Differences in Tumor Microenvironment Dictate T Helper Lineage Polarization and Response to Immune Checkpoint Therapy.

Immune checkpoint therapy (ICT) shows encouraging results in a subset of patients with metastatic castration-resistant prostate cancer (mCRPC) but still elicits a sub-optimal response among those with bone metastases. Analysis of patients' bone marrow samples revealed increased Th17 instead of Th1 subsets after ICT. To further evaluate the different tumor microenvironments, we injected mice with prostate tumor cells either subcutaneously or intraosseously. ICT in the subcutaneous CRPC model significantly increases intra-tumoral Th1 subsets and improves survival. However, ICT fails to elicit an anti-tumor response in the bone CRPC model despite an increase in the intra-tumoral CD4 T cells, which are polarized to Th17 rather than Th1 lineage. Mechanistically, tumors in the bone promote osteoclast-mediated bone resorption that releases TGF-ß, which restrains Th1 lineage development. Blocking TGF-ß along with ICT increases Th1 subsets and promotes clonal expansion of CD8 T cells and subsequent regression of bone CRPC and improves survival.

Mast cell-derived IL-13 downregulates IL-12 production by skin dendritic cells to inhibit the TH1 cell response to cutaneous antigen exposure.

BACKGROUND: Atopic dermatitis (AD) is characterized by a skin barrier defect aggravated by mechanical injury inflicted by scratching, a TH2 cell-dominated immune response, and susceptibility to viral skin infections that are normally restrained by a TH1 cell response. The signals leading to a TH2 cell-dominated immune response in AD are not completely understood. OBJECTIVE: Our aim was to determine the role of IL-13 in initiation of the TH cell response to cutaneously encountered antigens. METHODS: Wild-type, Il13-/-, Il1rl1-/-, and Il4ra-/- mice, as well as mice with selective deficiency of IL-13 in mast cells (MCs) were studied; in addition, dendritic cells (DCs) purified from the draining lymph nodes of tape-stripped and ovalbumin (OVA)-sensitized skin were examined for their ability to polarize naive OVA-TCR transgenic CD4+ T cells. Cytokine expression was examined by reverse-transcriptase quantitative PCR, intracellular flow cytometry, and ELISA. Contact hypersensitivity to dinitrofluorobenzene was examined. RESULTS: Tape stripping caused IL-33-driven upregulation of Il13 expression by skin MCs. MC-derived IL-13 acted on DCs from draining lymph nodes of OVA-sensitized skin to selectively suppress their ability to polarize naive OVA-TCR transgenic CD4+ T cells into IFN-γ-secreting cells. MC-derived IL-13 inhibited the TH1 cell response in contact hypersensitivity to dinitrofluorobenzene. IL-13 suppressed IL-12 production by mouse skin-derived DCs in vitro and in vivo. Scratching upregulated IL13 expression in human skin, and IL-13 suppressed the capacity of LPS-stimulated human skin DCs to express IL-12 and promote IFN-γ secretion by CD4+ T cells. CONCLUSION: Release of IL-13 by cutaneous MCs in response to mechanical skin injury inhibits the TH1 cell response to cutaneous antigen exposure in AD.

Tape strips detect distinct immune and barrier profiles in atopic dermatitis and psoriasis.

