Intestinal T-cell Research Articles

Generation of Non-Hodgkin Lymphoma Patient-Derived Xenografts and in Depth Characterization of a Monomorphic Epitheliotropic Intestinal T-Cell Lymphoma Model

Non-Hodgkin lymphomas (NHL) are a diverse group of blood cancers on the rising trend both worldwide and in Singapore. Better understanding of the pathogenesis and biology of these tumors is urgently required to improve the diagnosis and prognostication. Moreover, the outcome using currently available therapies is poor and there is an immediate need for better treatment options. With an increasing number of genomic studies published, there is a growing demand to bring these discoveries into in vitro and in vivo models. However, the lack of publicly available cell lines and/or animal models for the majority of the NHL subtypes makes these studies difficult to proceed.With that objective, we have started collecting fresh NHL tumor samples and implanting them into NOD scid gamma (NSG) mice to generate patient-derived xenograft (PDX) models. After tumor dissemination, the cells are injected either subcutaneously or intraperitoneally into 6-10 week old mice. Each passage is thoroughly immunohistochemically characterized by a senior hematopathologist and contrasted to the primary patient specimen. Whole exome sequencing (WES) is performed and compared to the primary patient data where possible. A model is considered stable if it has not changed its phenotype for at least 3 passages. A full list of currently available stable models is listed in Table 1. Efforts are also under way to generate stable cell lines based on these models. Strategies tested are spontaneous immortalization, human telomerase reverse transcriptase (TERT), Herpesvirus saimiri (HVS) or Epstein-Barr virus (EBV) transduction and feeder culture.We have recently reported a comprehensive molecular profiling of monomorphic epitheliotropic intestinal T-cell lymphoma (MEITL; previously also known as type II enteropathy-associated T-cell lymphoma; Nairismagi et al., Leukemia, 2016). MEITL, a newly recognized WHO entity, is a rare aggressive primary intestinal disease with poor prognosis and a median overall survival of only 7 months. Frequent alterations were identified in the JAK-STAT and G-protein-coupled receptor signaling pathways and a number of epigenetic regulators. Specifically, 60-30% mutation frequencies were found in the STAT5B, JAK3, SETD2, GNAI2 and CREBBP genes.Tumor tissue was collected from a 55 year old Chinese male with relapsed MEITL. The cells have been propagated in the NSG mouse by subcutaneous or intraperitoneal injection up to 6 and 3 passages, respectively. All passages have been histologically characterized and maintained their CD3+ CD8alpha-alpha+ cytotoxic granules+ MATK+ and EBER- phenotype with aberrant expression of both TCR beta and gamma. WES was performed both in the patient primary sample and in passage 1 (P1) PDX sample. There were 83 somatic mutations present in the primary sample, all of which were also identified in the P1 PDX sample. Furthermore, Sanger sequencing was used to validate the presence of all 83 mutations in the remaining passages. There was only 1 mutation lost in the P6 subcutaneous PDX model compared to the primary patient specimen demonstrating the outstanding stability of this model. Using peritoneal fluid cells from P2 we have previously performed ex vivo studies and identified novel treatment modalities for this deadly disease (Nairismagi et al., Leukemia, 2016). Efforts are currently under way to test these regimens also in in vivo setting.In summary, we have generated a number of NHL PDX models with the objective to enhance drug testing. MEITL presents a real life situation where clinical trials are not possible due to the rarity of the disease; however, the availability of PDX models allows for efficient testing of novel therapies and can lead to improved patient outcome. Efforts are under way to further characterize the existing models and tissue collection is ongoing to establish more models. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Enteropathy-Associated T-Cell Lymphoma.

Enteropathy-associated T-cell lymphoma is a rare neoplasm with uniformly aggressive features that arises from intestinal T-cells. There is strong evidence supporting its association as a dire complication of celiac disease. The clinical presentation can vary from malabsorption and abdominal pain to an acute abdominal emergency. Originally, it was divided into types I and II in World Health Organization (WHO) classification schemes, reflective of epidemiology and differences in clinicopathologic features. The debate over the degree of separation of the two types is ongoing as new data emerges regarding the pathogenetics. The low incidence and variable patient factors are major barriers in conducting clinical trials and establishing standard treatment regimens. Yet, the collective experience demonstrates favorable outcomes with combination chemotherapy followed by an autologous hematopoietic stem cell transplant in patients who can tolerate such treatment. The prognosis remains dismal; thus, future research studies are warranted to identify effective novel therapies that can improve outcomes in this rare disease entity.

