Allogeneic bone marrow transplantation for patients with treatment-refractory Crohn's Disease.

Background & aims: Durable remissions of Crohn's Disease (CD) have followed myeloablative conditioning therapy and allogeneic marrow transplantation. For patients with treatment-refractory disease, we used reduced-intensity conditioning to minimize toxicity, marrow from donors with low Polygenic Risk Scores for CD as cell sources, and protracted immune suppression to lower the risk of graft-versus-host disease (GVHD). Our aim was to achieve durable CD remissions while minimizing transplant-related complications. Methods: DNA from patients and their HLA-matched unrelated donors was genotyped and Polygenic Risk Scores calculated. Donor marrow was infused following non-myeloablative conditioning. Patient symptoms and endoscopic findings were documented at intervals after transplant. Results: We screened 807 patients, 143 of whom met eligibility criteria; 2 patients received allografts. Patient 1 had multiple complications and died at day 332 from respiratory failure. Patient 2 had resolution of CD symptoms until day 178 when CD recurred, associated with persistent host chimerism in both peripheral blood and intestinal mucosa. Withdrawal of immune suppression was followed by dominant donor immune chimerism in peripheral blood and resolution of CD findings. Over time, mucosal T-cells became donor-dominant. At 5 years after allografting, Patient 2 remained off all medications but had mild symptoms related to a jejunal stricture that required stricturoplasty at 6 years. At 8 years, she remains stable off medications. Conclusions: The kinetics of immunologic chimerism after allogeneic marrow transplantation for CD patients depends on the intensity of the conditioning regimen and the magnitude of immune suppression. One patient achieved durable improvement of her previously refractory CD only after establishing donor immunologic chimerism in intestinal mucosa. Her course provides proof-of-principal for allografting as a potential treatment for refractory CD, but an immunoablative conditioning regimen should be considered for future studies.(ClinicalTrials.gov, NCT01570348).

Human small intestine contains 2 functionally distinct regulatory T-cell subsets.

BACKGROUND: Regulatory T (Treg) CD4 cells in mouse gut are mainly specific for intestinal antigens and play an important role in the suppression of immune responses against harmless dietary antigens and members of the microbiota. However, information about the phenotype and function of Treg cells in the human gut is limited. OBJECTIVE: We performed a detailed characterization of Foxp3+ CD4 Treg cells in human normal small intestine (SI) as well as from transplanted duodenum and celiac disease lesions. METHODS: Treg cells and conventional CD4 T cells derived from SI were subjected to extensive immunophenotyping and their suppressive activity and ability to produce cytokines assessed. RESULTS: SI Foxp3+ CD4 T cells were CD45RA-CD127-CTLA-4+ and suppressed proliferation of autologous T cells. Approximately 60% of Treg cells expressed the transcription factor Helios. When stimulated, Helios-negative Treg cells produced IL-17, IFN-γ, and IL-10, whereas Helios-positive Treg cells produced very low levels of these cytokines. By sampling mucosal tissue from transplanted human duodenum, we demonstrated that donor Helios-negative Treg cells persisted for at least 1 year after transplantation. In normal SI, Foxp3+ Treg cells constituted only 2% of all CD4 T cells, while in active celiac disease, both Helios-negative and Helios-positive subsets expanded 5- to 10-fold. CONCLUSION: The SI contains 2 subsets of Treg cells with different phenotypes and functional capacities. Both subsets are scarce in healthy gut but increase dramatically in active celiac disease.

Exploring the role of the multiple sclerosis susceptibility gene CLEC16A in T cells.