BACKGROUND: Our current understanding of atopic dermatitis (AD) and psoriasis pathophysiology is largely derived from skin biopsy studies that cause scarring and may be impractical in large-scale clinical trials. Although tape strips show promise as a minimally invasive technique in these common diseases, a comprehensive molecular profiling characterizing and differentiating the 2 diseases in tape strips is unavailable. OBJECTIVE: Our aim was to construct a global transcriptome of tape strips from lesional and nonlesional skin of adults with moderate-to-severe AD and psoriasis. METHODS: A total of 20 tape strips were obtained from lesional and nonlesional skin of patients with AD and psoriasis and skin from controls (n = 20 each); the strips were subjected to RNA sequencing (RNA-seq), with quantitative RT-PCR validation of immune and barrier biomarkers. RESULTS: We detected RNA-seq profiles in 96 of 100 of samples (96%), with 4123 and 5390 genes differentially expressed in AD and psoriasis lesions versus in controls, respectively (fold change ≥ 2; false discovery rate [FDR] < 0.05). Nonlesional tape-stripped skin from patients with AD was more similar to lesional skin than to nonlesional skin of patients with psoriasis, which showed larger differentiation from lesions. AD and psoriasis tissues shared increases in levels of dendritic cell and T-cell markers (CD3, ITGAX/CD11c, and CD83), but AD tissues showed preferential TH2 skewing (IL-13, CCL17/TARC, and CCL18), whereas psoriasis was characterized by higher levels of expression of TH17-related (IL-17A/F and IL-36A/IL-36G), TH1-related (IFN-γ and CXCL9/CXCL10), and innate immunity-related (nitric oxide synthase 2/inducible nitric oxide synthase and IL-17C) products (FDR < 0.05). Terminal differentiation (FLG2 and LCE5A), tight junction (CLDN8), and lipid biosynthesis and metabolism (FA2H and ALOXE3) products were significantly downregulated in both AD and psoriasis (FDR < 0.05). Nitric oxide synthase 2/inducible nitric oxide synthase expression (determined by quantitative PCR) differentiated AD and psoriasis with 100% accuracy. CONCLUSION: RNA-seq tape strip profiling detected distinct immune and barrier signatures in lesional and nonlesional AD and psoriasis skin, suggesting their utility as a minimally invasive alternative to biopsies for detecting disease biomarkers.

Human TH1 and TH2 cells targeting rhinovirus and allergen coordinately promote allergic asthma.

BACKGROUND: Allergic asthmatic subjects are uniquely susceptible to acute wheezing episodes provoked by rhinovirus. However, the underlying immune mechanisms and interaction between rhinovirus and allergy remain enigmatic, and current paradigms are controversial. OBJECTIVE: We sought to perform a comprehensive analysis of type 1 and type 2 innate and adaptive responses in allergic asthmatic subjects infected with rhinovirus. METHODS: Circulating virus-specific TH1 cells and allergen-specific TH2 cells were precisely monitored before and after rhinovirus challenge in allergic asthmatic subjects (total IgE, 133-4692 IU/mL; n = 28) and healthy nonallergic controls (n = 12) using peptide/MHCII tetramers. T cells were sampled for up to 11 weeks to capture steady-state and postinfection phases. T-cell responses were analyzed in parallel with 18 cytokines in the nose, upper and lower airway symptoms, and lung function. The influence of in vivo IgE blockade was also examined. RESULTS: In uninfected asthmatic subjects, higher numbers of circulating virus-specific PD-1+ TH1 cells, but not allergen-specific TH2 cells, were linked to worse lung function. Rhinovirus infection induced an amplified antiviral TH1 response in asthmatic subjects versus controls, with synchronized allergen-specific TH2 expansion, and production of type 1 and 2 cytokines in the nose. In contrast, TH2 responses were absent in infected asthmatic subjects who had normal lung function, and in those receiving anti-IgE. Across all subjects, early induction of a minimal set of nasal cytokines that discriminated high responders (G-CSF, IFN-γ, TNF-α) correlated with both egress of circulating virus-specific TH1 cells and worse symptoms. CONCLUSIONS: Rhinovirus induces robust TH1 responses in allergic asthmatic subjects that may promote disease, even after the infection resolves.

Age-specific changes in the molecular phenotype of patients with moderate-to-severe atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) shows differential clinical presentation in older compared with younger patients. Nevertheless, changes in the AD molecular profile with age are unknown. OBJECTIVE: We sought to characterize age-related changes in the AD profile. METHODS: We evaluated age-specific changes in lesional and nonlesional tissues and blood from patients with moderate-to-severe AD (n = 246) and age-matched control subjects (n = 71) using immunohistochemistry, quantitative real-time PCR, and Singulex in a cross-sectional study. Patients were analyzed by age group (18-40, 41-60, and ≥61 years). RESULTS: Although disease severity/SCORAD scores were similar across AD age groups (mean, approximately 60 years; P = .873), dendritic cell infiltrates (CD1b+ and FcεRI+, P < .05) decreased with age. TH2 measures (IL5, IL13, CCL13, CCL18, and CCL26) significantly decreased with age in patients with AD, despite increasing with age in control subjects. Consistent with TH2 axis decreases, serum IgE levels and eosinophil counts negatively correlated with age in patients with AD (r = -0.24 and r = -0.23, respectively; P < .05). TH22-secreted IL22 expression levels also decreased with age uniquely in patients with AD (P < .05). Expression of TH1-related (IFNG, IL12/23p40, STAT1, and CXCL9; P < .05 for CXCL9) and TH17-related (IL17A and IL20; P < .05 for IL20) markers increased with age in both patients with AD and control subjects. Expression of terminal differentiation measures significantly increased in older patients with AD (loricrin [LOR] and filaggrin [FLG], P < .05), whereas expression of S100As (S100A8, P < .01) and hyperplasia markers (epidermal thickness, keratin 16, and Ki67; P < .05 for keratin 16) decreased. Serum trends in AD mimicked skin findings, with TH2 downregulation (CCL26; r = -0.32, P < .1) and TH1 upregulation (IFN-γ; r = 0.48, P < .01) with age. CONCLUSION: The adult AD profile varies with age. Although TH1/TH17 skewing increases in both patients with AD and control subjects, patients with AD show unique decreases in TH2/TH22 polarization and normalization of epithelial abnormalities. Thus age-specific treatment approaches might be beneficial for AD.

