Stem-like T cells are associated with the pathogenesis of ulcerative colitis in humans.

To understand the role of T cells in the pathogenesis of ulcerative colitis (UC), we analyzed colonic T cells isolated from patients with UC and controls. Here we identified colonic CD4+ and CD8+ T lymphocyte subsets with gene expression profiles resembling stem-like progenitors, previously reported in several mouse models of autoimmune disease. Stem-like T cells were increased in inflamed areas compared to non-inflamed regions from the same patients. Furthermore, TCR sequence analysis indicated stem-like T cells were clonally related to proinflammatory T cells, suggesting their involvement in sustaining effectors that drive inflammation. Using an adoptive transfer colitis model in mice, we demonstrated that CD4+ T cells deficient in either BCL-6 or TCF1, transcription factors that promote T cell stemness, had decreased colon T cells and diminished pathogenicity. Our results establish a strong association between stem-like T cell populations and UC pathogenesis, highlighting the potential of targeting this population to improve clinical outcomes.

A basophil-neuronal axis promotes itch.

Itch is an evolutionarily conserved sensation that facilitates expulsion of pathogens and noxious stimuli from the skin. However, in organ failure, cancer, and chronic inflammatory disorders such as atopic dermatitis (AD), itch becomes chronic, intractable, and debilitating. In addition to chronic itch, patients often experience intense acute itch exacerbations. Recent discoveries have unearthed the neuroimmune circuitry of itch, leading to the development of anti-itch treatments. However, mechanisms underlying acute itch exacerbations remain overlooked. Herein, we identify that a large proportion of patients with AD harbor allergen-specific immunoglobulin E (IgE) and exhibit a propensity for acute itch flares. In mice, while allergen-provoked acute itch is mediated by the mast cell-histamine axis in steady state, AD-associated inflammation renders this pathway dispensable. Instead, a previously unrecognized basophil-leukotriene (LT) axis emerges as critical for acute itch flares. By probing fundamental itch mechanisms, our study highlights a basophil-neuronal circuit that may underlie a variety of neuroimmune processes.

Commensal Cryptosporidium colonization elicits a cDC1-dependent Th1 response that promotes intestinal homeostasis and limits other infections.

Cryptosporidium can cause severe diarrhea and morbidity, but many infections are asymptomatic. Here, we studied the immune response to a commensal strain of Cryptosporidium tyzzeri (Ct-STL) serendipitously discovered when conventional type 1 dendritic cell (cDC1)-deficient mice developed cryptosporidiosis. Ct-STL was vertically transmitted without negative health effects in wild-type mice. Yet, Ct-STL provoked profound changes in the intestinal immune system, including induction of an IFN-γ-producing Th1 response. TCR sequencing coupled with in vitro and in vivo analysis of common Th1 TCRs revealed that Ct-STL elicited a dominant antigen-specific Th1 response. In contrast, deficiency in cDC1s skewed the Ct-STL CD4 T cell response toward Th17 and regulatory T cells. Although Ct-STL predominantly colonized the small intestine, colon Th1 responses were enhanced and associated with protection against Citrobacter rodentium infection and exacerbation of dextran sodium sulfate and anti-IL10R-triggered colitis. Thus, Ct-STL represents a commensal pathobiont that elicits Th1-mediated intestinal homeostasis that may reflect asymptomatic human Cryptosporidium infection.

Pitfalls of using polymerase chain reaction-based assays for JAK2 and CALR exon 9 variant testing in myeloproliferative neoplasms: Knowing when to go the extra mile!