C-type lectin-like domain family 16 member A (CLEC16A) is associated with autoimmune disorders, including multiple sclerosis (MS), but its functional relevance is not completely understood. CLEC16A is expressed in several immune cells, where it affects autophagic processes and receptor expression. Recently, we reported that the risk genotype of an MS-associated single nucleotide polymorphism in CLEC16A intron 19 is associated with higher expression of CLEC16A in CD4+ T cells. Here, we show that CLEC16A expression is induced in CD4+ T cells upon T cell activation. By the use of imaging flow cytometry and confocal microscopy, we demonstrate that CLEC16A is located in Rab4a-positive recycling endosomes in Jurkat TAg T cells. CLEC16A knock-down in Jurkat cells resulted in lower cell surface expression of the T cell receptor, however, this did not have a major impact on T cell activation response in vitro in Jurkat nor in human, primary CD4+ T cells.

CD4+ T cells persist for years in the human small intestine and display a TH1 cytokine profile.

Studies in mice and humans have shown that CD8+ T cell immunosurveillance in non-lymphoid tissues is dominated by resident populations. Whether CD4+ T cells use the same strategies to survey peripheral tissues is less clear. Here, examining the turnover of CD4+ T cells in transplanted duodenum in humans, we demonstrate that the majority of CD4+ T cells were still donor-derived one year after transplantation. In contrast to memory CD4+ T cells in peripheral blood, intestinal CD4+ TRM cells expressed CD69 and CD161, but only a minor fraction expressed CD103. Functionally, intestinal CD4+ TRM cells were very potent cytokine producers; the vast majority being polyfunctional TH1 cells, whereas a minor fraction produced IL-17. Interestingly, a fraction of intestinal CD4+ T cells produced granzyme-B and perforin after activation. Together, we show that the intestinal CD4+ T-cell compartment is dominated by resident populations that survive for more than 1 year. This finding is of high relevance for the development of oral vaccines and therapies for diseases in the gut.

WDR82/PNUTS-PP1 Prevents Transcription-Replication Conflicts by Promoting RNA Polymerase II Degradation on Chromatin.

Transcription-replication (T-R) conflicts cause replication stress and loss of genome integrity. However, the transcription-related processes that restrain such conflicts are poorly understood. Here, we demonstrate that the RNA polymerase II (RNAPII) C-terminal domain (CTD) phosphatase protein phosphatase 1 (PP1) nuclear targeting subunit (PNUTS)-PP1 inhibits replication stress. Depletion of PNUTS causes lower EdU uptake, S phase accumulation, and slower replication fork rates. In addition, the PNUTS binding partner WDR82 also promotes RNAPII-CTD dephosphorylation and suppresses replication stress. RNAPII has a longer residence time on chromatin after depletion of PNUTS or WDR82. Furthermore, the RNAPII residence time is greatly enhanced by proteasome inhibition in control cells but less so in PNUTS- or WDR82-depleted cells, indicating that PNUTS and WDR82 promote degradation of RNAPII on chromatin. Notably, reduced replication is dependent on transcription and the phospho-CTD binding protein CDC73 after depletion of PNUTS/WDR82. Altogether, our results suggest that RNAPII-CTD dephosphorylation is required for the continuous turnover of RNAPII on chromatin, thereby preventing T-R conflicts.

Resident memory CD8 T cells persist for years in human small intestine.

Resident memory CD8 T (Trm) cells have been shown to provide effective protective responses in the small intestine (SI) in mice. A better understanding of the generation and persistence of SI CD8 Trm cells in humans may have implications for intestinal immune-mediated diseases and vaccine development. Analyzing normal and transplanted human SI, we demonstrated that the majority of SI CD8 T cells were bona fide CD8 Trm cells that survived for >1 yr in the graft. Intraepithelial and lamina propria CD8 Trm cells showed a high clonal overlap and a repertoire dominated by expanded clones, conserved both spatially in the intestine and over time. Functionally, lamina propria CD8 Trm cells were potent cytokine producers, exhibiting a polyfunctional (IFN-γ+ IL-2+ TNF-α+) profile, and efficiently expressed cytotoxic mediators after stimulation. These results suggest that SI CD8 Trm cells could be relevant targets for future oral vaccines and therapeutic strategies for gut disorders.

Plasma Cells Are the Most Abundant Gluten Peptide MHC-expressing Cells in Inflamed Intestinal Tissues From Patients With Celiac Disease.