GBR 830, an anti-OX40, improves skin gene signatures and clinical scores in patients with atopic dermatitis.

BACKGROUND: GBR 830 is a humanized mAb against OX40, a costimulatory receptor on activated T cells. OX40 inhibition might have a therapeutic role in T cell-mediated diseases, including atopic dermatitis (AD). OBJECTIVE: This exploratory phase 2a study investigated the safety, efficacy, and tissue effects of GBR 830 in patients with AD. METHODS: Patients with moderate-to-severe AD (affected body surface area, ≥10%; Eczema Area and Severity Index score, ≥12; and inadequate response to topical treatments) were randomized 3:1 to 10 mg/kg intravenous GBR 830 or placebo on day 1 (baseline) and day 29. Biopsy specimens were collected (n = 40) at days 1, 29, and 71. Primary end points included treatment-emergent adverse events (TEAEs) and changes from baseline in biomarkers (epidermal hyperplasia/cytokines) at days 29 and 71. RESULTS: GBR 830 was well tolerated, with equal TEAE distribution (GBR 830, 63.0% [29/46]; placebo, 63.0% [10/16]). One serious TEAE in the GBR 830 group was deemed unrelated to study drug. At day 71, the proportion of intent-to-treat subjects achieving 50% or greater improvement in Eczema Area and Severity Index score was greater with GBR 830 (76.9% [20/26]) versus placebo (37.5% [3/8]). GBR 830 induced significant progressive reductions in TH1 (IFN-γ/CXCL10), TH2 (IL-31/CCL11/CCL17), and TH17/TH22 (IL-23p19/IL-8/S100A12) mRNA expression in lesional skin. Significant progressive reductions until day 71 in the drug group were seen in OX40+ T cells and OX40L+ dendritic cells (P < .001). Hyperplasia measures (thickness/keratin 16/Ki67) showed greater reductions with GBR 830 (P < .001). CONCLUSIONS: Two doses of GBR 830 administered 4 weeks apart were well tolerated and induced significant progressive tissue and clinical changes until day 71 (42 days after the last dose), highlighting the potential of OX40 targeting in patients with AD.

Blood endotyping distinguishes the profile of vitiligo from that of other inflammatory and autoimmune skin diseases.

BACKGROUND: Peripheral blood skin-homing/cutaneous lymphocyte antigen (CLA)+ T cells emerge as biomarkers of cutaneous immune activation in patients with inflammatory skin diseases (atopic dermatitis [AD] and alopecia areata [AA]). However, blood phenotyping across these subsets is not yet available in patients with vitiligo. OBJECTIVE: We sought to measure cytokine production by circulating skin-homing (CLA+) versus systemic (CLA-) "polar" CD4+/CD8+ ratio and activated T-cell subsets in patients with vitiligo compared with patients with AA, AD, or psoriasis and control subjects. METHODS: Flow cytometry was used to measure levels of the cytokines IFN-γ, IL-13, IL-9, IL-17, and IL-22 in CD4+/CD8+ T cells in the blood of 19 patients with moderate-to-severe nonsegmental/generalized vitiligo, moderate-to-severe AA (n = 32), psoriasis (n = 24), or AD (n = 43) and control subjects (n = 30). Unsupervised clustering differentiated subjects into groups based on cellular frequencies. RESULTS: Patients with Vitiligo showed the highest CLA+/CLA- TH1/type 1 cytotoxic T-cell polarization, with parallel TH2/TH9/TH17/TH22 level increases to levels often greater than those seen in patients with AA, AD, or psoriasis (P < .05). Total regulatory T-cell counts were lower in patients with vitiligo than in control subjects and patients with AD or psoriasis (P < .001). Vitiligo severity correlated with levels of multiple cytokines (P < .1), whereas duration was linked with IFN-γ and IL-17 levels (P < .04). Patients and control subjects grouped into separate clusters based on blood biomarkers. CONCLUSIONS: Vitiligo is characterized by a multicytokine polarization among circulating skin-homing and systemic subsets, which differentiates it from other inflammatory/autoimmune skin diseases. Future targeted therapies should delineate the relative contribution of each cytokine axis to disease perpetuation.

