapy but fewer cytotoxic T cells were degranulated based on CD107a expression. In summary, IVIG treatment in patients with immune dysregulation has multiple effects, especially on NK cell subsets and CD4+ T cells, which are compatible with promoting resolution of inflammation. These novel findings provide insight into the immunomodulatory actions of IVIG in autoimmune and inflammatory conditions.