BACKGROUND & AIMS: Development of celiac disease is believed to involve the transglutaminase-dependent response of CD4+ T cells toward deamidated gluten peptides in the intestinal mucosa of individuals with specific HLA-DQ haplotypes. We investigated the antigen presentation process during this mucosal immune response. METHODS: We generated monoclonal antibodies (mAbs) specific for the peptide-MHC (pMHC) complex of HLA-DQ2.5 and the immunodominant gluten epitope DQ2.5-glia-α1a using phage display. We used these mAbs to assess gluten peptide presentation and phenotypes of presenting cells by flow cytometry and enzyme-linked immune absorbent spot (ELISPOT) in freshly prepared single-cell suspensions from intestinal biopsies from 40 patients with celiac disease (35 untreated and 5 on a gluten-free diet) as well as 18 subjects with confirmed noninflamed gut mucosa (controls, 12 presumed healthy, 5 undergoing pancreatoduodenectomy, and 1 with potential celiac disease). RESULTS: Using the mAbs, we detected MHC complexes on cells from intestinal biopsies from patients with celiac disease who consume gluten, but not from patients on gluten-free diets. We found B cells and plasma cells to be the most abundant cells that present DQ2.5-glia-α1a in the inflamed mucosa. We identified a subset of plasma cells that expresses B-cell receptors (BCR) specific for gluten peptides or the autoantigen transglutaminase 2 (TG2). Expression of MHC class II (MHCII) was not restricted to these specific plasma cells in patients with celiac disease but was observed in an average 30% of gut plasma cells from patients and controls. CONCLUSIONS: A population of plasma cells from intestinal biopsies of patients with celiac disease express MHCII; this is the most abundant cell type presenting the immunodominant gluten peptide DQ2.5-glia-α1a in the tissues from these patients. These results indicate that plasma cells in the gut can function as antigen-presenting cells and might promote and maintain intestinal inflammation in patients with celiac disease or other inflammatory disorders.

Transcriptional and functional profiling defines human small intestinal macrophage subsets.

Macrophages (Mfs) are instrumental in maintaining immune homeostasis in the intestine, yet studies on the origin and heterogeneity of human intestinal Mfs are scarce. Here, we identified four distinct Mf subpopulations in human small intestine (SI). Assessment of their turnover in duodenal transplants revealed that all Mf subsets were completely replaced over time; Mf1 and Mf2, phenotypically similar to peripheral blood monocytes (PBMos), were largely replaced within 3 wk, whereas two subsets with features of mature Mfs, Mf3 and Mf4, exhibited significantly slower replacement. Mf3 and Mf4 localized differently in SI; Mf3 formed a dense network in mucosal lamina propria, whereas Mf4 was enriched in submucosa. Transcriptional analysis showed that all Mf subsets were markedly distinct from PBMos and dendritic cells. Compared with PBMos, Mf subpopulations showed reduced responsiveness to proinflammatory stimuli but were proficient at endocytosis of particulate and soluble material. These data provide a comprehensive analysis of human SI Mf population and suggest a precursor-progeny relationship with PBMos.

Antibody-secreting plasma cells persist for decades in human intestine.

Plasma cells (PCs) produce antibodies that mediate immunity after infection or vaccination. In contrast to PCs in the bone marrow, PCs in the gut have been considered short lived. In this study, we studied PC dynamics in the human small intestine by cell-turnover analysis in organ transplants and by retrospective cell birth dating measuring carbon-14 in genomic DNA. We identified three distinct PC subsets: a CD19+ PC subset was dynamically exchanged, whereas of two CD19- PC subsets, CD45+ PCs exhibited little and CD45- PCs no replacement and had a median age of 11 and 22 yr, respectively. Accumulation of CD45- PCs during ageing and the presence of rotavirus-specific clones entirely within the CD19- PC subsets support selection and maintenance of protective PCs for life in human intestine.

CD1c-Expression by Monocytes - Implications for the Use of Commercial CD1c+ Dendritic Cell Isolation Kits.