Inflammatory cues enhance TGF\u03b2 activation by distinct subsets of human intestinal dendritic cells via integrin \u03b1v\u03b28

Regulation of intestinal T-cell responses is crucial for immune homeostasis and prevention of inflammatory bowel disease (IBD). A vital cytokine in regulating intestinal T cells is transforming growth factor-β (TGFβ), which is secreted by cells as a latent complex that requires activation to function. However, how TGFβ activation is regulated in the human intestine, and how such pathways are altered in IBD is completely unknown. Here we show that a key activator of TGFβ, integrin αvβ8, is highly expressed on human intestinal dendritic cells (DCs), specifically on the CD1c+ but not the CD141+ intestinal DC subset. Expression was significantly upregulated on intestinal DC from IBD patients, indicating that inflammatory signals may upregulate expression of this key TGFβ-activating molecule. Indeed, we found that the Toll-like receptor 4 ligand lipopolysaccharide upregulates integrin αvβ8 expression and TGFβ activation by human DC. We also show that DC expression of integrin αvβ8 enhanced induction of FOXP3 in CD4+ T cells, suggesting functional importance of integrin αvβ8 expression by human DC. These results show that microbial signals enhance the TGFβ-activating ability of human DC via regulation of integrin αvβ8 expression, and that intestinal inflammation may drive this pathway in patients with IBD.

MTOR is critical for intestinal T-cell homeostasis and resistance to Citrobacter rodentium

T-cells play an important role in promoting mucosal immunity against pathogens, but the mechanistic basis for their homeostasis in the intestine is still poorly understood. We report here that T-cell-specific deletion of mTOR results in dramatically decreased CD4 and CD8 T-cell numbers in the lamina propria of both small and large intestines under both steady-state and inflammatory conditions. These defects result in defective host resistance against a murine enteropathogen, Citrobacter rodentium, leading to the death of the animals. We further demonstrated that mTOR deficiency reduces the generation of gut-homing effector T-cells in both mesenteric lymph nodes and Peyer’s patches without obviously affecting expression of gut-homing molecules on those effector T-cells. Using mice with T-cell-specific ablation of Raptor/mTORC1 or Rictor/mTORC2, we revealed that both mTORC1 and, to a lesser extent, mTORC2 contribute to both CD4 and CD8 T-cell accumulation in the gastrointestinal (GI) tract. Additionally, mTORC1 but not mTORC2 plays an important role regulating the proliferative renewal of both CD4 and CD8 T-cells in the intestines. Our data thus reveal that mTOR is crucial for T-cell accumulation in the GI tract and for establishing local adaptive immunity against pathogens.

Sinomenine induces the generation of intestinal Treg cells and attenuates arthritis via activation of aryl hydrocarbon receptor

Sinomenine (SIN), an anti-arthritis drug, has previously been proven to exert immunomodulatory activity in rats by inducing intestinal regulatory T-cells (Treg cells). Here, we assessed the effect of SIN on the generation and function of Treg cells in autoimmune arthritis, and the underlying mechanisms in view of aryl hydrocarbon receptor (AhR). The proportions of Treg cells and IL-17-producing T-cells (Th17 cells) differentiated from naive T-cells were analyzed by flow cytometric analysis. The AhR agonistic effect of SIN was tested by analyzing the activation of downstream signaling pathways and target genes. The dependence of intestinal Treg cell induction and arthritis alleviation by SIN on AhR activation was confirmed in a mouse collagen-induced arthritis (CIA) model. SIN promoted the differentiation and function of intestinal Treg cells in vitro. It induced the expression and activity of AhR target gene, promoted AhR/Hsp90 dissociation and AhR nuclear translocation, induced XRE reporter activity, and facilitated AhR/XRE binding in vitro, displaying the potential to be an agonist of AhR. In CIA mice, SIN induced the generation of intestinal Treg cells, and facilitated the immunosuppressive function of these Treg cells as shown by an adoptive transfer test. In addition, the induction of intestinal Treg cells and the anti-arthritic effect of SIN in CIA mice could be largely diminished by the AhR antagonist resveratrol. SIN attenuates arthritis by promoting the generation and function of Treg cells in an AhR-dependent manner.