OBJECTIVES: The BCR::ABL1 negative myeloproliferative neoplasms are sequentially tested for JAK2 p.V617F, followed by CALR exon 9 pathogenic variants. Historically, these variants were thought to be mutually exclusive. However, recent reports indicate coexisting JAK2 p.V617F and CALR exon 9 somatic variants. METHODS: Analysis of JAK2 p.V617F and CALR exon 9 variant was performed by polymerase chain reaction (PCR)-based assays. Subsequent testing was performed on the Genexus integrated sequencer (ThermoFisher) using the Oncomine myeloid assay GX v2. RESULTS: CALR exon 9 variants were positive in 3 cases, while 2 were positive for JAK2 p.V617F on PCR-based assays. Next-generation sequencing confirmed the JAK2 P.V617F status in all cases. CALR variants resulting in in-frame deletions were identified in 2 cases at a variant allele frequency of 52.16% and 50.91%, while the third case had an intronic CALR variant c.-48G>A at a variant allele frequency of 51.1%. Thus, CALR variants in all 3 cases were interpreted as potentially germline. Of the 228 cases that underwent JAK2 p.V617F and CALR cotesting in the past 2 years, only these 2 cases were positive for both JAK2 p.V617F and CALR exon 9 variants. CONCLUSIONS: These cases highlight the importance of understanding the pitfalls of molecular techniques in current practice.

Upregulation of Enhancer of Zeste Homolog 2 (EZH2) with Associated pERK Co-Expression and PRC2 Complex Protein SUZ12 Correlation in Adult T-Cell Leukemia/Lymphoma.

EZH2, a subunit of the polycomb repressive complex 2 (PRC2), is an important methyltransferase that catalyzes the trimethylation of histone H3 at lysine 27 (H3K27me3). EZH2 is overexpressed in various malignancies. Here, we investigated EZH2 expression and potential signaling molecules that correlate with EZH2 expression in ATLL and other T-cell neoplasms. Immunohistochemical staining (IHC) was performed for EZH2, pERK, MYC, and pSTAT3 on 43 ATLL cases and 104 cases of other T-cell neoplasms. Further IHC studies were conducted for Ki-67, SUZ12, and H3K27me3 on ATLL cases. All ATLL cases showed EZH2 overexpression. In other T-cell neoplasms, a high prevalence of EZH2 overexpression was identified (86%), except for T-PLL (33%). In ATLL, EZH2 overexpression correlated with pERK co-expression (86%), while only a small subset of cases showed MYC (7%) or pSTAT3 (14%) co-expression. In the other T-cell neoplasms, there was a variable, but higher, co-expression of EZH2 with pERK, MYC, and pSTAT3. In ATLL, enhanced EZH2 expression correlated with higher Ki-67 staining, SUZ12 (another PRC2 subunit), and H3K27me3 co-expression. In conclusion, EZH2 is overexpressed in ATLL and is associated with pERK expression. It correlates with an increased proliferation index, indicating an aggressive clinical course. EZH2 also correlates with SUZ12 and H3K27me3 co-expression, suggesting its PRC2-dependent catalytic activity through trimethylation. Additionally, EZH2 is overexpressed in most T-cell neoplasms, suggesting that EZH2 could function as an oncogenic protein in T-cell tumorigenesis. EZH2 and pERK could serve as potential therapeutic targets for treating aggressive ATLL. EZH2 could also be targeted in other T-cell neoplasms.

The Counteracting Effects of Ang II and Ang-(1-7) on the Function and Growth of Insulin-secreting NIT-1 Cells.

INTRODUCTION: China now has the highest number of diabetes in the world. Angiotensin II (Ang II) causes insulin resistance by acting on the insulin signaling pathway of peripheral target tissues. However, its effect on islet ß-cells remains unclear. The possible role of Angiotensin-(1-7) [Ang-(1-7)] as an antagonist to the effects of Ang II and in treating diabetes needs to be elucidated. OBJECTIVES: To assess the effects of Ang II and Ang-(1-7) on the function and growth of islet ß cell line NIT-1, which is derived from the islets of non-obese diabetic/large T-antigen (NOD/LT) mice with insulinoma. METHODS: NIT-1 cells were treated with Ang II, Ang-(1-7) and their respective receptor antagonists. The impact on cell function and growth was then evaluated. RESULTS: Ang II significantly reduced insulin-stimulated IR-ß-Tyr and Akt-Ser; while Ang-(1-7), saralasin (an Ang II receptor antagonist), and diphenyleneiodonium [DPI, a nicotinamide adenine dinucleotide phosphate oxidase (NOX) antagonist] reversed the inhibiting effect. Conversely, Ang II significantly increased insulin-stimulated intracellular H2O2 and P47 phox, while saralasin and DPI reverted the effect. Furthermore, Ang-(1-7) reduced the elevated concentrations of ROS and MDA while increasing the proliferation rate that was reduced by high glucose, all of which were reversed by A-779, an antagonist of the Mas receptor (MasR). CONCLUSION: Angiotensin II poses a negative regulatory effect on insulin signal transduction, increases oxidative stress, and may inhibit the transcription of insulin genes stimulated by insulin in NIT-1 cells. Meanwhile, angiotensin-(1-7) blocked these effects via MasR. These results corroborate the rising potential of the renin-angiotensin system (RAS) in treating diabetes.