Tripartite motif-containing (TRIM) 21 negatively regulates intestinal mucosal inflammation through inhibiting TH1/TH17 cell differentiation in patients with inflammatory bowel diseases.

BACKGROUND: Tripartite motif-containing (TRIM) 21 has been implicated in the pathogenesis of several types of autoimmune diseases. OBJECTIVE: We sought to elucidate TRIM21 expression in patients with inflammatory bowel diseases (IBDs) and its role in regulating intestinal mucosal inflammation. METHODS: TRIM21 expression was analyzed in the inflamed mucosa of patients with IBDs by means of quantitative RT-PCR and immunohistochemistry. Peripheral blood CD4+ T cells were transfected with lentivirus-expressing TRIM21 (LV-TRIM21) or LV-sh-TRIM21, and cytokine expression was determined by using quantitative RT-PCR and ELISA. TRIM21-/- mice were generated, and trinitrobenzene sulfonic acid- and CD45RBhighCD4+ T cell-induced colitis models were established to determine its role in induction of intestinal inflammation. RESULTS: TRIM21 was expressed predominantly in CD4+ T cells and decreased markedly in the inflamed mucosa of patients with IBDs compared with healthy control subjects. Ectopic expression of TRIM21 inhibited IBD CD4+ T cells to differentiate into TH1 and TH17 cells, whereas downregulation of TRIM21 had the opposite effects. TRIM21-/- mice had more severe colitis after administration of trinitrobenzene sulfonic acid compared with wild-type mice, which was characterized by increased expression of IFN-γ, TNF-α, and IL-17A in the colon. TRIM21-/-CD45RBhighCD4+ T cells reconstituted into recombination-activating gene (Rag1)-/- mice induced more severe colitis than in wild-type control mice. Mechanistically, interferon regulatory factor 3 was identified as a functional downstream target of TRIM21 in that silencing of interferon regulatory factor 3 suppressed TRIM21-/-CD4+ T-cell differentiation into TH1 and TH17 cells. CONCLUSIONS: TRIM21 plays a protective role in mucosal inflammation through inhibiting TH1 and TH17 cell differentiation. Thus TRIM21 might serve as a potential therapeutic target for the treatment of IBDs.

R-loops cause genomic instability in T helper lymphocytes from patients with Wiskott-Aldrich syndrome.

BACKGROUND: Wiskott-Aldrich syndrome (WAS), X-linked thrombocytopenia (XLT), and X-linked neutropenia, which are caused by WAS mutations affecting Wiskott-Aldrich syndrome protein (WASp) expression or activity, manifest in immunodeficiency, autoimmunity, genomic instability, and lymphoid and other cancers. WASp supports filamentous actin formation in the cytoplasm and gene transcription in the nucleus. Although the genetic basis for XLT/WAS has been clarified, the relationships between mutant forms of WASp and the diverse features of these disorders remain ill-defined. OBJECTIVE: We sought to define how dysfunctional gene transcription is causally linked to the degree of TH cell deficiency and genomic instability in the XLT/WAS clinical spectrum. METHODS: In human TH1- or TH2-skewing cell culture systems, cotranscriptional R-loops (RNA/DNA duplex and displaced single-stranded DNA) and DNA double-strand breaks (DSBs) were monitored in multiple samples from patients with XLT and WAS and in normal T cells depleted of WASp. RESULTS: WASp deficiency provokes increased R-loops and R-loop-mediated DSBs in TH1 cells relative to TH2 cells. Mechanistically, chromatin occupancy of serine 2-unphosphorylated RNA polymerase II is increased, and that of topoisomerase 1, an R-loop preventing factor, is decreased at R-loop-enriched regions of IFNG and TBX21 (TH1 genes) in TH1 cells. These aberrations accompany increased unspliced (intron-retained) and decreased spliced mRNA of IFNG and TBX21 but not IL13 (TH2 gene). Significantly, increased cellular load of R-loops and DSBs, which are normalized on RNaseH1-mediated suppression of ectopic R-loops, inversely correlates with disease severity scores. CONCLUSION: Transcriptional R-loop imbalance is a novel molecular defect causative in TH1 immunodeficiency and genomic instability in patients with WAS. The study proposes that cellular R-loop load could be used as a potential biomarker for monitoring symptom severity and prognostic outcome in the XLT-WAS clinical spectrum and could be targeted therapeutically.