Does categorisation of lymphoma subtypes according to the World Health Organization classification predict clinical outcome in cats?

Objectives The purpose of this study was to specify lymphoma subtypes according to the World Health Organization (WHO) classification in a group of cats and to investigate their potential prognostic value. Methods Records of cats from the University of Veterinary Medicine Vienna suffering from lymphoma were reviewed in this retrospective study. To diagnose various subtypes specified in the WHO classification, histopathological and immunohistochemical examinations, as well as clonality assays in some cases, were performed. Results Of the 30 cats included in this study and classified according to the WHO guidelines, peripheral T-cell lymphoma was the most prevalent lymphoma subtype (37% of cases; n = 11), followed by diffuse large B-cell (23%; n = 7), intestinal T-cell (10%; n = 3), T-cell-rich B-cell (10%; n = 3), large granular lymphocytic (7%; n = 2), anaplastic large T-cell (7%; n = 2), B-cell small lymphocytic (3%; n = 1) and T-cell angiotropic lymphoma (3%; n = 1). The median survival time (MST) was 5.4 months (range 6 days to 2.2 years), with two cats still alive after 1.7 and 2.0 years, respectively. Treating cats prior to chemotherapy with glucocorticoids did not worsen their prognosis. Adding to chemotherapy, radiotherapy or surgery did not improve the clinical outcome. We observed that patients with intestinal T-cell lymphoma lived significantly longer (MST 1.7 years) than those with a diffuse large B-cell (MST 4.5 months) or peripheral T-cell lymphoma (MST 6.1 months). Cats with T-cell-rich B-cell lymphoma survived significantly longer (MST 1.2 years) than those with a diffuse large B-cell lymphoma. Conclusions and relevance A detailed diagnosis of feline lymphoma can be obtained by allocating different subtypes according to the WHO classification. From the eight detected lymphoma subtypes, two, intestinal T-cell lymphoma and T-cell-rich B-cell lymphoma, showed promising survival times in cats.

Antibiotic-mediated gut microbiome perturbation accelerates development of type 1 diabetes in mice.

The early life microbiome plays important roles in host immunological and metabolic development. Because the incidence of type 1 diabetes (T1D) has been increasing substantially in recent decades, we hypothesized that early-life antibiotic use alters gut microbiota, which predisposes to disease. Using non-obese diabetic mice that are genetically susceptible to T1D, we examined the effects of exposure to either continuous low-dose antibiotics or pulsed therapeutic antibiotics (PAT) early in life, mimicking childhood exposures. We found that in mice receiving PAT, T1D incidence was significantly higher, and microbial community composition and structure differed compared with controls. In pre-diabetic male PAT mice, the intestinal lamina propria had lower Th17 and Treg proportions and intestinal SAA expression than in controls, suggesting key roles in transducing the altered microbiota signals. PAT affected microbial lipid metabolism and host cholesterol biosynthetic gene expression. These findings show that early-life antibiotic treatments alter the gut microbiota and its metabolic capacities, intestinal gene expression and T-cell populations, accelerating T1D onset in non-obese diabetic mice.

A retrospective analysis of clinical pathological characteristics and prognosis of 82 patients of primary intestinal lymphoma