Conventional dendritic cells (cDCs) comprise a heterogeneous population of cells that are important regulators of immunity and homeostasis. CD1c+ cDCs are present in human blood and tissues, and found to efficiently activate naïve CD4+ T cells. While CD1c is thought to specifically identify this subset of human cDCs, we show here that also classical and intermediate monocytes express CD1c. Accordingly, the commercial CD1c (BDCA-1)+ Dendritic Cell Isolation Kit isolates two distinct cell populations from blood: CD1c+CD14- cDCs and CD1c+CD14+ monocytes. CD1c+ cDCs and CD1c+ monocytes exhibited strikingly different properties, including their differential regulation of surface marker expression, their levels of cytokine production, and their ability to stimulate naïve CD4+ T cells. These results demonstrate that a commercial CD1c (BDCA-1)+ Dendritic Cell Isolation Kit isolates two functionally different cell populations, which has important implications for the interpretation of previously generated data using this kit to characterize CD1c+ cDCs.

Secretoneurin is a novel prognostic cardiovascular biomarker associated with cardiomyocyte calcium handling.

BACKGROUND: Secretoneurin (SN) levels are increased in patients with heart failure (HF), but whether SN provides prognostic information and influences cardiomyocyte function is unknown. OBJECTIVES: This study sought to evaluate the merit of SN as a cardiovascular biomarker and assess effects of SN on cardiomyocyte Ca(2+) handling. METHODS: We assessed the association between circulating SN levels and mortality in 2 patient cohorts and the functional properties of SN in experimental models. RESULTS: In 143 patients hospitalized for acute HF, SN levels were closely associated with mortality (n = 66) during follow-up (median 776 days; hazard ratio [lnSN]: 4.63; 95% confidence interval: 1.93 to 11.11; p = 0.001 in multivariate analysis). SN reclassified patients to their correct risk strata on top of other predictors of mortality. In 155 patients with ventricular arrhythmia-induced cardiac arrest, SN levels were also associated with short-term mortality (n = 51; hazard ratio [lnSN]: 3.33; 95% confidence interval: 1.83 to 6.05; p < 0.001 in multivariate analysis). Perfusing hearts with SN yielded markedly increased myocardial levels and SN internalized into cardiomyocytes by endocytosis. Intracellularly, SN reduced Ca(2+)/calmodulin (CaM)-dependent protein kinase II δ (CaMKIIδ) activity via direct SN-CaM and SN-CaMKII binding and attenuated CaMKIIδ-dependent phosphorylation of the ryanodine receptor. SN also reduced sarcoplasmic reticulum Ca(2+) leak, augmented sarcoplasmic reticulum Ca(2+) content, increased the magnitude and kinetics of cardiomyocyte Ca(2+) transients and contractions, and attenuated Ca(2+) sparks and waves in HF cardiomyocytes. CONCLUSIONS: SN provided incremental prognostic information to established risk indices in acute HF and ventricular arrhythmia-induced cardiac arrest.

The human-specific invariant chain isoform Iip35 modulates Iip33 trafficking and function.

The invariant chain (Ii) is a multifunctional protein, which has an essential role in the assembly and transport of major histocompatibility complex class II (MHC II) molecules. From a single gene, Ii is synthesized as four different isoforms: Iip33, Iip35, Iip41 and Iip43. Iip35 and Iip43 are specific to humans, and are formed due to an upstream alternative translation site, resulting in an N-terminal extension of 16 amino acids. This extension harbors a strong endoplasmic reticulum (ER) retention motif. Consequently, Iip35 or Iip43 expressed alone are retained in the ER, whereas Iip33 and Iip41 rapidly traffic to the endosomal pathway. Endogenously expressed, the four isoforms form mixed heterotrimers in the ER; however, mainly due to the absence of the Iip35/p43 isoforms in mice, little is known about how they influence general Ii function. In this study, we have co-expressed Iip33 and Iip35 in human cells with and without MHC II to gain a better understanding of how Iip35 isoform influences the cellular properties of Iip33. We find that Iip35 significantly affects the properties of Iip33. In the presence of Iip35, the transport of Iip33 out of the ER is delayed, its half-life is dramatically prolonged and its ability to induce enlarged endosomes and delayed endosomal maturation is abrogated.