TH1 signatures are present in the lower airways of children with severe asthma, regardless of allergic status.

BACKGROUND: The pathogenesis of severe asthma in childhood remains poorly understood. OBJECTIVE: We sought to construct the immunologic landscape in the airways of children with severe asthma. METHODS: Comprehensive analysis of multiple cell types and mediators was performed by using flow cytometry and a multiplex assay with bronchoalveolar lavage (BAL) specimens (n = 68) from 52 highly characterized allergic and nonallergic children (0.5-17 years) with severe treatment-refractory asthma. Multiple relationships were tested by using linear mixed-effects modeling. RESULTS: Memory CCR5+ TH1 cells were enriched in BAL fluid versus blood, and pathogenic respiratory viruses and bacteria were readily detected. IFN-γ+IL-17+ and IFN-γ-IL-17+ subsets constituted secondary TH types, and BAL fluid CD8+ T cells were almost exclusively IFN-γ+. The TH17-associated mediators IL-23 and macrophage inflammatory protein 3α/CCL20 were highly expressed. Despite low TH2 numbers, TH2 cytokines were detected, and TH2 skewing correlated with total IgE levels. Type 2 innate lymphoid cells and basophils were scarce in BAL fluid. Levels of IL-5, IL-33, and IL-28A/IFN-λ2 were increased in multisensitized children and correlated with IgE levels to dust mite, ryegrass, and fungi but not cat, ragweed, or food sources. Additionally, levels of IL-5, but no other cytokine, increased with age and correlated with eosinophil numbers in BAL fluid and blood. Both plasmacytoid and IgE+FcεRI+ myeloid dendritic cells were present in BAL fluid. CONCLUSIONS: The lower airways of children with severe asthma display a dominant TH1 signature and atypical cytokine profiles that link to allergic status. Our findings deviate from established paradigms and warrant further assessment of the pathogenicity of TH1 cells in patients with severe asthma.

Major differences between human atopic dermatitis and murine models, as determined by using global transcriptomic profiling.

BACKGROUND: Atopic dermatitis (AD) is caused by a complex interplay between immune and barrier abnormalities. Murine models of AD are essential for preclinical assessments of new treatments. Although many models have been used to simulate AD, their transcriptomic profiles are not fully understood, and a comparison of these models with the human AD transcriptomic fingerprint is lacking. OBJECTIVE: We sought to evaluate the transcriptomic profiles of 6 common murine models and determine how they relate to human AD skin. METHODS: Transcriptomic profiling was performed by using microarrays and quantitative RT-PCR on biopsy specimens from NC/Nga, flaky tail, Flg-mutated, ovalbumin-challenged, oxazolone-challenged, and IL-23-injected mice. Gene expression data of patients with AD, psoriasis, and contact dermatitis were obtained from previous patient cohorts. Criteria of a fold change of 2 or greater and a false discovery rate of 0.05 or less were used for gene arrays. RESULTS: IL-23-injected, NC/Nga, and oxazolone-challenged mice show the largest homology with our human meta-analysis-derived AD transcriptome (37%, 18%, 17%, respectively). Similar to human AD, robust TH1, TH2, and also TH17 activation are seen in IL-23-injected and NC/Nga mice, with similar but weaker inflammation in ovalbumin-challenged mice. Oxazolone-challenged mice show a TH1-centered reaction, and flaky tail mice demonstrate a strong TH17 polarization. Flg-mutated mice display filaggrin downregulation without significant inflammation. CONCLUSION: No single murine model fully captures all aspects of the AD profile; instead, each model reflects different immune or barrier disease aspects. Overall, among the 6 murine models, IL-23-injected mice best simulate human AD; still, the translational focus of the investigation should determine which model is most applicable.