Background: To evaluate the histological type, clinical presentation, treatment and prognosis of primary intestinal lymphoma (PIL). Methods: From 2003 to 2015, in a single institution in China, 82 patients with a diagnosis of PIL were enrolled in our study. According to World Health Organization (WHO) classification and Lugano staging system for gastrointestinal lymphoma, we divided patients into different subtypes and stages. Results: Male to female ratio was 1.8:1 and the median age was 59.5 years. Of the 82 patients, 68 patients (82.9%) suffered from intestinal B-cell lymphoma (IBCL) and 14 patients (17.1%) suffered from intestinal T-cell lymphoma (ITCL). Diffuse large B cell lymphoma (DLBCL) was the predominant histological subtype (59.8%) in all of the PIL cases. Mucosa-associated lymphoid tissue-type lymphoma (MALT) was the second most common subtype (15.9%). From the study we also concluded that PIL mostly occurred in the small intestine (45.1%), followed by colon and rectum (26.8%). The univariate prognosis analysis revealed that Lugano stage I/II, B-cell phenotype and treatment based on surgery plus chemotherapy were independent prognostic factors (P=0.003, 0.000 and 0.000, respectively). According to multivariate analysis, age of onset, Lugano stage, immunophenotype and the method of treatment were significant predictive factors for better survival (P=0.005, 0.005, 0.000 and 0.000, respectively). Conclusions: The patients with younger age of onset, B cell lymphoma, earlier stages, and treatment based on surgery plus chemotherapy have a better prognosis. To ensure the diagnosis of PIL to be made as soon as possible, we should make efforts to select the proper laboratory parameters and imaging findings of PIL.

Immune-mediated hemolytic anemia in a hypereosinophilic dog with intestinal T-cell lymphoma

A 13-year-old, 21-kg, female, mixed breed dog was presented with multifocal skin rashes, abdominal distension, and vomiting for several days. Radiographic images displayed the small intestine thickening. On exploratory laparotomy, a wall thickness of the jejunum and ileum about 15 cm long, with an adhesive 1-cm-diameter nodular mass was found. Histopathologically, the mass was diagnosed as lymphoma. The lymphoid cells were positive for CD 3, but were negative for CD 79α on immunohistochemical staining. Hematological profiles revealed severe hypochromia, autoagglutination, and eosinophilic left shift. A protein electrophoresis revealed monoclonal gammopathy. All of the results, an immune-mediated hemolytic anemia presenting with hypereosinophilia and intestinal T-cell lymphoma was diagnosis.

Influence of orally fed a select mixture of Bacillus probiotics on intestinal T-cell migration in weaned MUC4 resistant pigs following Escherichia coli challenge

Efficient strategies for treating enteritis caused by F4+ enterotoxigenic Escherichia coli (ETEC)/verocytotoxigenic Escherichia coli (VTEC)/enteropathogenic E. coli (EPEC) in mucin 4 resistant (MUC4 RR; supposed to be F4ab/ac receptor–negative [F4ab/acR−]) pigs remain elusive. A low (3.9 × 108 CFU/day) or high (7.8 × 108 CFU/day) dose of Bacillus licheniformis and Bacillus subtilis spore mixture (BLS-mix) was orally administered to MUC4 RR piglets for 1 week before F4+ ETEC/VTEC/EPEC challenge. Orally fed BLS-mix upregulated the expression of TLR4, NOD2, iNOS, IL-8, and IL-22 mRNAs in the small intestine of pigs challenged with E. coli. Expression of chemokine CCL28 and its receptor CCR10 mRNAs was upregulated in the jejunum of pigs pretreated with high-dose BLS-mix. Low-dose BLS-mix pretreatment induced an increase in the proportion of peripheral blood CD4−CD8− T-cell subpopulations and high-dose BLS-mix induced the expansion of CD4−CD8− T cells in the inflamed intestine. Immunostaining revealed that considerable IL-7Rα–expressing cells accumulated at the lamina propria of the inflamed intestines after E. coli challenge, even in pigs pretreated with either low- or high-dose BLS-mix, although Western blot analysis of IL-7Rα expression in the intestinal mucosa did not show any change. Our data indicate that oral administration of the probiotic BLS-mix partially ameliorates E. coli-induced enteritis through facilitating upregulation of intestinal IL-22 and IκBα expression, and preventing loss of intestinal epithelial barrier integrity via elevating ZO-1 expression. However, IL-22 also elicits an inflammatory response in inflamed intestines as a result of infection with enteropathogenic bacteria.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-016-0355-8) contains supplementary material, which is available to authorized users.

Clinicopathologic Spectrum of Gastrointestinal T-cell Lymphoma: Reappraisal Based on T-cell Receptor Immunophenotypes.