Invariant chain as a vehicle to load antigenic peptides on human MHC class I for cytotoxic T-cell activation.

Protective T-cell responses depend on efficient presentation of antigen (Ag) in the context of major histocompatibility complex class I (MHCI) and class II (MHCII) molecules. Invariant chain (Ii) serves as a chaperone for MHCII molecules and mediates trafficking to the endosomal pathway. The genetic exchange of the class II-associated Ii peptide (CLIP) with antigenic peptides has proven efficient for loading of MHCII and activation of specific CD4(+) T cells. Here, we investigated if Ii could similarly activate human CD8(+) T cells when used as a vehicle for cytotoxic T-cell (CTL) epitopes. The results show that wild type Ii, and Ii in which CLIP was replaced by known CTL epitopes from the cancer targets MART-1 or CD20, coprecipitated with HLA-A*02:01 and mediated colocalization in the endosomal pathway. Furthermore, HLA-A*02:01-positive cells expressing CLIP-replaced Ii efficiently activated Ag-specific CD8(+) T cells in a TAP- and proteasome-independent manner. Finally, dendritic cells transfected with mRNA encoding IiMART-1 or IiCD20 primed naïve CD8(+) T cells. The results show that Ii carrying antigenic peptides in the CLIP region can promote efficient presentation of the epitopes to CTLs independently of the classical MHCI peptide loading machinery, facilitating novel vaccination strategies against cancer.

Differential regulation of Rab GTPase expression in monocyte-derived dendritic cells upon lipopolysaccharide activation: a correlation to maturation-dependent functional properties.

The regulation of Rab expression to modulate cellular function has recently been proposed. Dendritic cells are a prototypic example of cells that drastically alter their function in response to environmental cues by reducing endocytosis, secreting cytokines, changing surface protein repertoires and altering morphology and migration. This is not a binary event, but is subject to fluctuations through the activation process, termed maturation. Consequently, DCs transiently increase endocytosis and production of major histocompatibility complex class II molecules, and secrete inflammatory cytokines in infected tissues before migrating to secondary lymph nodes and releasing T cell polarizing factors. All these cellular processes rely on intracellular membrane transport, which is regulated by Rab family GTPases and their diverse effectors. Here we examine how the Rabs likely to be involved in these functions are regulated throughout DC maturation. We find that Rab expression is altered upon lipopolysaccharide-induced activation, and discuss how this correlates to the reported functions of these cells during maturation.

Selection of nanobodies that target human neonatal Fc receptor.

FcRn is a key player in several immunological and non-immunological processes, as it mediates maternal-fetal transfer of IgG, regulates the serum persistence of IgG and albumin, and transports both ligands between different cellular compartments. In addition, FcRn enhances antigen presentation. Thus, there is an intense interest in studies of how FcRn binds and transports its cargo within and across several types of cells, and FcRn detection reagents are in high demand. Here we report on phage display-selected Nanobodies that target human FcRn. The Nanobodies were obtained from a variable-domain repertoire library isolated from a llama immunized with recombinant human FcRn. One candidate, Nb218-H4, was shown to bind FcRn with high affinity at both acidic and neutral pH, without competing ligand binding and interfering with FcRn functions, such as transcytosis of IgG. Thus, Nb218-H4 can be used as a detection probe and as a tracker for visualization of FcRn-mediated cellular transport.

Differential regulation of MHC II and invariant chain expression during maturation of monocyte-derived dendritic cells.