Notch signaling in T cells is essential for allergic airway inflammation, but expression of the Notch ligands Jagged 1 and Jagged 2 on dendritic cells is dispensable.

BACKGROUND: Allergic asthma is characterized by a TH2 response induced by dendritic cells (DCs) that present inhaled allergen. Although the mechanisms by which they instruct TH2 differentiation are still poorly understood, expression of the Notch ligand Jagged on DCs has been implicated in this process. OBJECTIVE: We sought to establish whether Notch signaling induced by DCs is critical for house dust mite (HDM)-driven allergic airway inflammation (AAI) in vivo. METHODS: The induction of Notch ligand expression on DC subsets by HDM was quantified by using quantitative real-time PCR. We used an HDM-driven asthma mouse model to compare the capacity of Jagged 1 and Jagged 2 single- and double-deficient DCs to induce AAI. In addition, we studied AAI in mice with a T cell-specific deletion of recombination signal-binding protein for immunoglobulin Jκ region (RBPJκ), a downstream effector of Notch signaling. RESULTS: HDM exposure promoted expression of Jagged 1, but not Jagged 2, on DCs. In agreement with published findings, in vitro-differentiated and HDM-pulsed Jagged 1 and Jagged 2 double-deficient DCs lacked the capacity to induce AAI. However, after in vivo intranasal sensitization and challenge with HDM, DC-specific Jagged 1 or Jagged 2 single- or double-deficient mice had eosinophilic airway inflammation and a TH2 cell activation phenotype that was not different from that in control littermates. In contrast, RBPJκ-deficient mice did not experience AAI and airway hyperreactivity. CONCLUSION: Our results show that the Notch signaling pathway in T cells is crucial for the induction of TH2-mediated AAI in an HDM-driven asthma model but that expression of Jagged 1 or Jagged 2 on DCs is not required.

Ruxolitinib reverses dysregulated T helper cell responses and controls autoimmunity caused by a novel signal transducer and activator of transcription 1 (STAT1) gain-of-function mutation.

BACKGROUND: Gain-of-function (GOF) mutations in the human signal transducer and activator of transcription 1 (STAT1) manifest in immunodeficiency and autoimmunity with impaired TH17 cell differentiation and exaggerated responsiveness to type I and II interferons. Allogeneic bone marrow transplantation has been attempted in severely affected patients, but outcomes have been poor. OBJECTIVE: We sought to define the effect of increased STAT1 activity on T helper cell polarization and to investigate the therapeutic potential of ruxolitinib in treating autoimmunity secondary to STAT1 GOF mutations. METHODS: We used in vitro polarization assays, as well as phenotypic and functional analysis of STAT1-mutated patient cells. RESULTS: We report a child with a novel mutation in the linker domain of STAT1 who had life-threatening autoimmune cytopenias and chronic mucocutaneous candidiasis. Naive lymphocytes from the affected patient displayed increased TH1 and follicular T helper cell and suppressed TH17 cell responses. The mutation augmented cytokine-induced STAT1 phosphorylation without affecting dephosphorylation kinetics. Treatment with the Janus kinase 1/2 inhibitor ruxolitinib reduced hyperresponsiveness to type I and II interferons, normalized TH1 and follicular T helper cell responses, improved TH17 differentiation, cured mucocutaneous candidiasis, and maintained remission of immune-mediated cytopenias. CONCLUSIONS: Autoimmunity and infection caused by STAT1 GOF mutations are the result of dysregulated T helper cell responses. Janus kinase inhibitor therapy could represent an effective targeted treatment for long-term disease control in severely affected patients for whom hematopoietic stem cell transplantation is not available.

Humoral and cellular responses to casein in patients with food protein-induced enterocolitis to cow's milk.