The differential diagnosis of primary gastrointestinal EBV T-cell lymphoma (GITCL) includes enteropathy-associated T-cell lymphoma (EATL), peripheral T-cell lymphoma, not otherwise specified, adult T-cell leukemia/lymphoma, and anaplastic large cell lymphoma. Type II EATL is considered to be a tumor of intraepithelial lymphocytes. However, the evaluation of intraepithelial lymphocytosis by biopsy specimens is challenging, which poses a diagnostic problem between the EATL and peripheral T-cell lymphoma, not otherwise specified. This situation requested us to establish a pragmatic diagnostic approach for the classification of GITCL. We identified 42 cases of GITCL and analyzed clinicopathologic features, especially addressing their T-cell receptor (TCR) phenotype. Nine (21%) of 42 GITCL cases were positive for TCRγ protein expression. Among these TCRγ cases, TCRβ expression or not was detected in 5 and 4, respectively, but resulted in no further clinicopathologic differences. TCRβ positivity without TCRγ expression (βγ) was seen in 9 GITCL patients (21%). Twenty-four patients (57%) were negative for TCRβ and γ expression (βγ). Compared with TCRβγ or βγ type, TCRγ cases were characterized by exclusive involvement of intestinal sites (100% vs. 11%, P<0.001; 100% vs. 58%, P=0.032, respectively), but not of stomach (0% vs. 78%, P=0.002; 0% vs. 38%, P=0.039, respectively). Notably, TCRγ positivity was an independent unfavorable prognostic factor among our GITCL patients (P<0.001). Considering our results, TCRγ GITCL, that is, intestinal γδ T-cell lymphoma, appears to constitute a distinct disease entity.

Regulation of human intestinal T-cell responses by type 1 interferon-STAT1 signaling is disrupted in inflammatory bowel disease.

Type 1 interferon (IFN-1) promotes regulatory T-cell function to suppress inflammation in the mouse intestine, but little is known about IFN-1 in the human gut. We therefore assessed the influence of IFN-1 on CD4+ T-cells isolated from human colon tissue obtained from healthy controls or patients with inflammatory bowel disease (IBD). Immunofluorescent imaging revealed constitutive expression of IFNβ in human intestinal tissue, and colonic T-cells were responsive to exogenous IFN-1 as assessed by phosphorylation of signal transduction and activator of transcription 1 (pSTAT1) and induction of interferon stimulated genes (ISGs). Unlike their blood counterparts, intestinal T-cells from non-inflamed regions of IBD colon displayed enhanced responsiveness to IFN-1, increased frequency of pSTAT1+ cells, and greater induction of ISGs upon IFN-1 exposure in vitro. In healthy tissue, antibody neutralization of IFNβ selectively reduced T-cell production of the pro-regulatory cytokine interleukin-10 (IL-10) and increased IFNγ synthesis. In contrast, neutralization of IFNβ in IBD tissue cultures increased the frequency of T-cells producing inflammatory cytokines but did not alter IL-10 expression. These data support a role for endogenous IFN-1 as a context-dependent modulator of T-cell function that promotes regulatory activity in healthy human intestine, but indicate that the IFN-1/STAT1 pathway is dysregulated in inflammatory bowel disease.

MyD88 in donor bone marrow cells is critical for protection from acute intestinal graft-vs.-host disease.

To understand the role of myeloid differentiation factor 88 (MyD88) expressed by donor bone marrow (BM) in the pathophysiology of graft-vs.-host disease (GVHD), we investigated the effects of transplantation of MyD88-deficient T cell-depleted BM (MyD88KO TCD-BM) on the severity of GVHD. Transplantation with MyD88KO TCD-BM aggravated GVHD; serious gut damage was evident, with high infiltration of T cells into the intestines of recipients and markedly reduced expansion of CD11b(+)Gr-1(+) myeloid-derived suppressor cells (MDSCs). MDSCs from MyD88KO mice were defective in inducing donor T-cell apoptosis and inhibiting T-cell proliferation. Supplementation of transplanted mice with MDSCs from wild-type mice, but not MyD88KO mice, attenuated GVHD severity with reduced intestinal T-cell infiltration in MyD88KO TCD-BM recipients. Pretreatment of BM donors with lipopolysaccharide to increase MDSC levels and MyD88 transcription in the TCD-BM transplant alleviated GVHD severity and intestinal T-cell infiltration. The T cell/MDSC ratios were correlated with intestinal GVHD severity in both animal models and human patients. This study indicates that MyD88-dependent MDSC expansion from donor BM is critical for protection against fatal intestinal GVHD.