Clinical validation of the Ion Torrent Oncomine Myeloid Assay GX v2 on the Genexus Integrated Sequencer as a stand-alone assay for single-nucleotide variants, insertions/deletions, and fusion genes: Challenges, performance, and perspectives.

OBJECTIVES: Myeloid neoplasms require comprehensive characterization of genetic abnormalities, including single-nucleotide variants, small insertions and deletions, and fusions and translocations for management. The Oncomine Myeloid Assay GX v2 (Thermo Fisher Scientific) analyzes 17 full genes, 28 hotspot genes, 30 fusion driver genes, and 5 expression genes. METHODS: The validation set included 192 DNA samples, 28 RNA samples, and 9 cell lines and contrived controls. The DNA and RNA were extracted from both peripheral blood and bone marrow. Library preparation, templating, and sequencing was performed on the fully automated Genexus Integrated Sequencer (Thermo Fisher Scientific). The sequencing data were analyzed by manual curation, default Oncomine filters and the Oncomine Reporter (Thermo Fisher Scientific). RESULTS: Of the 600 reference pathogenic DNA variants targeted by the assay, concordance was seen in 98.3% of unfiltered variant call format files. Precision and reproducibility were 100%, and the lower limit of detection was 2% variant allele frequency for DNA. Inability to detect variants in long homopolymer regions intrinsic to the Ion Torrent chemistry led to 7 missed variants; 100% concordance was seen with reference RNA samples. CONCLUSIONS: This extensive clinical validation of the Oncomine Myeloid Assay GX v2 on the Genexus Integrated Sequencer with its built-in bioinformatics pipeline and Ion Torrent Oncomine Reporter shows robust performance in terms of variant calling accuracy, precision, and reproducibility, with the advantage of a rapid turnaround time of 2 days. The greatest limitation is the inability to detect variants in long homopolymer regions.

Clinicopathologic characterization of cutaneous adult T-cell leukemia/lymphoma: A single tertiary care center experience in the United States.

OBJECTIVES: Adult T-cell leukemia/lymphoma (ATLL) is a rare aggressive T-cell leukemia/lymphoma associated with human T lymphotropic virus type 1 infection. The patients might present with skin rash before, at, or after the diagnosis. The dermatopathologic finding might be diagnostically very challenging. METHODS: We retrospectively identified 110 patients with ATLL at a single institution in a 19-year period, with 19 patients having skin biopsies. Clinical, dermatopathologic, immunophenotypic, and molecular findings were studied. RESULTS: The cohort included 13 skin-first (5 acute, 5 lymphomatous, 2 chronic, 1 smoldering), 6 skin-second (4 acute, 1 lymphomatous, 1 smoldering), and 91 patients without skin biopsy. Some nonphotoprotected areas of body such as the forearm and lower lip were also seen. Skin manifestations included papular (5), erythroderma (1), nodulotumoral (3), plaques (1), patches (1), and a combination of skin rashes (2). Histopathologic findings included large pleomorphic cells, angiocentrism, epidermal infiltration with large Pautrier-like microabscesses, and folliculotropism. Fifteen (78.9%) cases showed CD4+/CD7-/CD25+. Next-generation sequencing study was conducted on 5 patients using either blood or bone marrow samples, revealing multiple genetic mutations across multiple signaling pathways. CONCLUSIONS: Pleomorphic large, atypical cells with CD4+/CD25+/CD7- immunophenotype from a non-"bathing trunk" location, especially in a patient from endemic regions, raise suspicion for ATLL. T-cell receptor gene rearrangement is almost always positive, and the neoplasm usually demonstrates multiple mutations by next-generation sequencing study.