DCs are potent initiators of adaptive immune responses toward invading pathogens. Upon reception of pathogenic stimuli, DCs initiate a complex differentiation program, culminating in mature DCs with an extreme capacity to activate naïve T cells. During this maturation, DCs reduce the synthesis and turnover of MHC II molecules. This allows for a stable population of MHC II, presenting peptides captured at the time and place of activation, thus provoking specific immune responses toward the activating pathogen. The efficient loading of antigenic peptides onto MHC II is vitally dependent on the accessory molecule Ii, which aids in the assembly of the MHC II α- and ß-chains in the ER and directs their trafficking to the endocytic compartments, where they encounter endocytosed antigen. However, Ii plays additional roles in DC function by influencing migration, antigen uptake, and processing. To examine the biosynthetic background for diverse Ii functions in DCs, we investigated mRNA and protein levels of Ii compared with MHC II in human moDCs during maturation using various stimuli. We find that the production of Ii did not correlate with that of MHC II and that mature DCs maintain abundant levels of Ii despite a reduced production of new MHC II.

Invariant chain increases the half-life of MHC II by delaying endosomal maturation.

Mounting adaptive immune responses requires the cell surface expression of major histocompatibility class II molecules (MHC II) loaded with antigenic peptide. However, in the absence of antigenic stimuli, the surface population of MHC II is highly dynamic and exhibits a high turnover. Several studies have focused on the regulation of MHC II, and it is now recognized that ubiquitination is one key mechanism operating in the turnover of MHC II in B cells and dendritic cells. Here, we describe how the invariant chain (Ii) can prolong the half-life of MHC II through its action on the endocytic pathway. We find that in cells expressing intermediate-to-high levels of Ii, the half-life of MHC II is increased, with MHC II accumulating in slowly-maturing endosomes. The accumulation in endosomes is not due to retention of new MHC II directed from the endoplasmatic reticulum, as also mature, not Ii associated, MHC II is preserved. We suggest that this alternative endocytic pathway induced by Ii would serve to enhance the rate, quantity and diversity of MHC II antigen presentation by concentrating MHC II into specialized compartments and reducing the need for new MHC II synthesis upon antigen encounter.

The protein phosphatase 1 regulator PNUTS is a new component of the DNA damage response.

The function of protein phosphatase 1 nuclear-targeting subunit (PNUTS)--one of the most abundant nuclear-targeting subunits of protein phosphatase 1 (PP1c)--remains largely uncharacterized. We show that PNUTS depletion by small interfering RNA activates a G2 checkpoint in unperturbed cells and prolongs G2 checkpoint and Chk1 activation after ionizing-radiation-induced DNA damage. Overexpression of PNUTS-enhanced green fluorescent protein (EGFP)--which is rapidly and transiently recruited at DNA damage sites--inhibits G2 arrest. Finally, γH2AX, p53-binding protein 1, replication protein A and Rad51 foci are present for a prolonged period and clonogenic survival is decreased in PNUTS-depleted cells after ionizing radiation treatment. We identify the PP1c regulatory subunit PNUTS as a new and integral component of the DNA damage response involved in DNA repair.

Cell-cycle-dependent binding kinetics for the early endosomal tethering factor EEA1.

Early endosomal antigen 1 (EEA1) is a cytosolic protein that specifically binds to early endosomal membranes where it has a crucial role in the tethering process leading to homotypic endosome fusion. Green fluorescent protein-tagged EEA1 (EEA1-GFP) was bound to the endosomal membrane throughout the cell cycle, and measurements using fluorescent recovery after photobleaching showed two fractions: one rapidly exchanging with the cytosolic pool, and the other with a long half-life. The exchange consists of a release and binding process, and we have separated these two by using GFP and photoactivable GFP. The release rate was identical to the exchange rate, showing that the dissociation characteristics determine the cycling of this molecule. During mitosis, we found that the dissociation rate was markedly accelerated and, in addition, the long-lived fraction was markedly reduced. This indicates that a fusion arrest in mitosis is not the result of EEA1 not binding to early endosomes, but rather due to the marked shift in membrane-binding characteristics. This might be a general mechanism to fine-tune and control tethering and fusion of early endosomes.