BACKGROUND: Food protein-induced enterocolitis syndrome (FPIES) is a non-IgE-mediated food allergy manifesting within 1 to 4 hours of food ingestion with repetitive emesis and lethargy. OBJECTIVE: We sought to characterize immune responses to casein in children with FPIES caused by cow's milk (CM). METHODS: Total IgE and IgM, CM-specific IgG, and casein-specific IgE, IgG, IgG4, and IgM levels, as well as immunoglobulin free light chains, were measured in both patients with active and those with resolved CM-FPIES. Proliferating casein/T-effector cell counts were measured in children with CM-FPIES, children with IgE-mediated CM allergy, and those tolerating CM. Cytokine concentrations in the supernatants were quantified. Serum cytokine and tryptase levels were measured before and after a positive oral food challenge (OFC) result and compared with levels in those with a negative OFC result. RESULTS: We found low levels of CM and casein-specific IgG and casein-specific IgG4 in patients with CM-FPIES versus those tolerating CM (P < .05). Although we found both a high CD4+ T cell-proliferative response and TH2 cytokines production after casein stimulation in children with CM-FPIES, results were similar to those in control subjects. Significantly lower secretion of IL-10 and higher secretion of IL-9 by casein-stimulated T cells were found in patients with CM-FPIES versus those with IgE-mediated CM allergy. Lower baseline serum levels of IL-10 and higher tryptase levels were found in active CM-FPIES versus resolved CM-FPIES. We found a significant increase in serum IL-10 and IL-8 levels after a positive OFC result. CONCLUSIONS: We confirm the paucity of humoral response in patients with CM-FPIES. IL-10 might play a key role in acquisition of tolerance in patients with CM-FPIES. Increased serum IL-8 levels in patients with active FPIES suggest neutrophil involvement. Elevated baseline serum tryptase levels in patients with active FPIES suggest low-grade intestinal mast cell activation or increased mast cell load.

Recognition of viral and self-antigens by TH1 and TH1/TH17 central memory cells in patients with multiple sclerosis reveals distinct roles in immune surveillance and relapses.

BACKGROUND: Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) that is caused by autoreactive T cells and associated with viral infections. However, the phenotype of pathogenic T cells in peripheral blood remains to be defined, and how viruses promote MS is debated. OBJECTIVE: We aimed to identify and characterize potentially pathogenic autoreactive T cells, as well as protective antiviral T cells, in patients with MS. METHODS: We analyzed CD4+ helper T-cell subsets from peripheral blood or cerebrospinal fluid for cytokine production, gene expression, plasticity, homing potentials, and their reactivity to self-antigens and viral antigens in healthy subjects and patients with MS. Moreover, we monitored their frequencies in untreated and fingolimod- or natalizumab-treated patients with MS. RESULTS: TH1/TH17 central memory (TH1/TH17CM) cells were selectively increased in peripheral blood of patients with relapsing-remitting MS with a high disease score. TH1/TH17CM cells were closely related to conventional TH17 cells but had more pathogenic features. In particular, they could shuttle between lymph nodes and the CNS and produced encephalitogenic cytokines. The cerebrospinal fluid of patients with active MS was enriched for CXCL10 and contained mainly CXCR3-expressing TH1 and TH1/TH17 subsets. However, while TH1 cells responded consistently to viruses, TH1/TH17CM cells reacted strongly with John Cunningham virus in healthy subjects but responded instead to myelin-derived self-antigens in patients with MS. Fingolimod and natalizumab therapies efficiently targeted autoreactive TH1/TH17CM cells but also blocked virus-specific TH1 cells. CONCLUSIONS: We propose that autoreactive TH1/TH17CM cells expand in patients with MS and promote relapses after bystander recruitment to the CNS, whereas TH1 cells perform immune surveillance. Thus the selective targeting of TH1/TH17 cells could inhibit relapses without causing John Cunningham virus-dependent progressive multifocal encephalomyelitis.

Pathogenic CD4+ T cells in patients with asthma.