Acute myeloid leukemias with JAK2/STAT mutations are associated with PD-L1 upregulation.

Even though overexpression of the immune checkpoint protein, programmed cell death ligand-1 (PD-L1), is observed in solid tumors, its expression patterns in acute myeloid leukemia remain understudied. As activation of the JAK/STAT pathway has been shown to enhance PD-L1 expression in preclinical models, we evaluated biopsies from AML patients with activating mutations in JAK2/STATs. PD-L1 expression was significantly upregulated in JAK2/STAT mutant cases when compared to JAK2 wildtype controls as demonstrated by PD-L1 immunohistochemistry staining and quantified using the combined positive score (CPS) system. There is significant overexpression of phosphorylated STAT3 expression in patients with oncogenic JAK2 activation and a positive correlation between p-STAT3 and PD-L1 expression. In conclusion, we demonstrate the CPS scoring system could be applied as a quantitative measure of PD-L1 expression in leukemias and that JAK2/STATs mutant AML can be potential candidates for checkpoint inhibitor trials.

A Splice Site Mutation Associated with Congenital CD59 Deficiency.

Congenital CD59 deficiency is a recently described rare autosomal recessive disease associated with CD59 gene mutations that lead to deficient or dysfunctional CD59 protein on the cell surface. The disease is characterized by the early onset of chronic hemolysis, relapsing peripheral demyelinating neuropathy, and recurrent ischemic strokes. To date, there are 14 patients with 4 exon mutations reported globally. A young boy with early onset peripheral neuropathy and atypical hemolytic uremic syndrome is presented. Next-generation sequencing (NGS) identified a homozygous splice site variant in intron 1 of the CD59 gene (c.67 + 1G > T). This variant alters a consensus donor splicing site. Quantitative reverse transcription PCR showed that CD59 mRNA expression in the patient is significantly reduced to 0.017-fold compared to the controls. Flow cytometry showed the lack of CD59 protein on the surface of the patient's red blood cells. This variant is the first splice site mutation reported to be associated with congenital CD59 deficiency.

Gut Helicobacter presentation by multiple dendritic cell subsets enables context-specific regulatory T cell generation.

Generation of tolerogenic peripheral regulatory T (pTreg) cells is commonly thought to involve CD103+ gut dendritic cells (DCs), yet their role in commensal-reactive pTreg development is unclear. Using two Helicobacter-specific T cell receptor (TCR) transgenic mouse lines, we found that both CD103+ and CD103- migratory, but not resident, DCs from the colon-draining mesenteric lymph node presented Helicobacter antigens to T cells ex vivo. Loss of most CD103+ migratory DCs in vivo using murine genetic models did not affect the frequency of Helicobacter-specific pTreg cell generation or induce compensatory tolerogenic changes in the remaining CD103- DCs. By contrast, activation in a Th1-promoting niche in vivo blocked Helicobacter-specific pTreg generation. Thus, these data suggest a model where DC-mediated effector T cell differentiation is 'dominant', necessitating that all DC subsets presenting antigen are permissive for pTreg cell induction to maintain gut tolerance.

Unexpected High Prevalence of Lymphocytic Infiltrates in Myenteric Ganglions in Intestinal Inertia.