CD45 regulates GM-CSF, retinoic acid and T-cell homing in intestinal inflammation.

CD45 is a leukocyte-specific tyrosine phosphatase important for T-cell development, and as a result, CD45-/- mice have substantially reduced numbers of T cells. Here we show that, upon dextran sodium sulfate (DSS)-induced colitis, CD45-/- mice have equivalent intestinal pathology and T-cell numbers in their colon as C57BL/6 mice and show enhanced weight loss. CD45-/- mice have a greater percentage of α4β7+ T cells prior to and after colitis and an increased percentage of T cells producing inflammatory cytokines in the inflamed colon, suggesting that CD45-/- effector T cells preferentially home to the intestine. In DSS-induced colitis in CD45RAG-/- mice lacking an adaptive immune system, CD45 was required for optimal granulocyte-macrophage colony-stimulating factor (GM-CSF) and retinoic acid (RA) production by innate immune cells. Addition of CD45+/+ T cells led to greater weight loss in the RAG-/- mice compared with CD45RAG-/- mice that correlated with reduced α4β7+ T cells and lower recruitment to the colon of CD45RAG-/- mice in DSS-induced colitis. Addition of exogenous GM-CSF to CD45RAG-/- mice rescued RA production, increased colonic T-cell numbers, and increased weight loss. This demonstrates opposing effects of CD45 in innate and adaptive immune cells in proinflammatory responses and the expression of the gut-homing molecule, α4β7.

JAK-STAT and G-protein-coupled receptor signaling pathways are frequently altered in epitheliotropic intestinal T-cell lymphoma.

Epitheliotropic intestinal T-cell lymphoma (EITL, also known as type II enteropathy-associated T-cell lymphoma) is an aggressive intestinal disease with poor prognosis and its molecular alterations have not been comprehensively characterized. We aimed to identify actionable easy-to-screen alterations that would allow better diagnostics and/or treatment of this deadly disease. By performing whole-exome sequencing of four EITL tumor-normal pairs, followed by amplicon deep sequencing of 42 tumor samples, frequent alterations of the JAK-STAT and G-protein-coupled receptor (GPCR) signaling pathways were discovered in a large portion of samples. Specifically, STAT5B was mutated in a remarkable 63% of cases, JAK3 in 35% and GNAI2 in 24%, with the majority occurring at known activating hotspots in key functional domains. Moreover, STAT5B locus carried copy-neutral loss of heterozygosity resulting in the duplication of the mutant copy, suggesting the importance of mutant STAT5B dosage for the development of EITL. Dysregulation of the JAK-STAT and GPCR pathways was also supported by gene expression profiling and further verified in patient tumor samples. In vitro overexpression of GNAI2 mutants led to the upregulation of pERK1/2, a member of MEK-ERK pathway. Notably, inhibitors of both JAK-STAT and MEK-ERK pathways effectively reduced viability of patient-derived primary EITL cells, indicating potential therapeutic strategies for this neoplasm with no effective treatment currently available.

IL-36α expression is elevated in ulcerative colitis and promotes colonic inflammation.

A role for the IL-36 family of cytokines has been identified in the pathogenesis of psoriasis. Although significant mechanistic overlap can exist between psoriasis and inflammatory bowel disease (IBD), to date there have been no reports investigating the IL-36 family in gastrointestinal inflammation. Here we demonstrate that expression levels of IL-36α are specifically elevated in the colonic mucosa of ulcerative colitis patients. This elevated expression is mirrored in the inflamed colonic mucosa of mice, wherein IL-36 receptor deficiency confirmed this pathway as a mediator of mucosal inflammation. Il36r-/- mice exhibited reduced disease severity in an acute DSS-induced model of colitis in association with decreased innate inflammatory cell infiltration to the colon lamina propria. Consistent with these data, infection with the enteropathogenic bacteria Citrobacter rodentium, resulted in reduced innate inflammatory cell recruitment and increased bacterial colonization in the colons of il36r-/- mice. Il36r-/- mice also exhibited altered T helper cell responses in this model, with enhanced Th17 and reduced Th1 responses, demonstrating that IL-36R signaling also regulates intestinal mucosal T-cell responses. These data identify a novel role for IL-36 signaling in colonic inflammation and indicate that the IL-36R pathway may represent a novel target for therapeutic intervention in IBD.