Asthma encompasses a variety of clinical phenotypes that involve distinct T cell-driven inflammatory processes. Improved understanding of human T-cell biology and the influence of innate cytokines on T-cell responses at the epithelial barrier has led to new asthma paradigms. This review captures recent knowledge on pathogenic CD4+ T cells in asthmatic patients by drawing on observations in mouse models and human disease. In patients with allergic asthma, TH2 cells promote IgE-mediated sensitization, airway hyperreactivity, and eosinophilia. Here we discuss recent discoveries in the myriad molecular pathways that govern the induction of TH2 differentiation and the critical role of GATA-3 in this process. We elaborate on how cross-talk between epithelial cells, dendritic cells, and innate lymphoid cells translates to T-cell outcomes, with an emphasis on the actions of thymic stromal lymphopoietin, IL-25, and IL-33 at the epithelial barrier. New concepts on how T-cell skewing and epitope specificity are shaped by multiple environmental cues integrated by dendritic cell "hubs" are discussed. We also describe advances in understanding the origins of atypical TH2 cells in asthmatic patients, the role of TH1 cells and other non-TH2 types in asthmatic patients, and the features of T-cell pathogenicity at the single-cell level. Progress in technologies that enable highly multiplexed profiling of markers within a single cell promise to overcome barriers to T-cell discovery in human asthmatic patients that could transform our understanding of disease. These developments, along with novel T cell-based therapies, position us to expand the assortment of molecular targets that could facilitate personalized treatments.

Atrophying pityriasis versicolor as an idiosyncratic T cell-mediated response to Malassezia: A case series.

BACKGROUND: Atrophying pityriasis versicolor (PV), first described in 1971, is a rare variant in which lesions appear atrophic. OBJECTIVE: We sought to determine the pathophysiology of atrophying PV. METHODS: A retrospective chart review identified 6 cases of atrophying PV. In all cases, routine light microscopy, an elastic tissue stain, and immunohistochemical assessment for the expression of CD3, CD4, CD8, GATA3 and CXCR3 was performed. RESULTS: All cases demonstrated hyperkeratosis with intracorneal infiltration by pathogenic hyphal forms as well as epidermal attenuation and papillary dermal elastolysis. A supervening, mild-to-moderate, superficial lymphocytic infiltrate was noted and characterized by a focal CD8+ T cell-mediated interface dermatitis along with a mixed T-cell infiltrate composed of GATA3+ and CXCR3+ T cells. LIMITATIONS: Small sample size and the loss of some patients to follow-up. CONCLUSION: Atrophying PV represents the sequelae of a mixed helper T-cell (TH1 and TH2) idiosyncratic immune response to Malassezia and can present as a protracted dermatosis that may clinically mimic an atypical lymphocytic infiltrate. TH1 cytokines can recruit histiocytes, a source of elastases, and upregulate matrix metalloproteinase activity, which may contribute to epidermal atrophy.

Epicutaneous immunization with ovalbumin and CpG induces TH1/TH17 cytokines, which regulate IgE and IgG2a production.

BACKGROUND: Subcutaneous allergen-specific immunotherapy is a standard route for the immunotherapy of allergic diseases. It modulates the course of allergy and can generate long-term remission. However, subcutaneous allergen-specific immunotherapy can also induce anaphylaxis in some patients, and therefore additional routes of administration should be investigated to improve the safety and tolerability of immunotherapy. OBJECTIVE: We sought to determine whether epicutaneous treatment with antigen in the presence of a Toll-like receptor 9 agonist can suppress TH2-mediated responses in an antigen-specific manner. METHODS: Epicutaneous immunization was performed by applying a skin patch soaked with ovalbumin (OVA) plus CpG, and its suppressor activity was determined by using the mouse model of atopic dermatitis. Finally, adoptive cell transfers were implemented to characterize the regulatory cells that are induced by epicutaneous immunization. RESULTS: Epicutaneous immunization with OVA and CpG reduces the production of OVA-specific IgE and increases the synthesis of OVA-specific IgG2a antibodies in an antigen-specific manner. Moreover, eosinophil peroxidase activity in the skin and production of IL-4, IL-5, IL-10, and IL-13 are suppressed. The observed reduction of IgE synthesis is transferable with T-cell receptor (TCR) αß(+)CD4(+)CD25(-) cells, whereas IgG2a production is dependent on both TCRαß(+) and TCRγδ(+) T cells. Further experiments show that the described phenomenon is myeloid differentiation primary response 88, IFN-γ, and IL-17A dependent. Finally, the results suggest that epicutaneous immunization with OVA and CpG decreases the synthesis of OVA-specific IgE and skin eosinophil peroxidase activity in mice with ongoing skin allergy. CONCLUSION: Epicutaneous application of protein antigen in the presence of adjuvant could be an attractive needle-free and self-administered immunotherapy for allergic diseases.