Intestinal inertia is a severe form of gut dysmotility that may require surgical resection. Loss of myenteric ganglion cells has been proposed as a possible etiology. Preclinical models have also suggested that virus infection-associated ganglionitis may be an alternative pathogenic factor. We determined to the extent intestinal inertia is associated with the lack of myenteric ganglion cells or ganglionitis using resection specimens from 27 intestinal inertia and 28 colon cancer patients. A hot spot approach with 5 HPFs was used for quantifying inflammatory cells. CD3, CD8, and CD20 immunohistochemistry was used to quantify T and B lymphocytes, along with subtyping the T-lymphocyte population by CD8. None of the intestinal inertia nor control cases showed the absence of myenteric ganglion cells. A total of 15 (55.6%) of the intestinal inertia cases showed inflammatory cell infiltration in the myenteric ganglion cells, compared with only 1 of 28 (3.6%) control cases (P<0.0001 by Fisher exact test). The inertia cases with inflammatory infiltrates were all associated predominantly with lymphocytes, including 3 cases (11.1%) with concurrent eosinophil infiltration, and 1 case (3.7%) with concurrent neutrophil infiltration. Furthermore, all 15 inertia cases with myenteric lymphocytic ganglionitis were associated with T lymphocytes (100%), including 1 case with a subset of concurrent B lymphocytes. The average CD3 count was 3.8 cells/HPF. CD8 immunohistochemical stain showed positive staining in 12 of the 15 cases (80%) with CD8-positive cells ranging from 1 to 8/HPF. In contrast, the only control case with lymphocytic ganglionitis showed mixed B and T lymphocytes and eosinophils. The high prevalence of T-lymphocyte infiltration in the myenteric ganglion in intestinal inertia cases suggests a possible pathogenic role.

Airway Microbiota-Host Interactions Regulate Secretory Leukocyte Protease Inhibitor Levels and Influence Allergic Airway Inflammation.

Homeostatic mucosal immune responses are fine-tuned by naturally evolved interactions with native microbes, and integrating these relationships into experimental models can provide new insights into human diseases. Here, we leverage a murine-adapted airway microbe, Bordetella pseudohinzii (Bph), to investigate how chronic colonization impacts mucosal immunity and the development of allergic airway inflammation (AAI). Colonization with Bph induces the differentiation of interleukin-17A (IL-17A)-secreting T-helper cells that aid in controlling bacterial abundance. Bph colonization protects from AAI and is associated with increased production of secretory leukocyte protease inhibitor (SLPI), an antimicrobial peptide with anti-inflammatory properties. These findings are additionally supported by clinical data showing that higher levels of upper respiratory SLPI correlate both with greater asthma control and the presence of Haemophilus, a bacterial genus associated with AAI. We propose that SLPI could be used as a biomarker of beneficial host-commensal relationships in the airway.

A Potential Role for Stress-Induced Microbial Alterations in IgA-Associated Irritable Bowel Syndrome with Diarrhea.

Stress is a known trigger for flares of inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS); however, this process is not well understood. Here, we find that restraint stress in mice leads to signs of diarrhea, fecal dysbiosis, and a barrier defect via the opening of goblet-cell associated passages. Notably, stress increases host immunity to gut bacteria as assessed by immunoglobulin A (IgA)-bound gut bacteria. Stress-induced microbial changes are necessary and sufficient to elicit these effects. Moreover, similar to mice, many diarrhea-predominant IBS (IBS-D) patients from two cohorts display increased antibacterial immunity as assessed by IgA-bound fecal bacteria. This antibacterial IgA response in IBS-D correlates with somatic symptom severity and was distinct from healthy controls or IBD patients. These findings suggest that stress may play an important role in patients with IgA-associated IBS-D by disrupting the intestinal microbial community that alters gastrointestinal function and host immunity to commensal bacteria.

CRTAM Protects Against Intestinal Dysbiosis During Pathogenic Parasitic Infection by Enabling Th17 Maturation.

The gastrointestinal tract hosts the largest collection of commensal microbes in the body. Infections at this site can cause significant perturbations in the microbiota, known as dysbiosis, that facilitate the expansion of pathobionts, and can elicit inappropriate immune responses that impair the intestinal barrier function. Dysbiosis typically occurs during intestinal infection with Toxoplasma gondii. Host resistance to T. gondii depends on a potent Th1 response. In addition, a Th17 response is also elicited. How Th17 cells contribute to the host response to T. gondii remains unclear. Here we show that class I-restricted T cell-associated molecule (CRTAM) expression on T cells is required for an optimal IL-17 production during T. gondii infection. Moreover, that the lack of IL-17, results in increased immunopathology caused by an impaired antimicrobial peptide production and bacterial translocation from the intestinal lumen to the mesenteric lymph nodes and spleen.

Lactobacillus reuteri induces gut intraepithelial CD4+CD8αα+ T cells.