CD19xCD3 DART protein mediates human B-cell depletion in vivo in humanized BLT mice

Novel therapeutic strategies are needed for the treatment of hematologic malignancies; and bispecific antibody-derived molecules, such as dual-affinity re-targeting (DART) proteins, are being developed to redirect T cells to kill target cells expressing tumor or viral antigens. Here we present our findings of specific and systemic human B-cell depletion by a CD19xCD3 DART protein in humanized BLT mice. Administration of the CD19xCD3 DART protein resulted in a dramatic sustained depletion of human CD19+ B cells from the peripheral blood, as well as a dramatic systemic reduction of human CD19+ B-cell levels in all tissues (bone marrow, spleen, liver, lung) analyzed. When human CD8+ T cells were depleted from the mice, no significant B-cell depletion was observed in response to CD19xCD3 DART protein treatment, confirming that human CD8+ T cells are the primary effector cells in this in vivo model. These studies validate the use of BLT humanized mice for the in vivo evaluation and preclinical development of bispecific molecules that redirect human T cells to selectively deplete target cells.

Colonic tolerance develops in the iliac lymph nodes and can be established independent of CD103(+) dendritic cells.

Tolerance to harmless exogenous antigens is the default immune response in the gastrointestinal tract. Although extensive studies have demonstrated the importance of the mesenteric lymph nodes (MLN) and intestinal CD103+ dendritic cells (DCs) in driving small intestinal tolerance to protein antigen, the structural and immunological basis of colonic tolerance remain poorly understood. We show here that the caudal and iliac lymph nodes (ILN) are inductive sites for distal colonic immune responses and that colonic T cell-mediated tolerance induction to protein antigen is initiated in these draining lymph nodes and not in MLN. In agreement, colonic tolerance induction was not altered by mesenteric lymphadenectomy. Despite tolerance development, CD103+CD11b+ DCs which are the major migratory DC population in the MLN, and the tolerance-related retinoic acid-generating enzyme RALDH2 were virtually absent from the ILN. Administration of ovalbumin (OVA) to the distal colon did increase the number of CD11c+MHCIIhi migratory CD103−CD11b+ and CD103+CD11b− DCs in the ILN. Strikingly, colonic tolerance was intact in Batf3-deficient mice specifically lacking CD103+CD11b− DCs, suggesting that CD103− DCs in the ILN are sufficient to drive tolerance induction after protein antigen encounter in the distal colon. Altogether, we identify different inductive sites for small intestinal and colonic T-cell responses and reveal that distinct cellular mechanisms are operative to maintain tolerance at these sites.

A Case of Intestinal T Cell Lymphoma Presenting With Gastrointestinal Bleeding

Intestinal non-Hodgkin's T-cell lymphoma (NHTCL) is a rare, extranodal manifestation of lymphoma. We report a case of a 67 year old man with a history of dyslipidemia, drug-induced neutropenia and low grade B-cell lymphoproliferative disorder (most consistent with B cell chronic lymphocytic leukemia) who presented with a two week history of melena and abdominal pain. An EGD was performed and did not reveal a bleeding source. The patient continued to have dark stool, hematochezia and progressive anemia, thus a colonoscopy was performed. Multiple 5mm ulcers were noted in the cecum, ascending and transverse colon on colonoscopy. Biopsies revealed an aggressive NHTCL. Cytology showed strong expression of CD2 and CD3 with absent expression of CD5, CD7, CD34 and TdT. An aggressive proliferation index of 75-80% was noted.Figure 1Figure 2Figure 3FDG-PET scan of the abdomen/pelvis revealed multiple foci of increased metabolic activity located in the abdomen, particularly associated with small bowel loops and mesenteric lymph nodes. A bone marrow biopsy was obtained and displayed kappa restricted CD5 coexpression consistent with history of small B-cell lymphoma. Bone marrow showed no immunohistochemical evidence of NHTCL, confirming the diagnosis of primary intestinal lymphoma in accordance with Dawson criteria.1 The patient was evaluated by oncology services at two separate centers, and it was agreed that the patient had primary NHTCL. He was placed on a clinical trial of CHOP-Belinostat. Three months after diagnosis, the patient developed severe, acute abdominal pain and was found to have bowel perforation. Laparotomy revealed extensive tumors in the small bowel, of which several had perforated. His clinical course was complicated and after a prolonged ICU stay, he was transitioned to comfort measures only and died shortly thereafter. Primary gastrointestinal (GI) lymphoma is rare, comprising 1-4% of all GI malignancies, of which less than 10% are of T-cell origin.1 We suspect this patient had Enteropathy Associated T Cell Lymphoma (EATL). Patients with EATL typically present at age 60 with lesions predominantly affecting the small bowel (especially jejunum), and less frequently the colon and stomach.1,2 There is a strong association with celiac disease, seen in at least one third of patients diagnosed with EATL.2 This patient had no known history of celiac disease and no villous atrophy was seen on EGD. EATL is aggressive and carries a poor prognosis, optimal treatment is not known.3