The small intestine contains CD4+CD8αα+ double-positive intraepithelial lymphocytes (DP IELs), which originate from intestinal CD4+ T cells through down-regulation of the transcription factor Thpok and have regulatory functions. DP IELs are absent in germ-free mice, which suggests that their differentiation depends on microbial factors. We found that DP IEL numbers in mice varied in different vivaria, correlating with the presence of Lactobacillus reuteri This species induced DP IELs in germ-free mice and conventionally-raised mice lacking these cells. L. reuteri did not shape the DP-IEL-TCR (TCR, T cell receptor) repertoire but generated indole derivatives of tryptophan that activated the aryl-hydrocarbon receptor in CD4+ T cells, allowing Thpok down-regulation and differentiation into DP IELs. Thus, L. reuteri, together with a tryptophan-rich diet, can reprogram intraepithelial CD4+ T cells into immunoregulatory T cells.

Zika virus has oncolytic activity against glioblastoma stem cells.

Glioblastoma is a highly lethal brain cancer that frequently recurs in proximity to the original resection cavity. We explored the use of oncolytic virus therapy against glioblastoma with Zika virus (ZIKV), a flavivirus that induces cell death and differentiation of neural precursor cells in the developing fetus. ZIKV preferentially infected and killed glioblastoma stem cells (GSCs) relative to differentiated tumor progeny or normal neuronal cells. The effects against GSCs were not a general property of neurotropic flaviviruses, as West Nile virus indiscriminately killed both tumor and normal neural cells. ZIKV potently depleted patient-derived GSCs grown in culture and in organoids. Moreover, mice with glioblastoma survived substantially longer and at greater rates when the tumor was inoculated with a mouse-adapted strain of ZIKV. Our results suggest that ZIKV is an oncolytic virus that can preferentially target GSCs; thus, genetically modified strains that further optimize safety could have therapeutic efficacy for adult glioblastoma patients.

Helicobacter species are potent drivers of colonic T cell responses in homeostasis and inflammation.

Specific gut commensal bacteria improve host health by eliciting mutualistic regulatory T (Treg) cell responses. However, the bacteria that induce effector T (Teff) cells during inflammation are unclear. We addressed this by analyzing bacterial-reactive T cell receptor (TCR) transgenic cells and TCR repertoires in a murine colitis model. Unexpectedly, we found that mucosal-associated Helicobacter species triggered both Treg cell responses during homeostasis and Teff cell responses during colitis, as suggested by an increased overlap between the Teff/Treg TCR repertoires with colitis. Four of six Treg TCRs tested recognized mucosal-associated Helicobacter species in vitro and in vivo. By contrast, the marked expansion of luminal Bacteroides species seen during colitis did not trigger a commensurate Teff cell response. Unlike other Treg cell-inducing bacteria, Helicobacter species are known pathobionts and cause disease in immunodeficient mice. Thus, our study suggests a model in which mucosal bacteria elicit context-dependent Treg or Teff cell responses to facilitate intestinal tolerance or inflammation.

Rapid and Efficient Generation of Regulatory T Cells to Commensal Antigens in the Periphery.

Commensal bacteria shape the colonic regulatory T (Treg) cell population required for intestinal tolerance. However, little is known about this process. Here, we use the transfer of naive commensal-reactive transgenic T cells expressing colonic Treg T cell receptors (TCRs) to study peripheral Treg (pTreg) cell development in normal hosts. We found that T cells were activated primarily in the distal mesenteric lymph node. Treg cell induction was rapid, generating >40% Foxp3(+) cells 1 week after transfer. Contrary to prior reports, Foxp3(+) cells underwent the most cell divisions, demonstrating that pTreg cell generation can be the dominant outcome from naive T cell activation. Moreover, Notch2-dependent, but not Batf3-dependent, dendritic cells were involved in Treg cell selection. Finally, neither deletion of the conserved nucleotide sequence 1 (CNS1) region in Foxp3 nor blockade of TGF-ß (transforming growth factor-ß)-receptor signaling completely abrogated Foxp3 induction. Thus, these data show that pTreg cell selection to commensal bacteria is rapid, is robust, and may be specified by TGF-ß-independent signals.