Clinical analysis of 18 primary intestinal T cell lymphoma misdiagnosed as Crohn's disease

Objective To analyze clinical features and prognosis of primary intestinal T cell lymphoma (ITCL) which was misdiagnosed as Crohn's disease (CD), and summarized the key points of differentiation between ITCL and CD. Methods From January 2003 to January 2014, clinical data of patients with ITCL once misdiagnosed as CD were retrospectively analyzed, which included demographic, clinical, pathological and prognostic data. The data of 177 patients diagnosed as CD from January 2012 to January 2014 were collected. The demographic, clinical, pathological and prognostic data of these two groups were analyzed and compared. The continuous variables were compared with t test or Mann-Whitney U test, and the differences of classification variables between two groups were analyzed by Chi-square test or Fisher exact probability method. Results A total of 18 patients (17 males and one female) with ITCL misdiagnosed as CD were enrolled in the study, and the median age at diagnosis was 38.5 (28.8 to 42.5) years and the median duration of diagnosis was 6.00 (3.75 to 13.25) months. The common primary symptoms were abdominal pain (12/18), diarrhea (13/18) and anemia (13/18). Intestinal perforation was primary symptom in two cases (2/18). However, B symptoms of lymphoma was observed in 16 patients, which included fever in 13 patients, weight loss in 16 patients and night sweat in one patient. One or more serious complications appeared in 12 patients, which included intestinal perforation in nine patients, severe gastrointestinal bleeding in seven patients and intestinal obstruction in two patients. In 177 patients with CD, 104 patients were male (58.8%), and the median age at diagnosis was 22.0 (18.0 to 29.0) years. The primary symptoms were abdominal pain (88.7%, 157/177), diarrhea (55.9%, 99/177), anemia (63.8%, 113/177), fever (33.3%, 59/177) and weight loss (59.9%, 106/177). During the disease course, 30 patients (16.9%) had intestinal perforation (mainly chronic), 12 patients (6.8%) had intestinal obstruction and seven patients (4.0%) had severe gastrointestinal bleeding. Compared to CD patients, male patients were more common in ITCL (χ2=8.837, P<0.01), age at diagnosis was older (χ2=314.5, P<0.01), the disease course was shorter (U=385.0, P<0.01), weight loss (χ2=5.867, P<0.05) and fever (χ2=10.609, P<0.01) were more common in clinical symptoms and intestinal perforation and severe gastrointestinal bleeding were more common in complications (χ2=9.185, 24.908, both P <0.01). The lesions of ITCL were multiple lesions, small bowel involved in eight cases, colon involved in 14 cases and one case with esophagus involved. Under endoscopy examination, most lesions appeared as ulcerations and were segmentally distributed. Compared to CD, lymphocyte proliferation was more common in the intestinal histopathological findings of ITCL (17/18 vs 19.7%(35/177); χ2=42.844, P<0.01) and granuloma was rare (0 vs 42.8%(76/177), χ2=12.665, P<0.01). Among 18 patients with ITCL, nine received chemotherapy and the median survival time was two months. Conclusions Primary ITCL had non-specific symptoms and was easily misdiagnosed as CD. More attention should be paid to the differential diagnosis of the two disease. Key words: Primary intestinal T-cell lymphoma; Crohn disease; Clinical features