Itamar Goldstein

The immunogenic part of infliximab is the F(ab′)2, but measuring antibodies to the intact infliximab molecule is more clinically useful

Objectives To localise the immunogenic part of infliximab and evaluate the clinical usefulness of measuring antibodies against infliximab fragments. Design Observational study. Settings A specialised inflammatory bowel disease (IBD) centre in a tertiary hospital. Interventions Serum was collected from patients with IBD and controls. Antibodies against whole infliximab (ATI) and against the digested Fc, F(ab′)2 and F(ab′) fragments were measured by a specifically developed ELISA and by western blotting. A separate ELISA was used to determine infliximab levels in serum. Results 109 serum samples from 62 infliximab-treated patients were tested along with 64 control samples. Anti-F(ab′)2 antibodies were found in 28/42 (67%) samples with positive ATI, all from infliximab-exposed patients. Anti-F(ab′)2 antibodies were also present in 26 of the remaining 67 (39%) samples from exposed patients despite absent ATI. No specific anti-Fc antibodies were detected. Low trough infliximab level and high ATI level was found in 10/12 patients (83%) with complete loss of response to infliximab, but in only 5/14 patients (36%, p=0.02) who regained response to intensified infliximab regimen and in 2/24 patients (8%, p<0.001) in maintained remission while on 5 mg/kg/8 week infliximab treatment. Although Anti-F(ab′)2 antibodies showed similar test characteristics to ATI in patients losing response to infliximab, they were also detected in 61% of patients in maintained remission, thereby limiting their clinical usefulness. No cross reactivity to adalimumab was noted. Conclusions F(ab′)2 is the immunogenic fragment of infliximab. However, ATI level in serum—combined with measurement of trough infliximab level—is better correlated with the clinical response to infliximab or with its loss.

Transendothelial migration of lymphocytes mediated by intraendothelial vesicle stores rather than by extracellular chemokine depots

Chemokines presented by the endothelium are critical for integrin-dependent adhesion and transendothelial migration of naive and memory lymphocytes. Here we found that effector lymphocytes of the type 1 helper T cell (TH1 cell) and type 1 cytotoxic T cell (TC1 cell) subtypes expressed adhesive integrins that bypassed chemokine signals and established firm arrests on variably inflamed endothelial barriers. Nevertheless, the transendothelial migration of these lymphocytes strictly depended on signals from guanine nucleotide–binding proteins of the Gi type and was promoted by multiple endothelium-derived inflammatory chemokines, even without outer endothelial surface exposure. Instead, transendothelial migration–promoting endothelial chemokines were stored in vesicles docked on actin fibers beneath the plasma membranes and were locally released within tight lymphocyte-endothelial synapses. Thus, effector T lymphocytes can cross inflamed barriers through contact-guided consumption of intraendothelial chemokines without surface-deposited chemokines or extraendothelial chemokine gradients.

MDR1 expression identifies human melanoma stem cells

ABC transporters are often found to be inherently expressed in a wide variety of stem cells, where they provide improved protection from toxins. We found a subpopulation of human melanoma cells expressing multidrug-resistance gene product 1 (MDR1). This fraction co-expresses the ABC transporters, ABCB5 and ABCC2 in addition to the stem cell markers, nanog and human telomerase reverse transcriptase (hTERT). The clonogenicity and self-renewal capacity of MDR1(+) melanoma cells were investigated in single cell settings using the limiting dilution assay. We found that the MDR1(+) cells, isolated by FACS sorting, demonstrated a higher self-renewal capacity than the MDR1(-) fraction, a key stem cell feature. Moreover, MDR1(+) cells had higher ability to form spheres in low attachment conditions, a hallmark of cancer. In conclusion, these novel findings imply that the MDR1(+) cells represent melanoma stem cells and thus should be considered as a unique cellular target for future anti-melanoma therapies.

Cell contact-dependent acquisition of cellular and viral nonautonomously encoded small RNAs

In some organisms, small RNA pathways can act nonautonomously, with responses spreading from cell to cell. Dedicated intercellular RNA delivery pathways have not yet been characterized in mammals, although secretory compartments have been found to contain RNA. Here we show that, upon cell contact, T cells acquire from B cells small RNAs that can impact the expression of target genes in the recipient T cells. Synthetic microRNA (miRNA) mimetics, viral miRNAs expressed by infected B cells, and endogenous miRNAs could all be transferred into T cells. These mechanisms may allow small RNA-mediated communication between immune cells. The documented transfer of viral miRNAs raises the possible exploitation of these pathways for viral manipulation of the host immune response.

Intercellular exchange of proteins: The immune cell habit of sharing

The recent recognition of new types of cell–cell communication pathways challenges classic theories of cell autonomy. Evidence of functional “proteome mixing” among interacting cells, particularly immune cells, supports the notion that no cell is an island, and that even these “unsplittable” units are actually non‐autonomous. We summarize various mechanisms of intercellular transfer of proteins—trans‐endocytosis, trogocytosis, exosomal transport, shuttle through nanotubes, and cell‐contact‐dependent intercellular transfer of intracellular proteins including oncogenic Ras. These phenomena suggest exciting new possibilities for proteome research, focusing on system‐level proteomics that characterize cell contents and functions in the context of intercellular protein transfer.

Developmental tumourigenesis: NCAM as a putative marker for the malignant renal stem/progenitor cell population

During development, renal stem cells reside in the nephrogenic blastema. Wilms’ tumour (WT), a common childhood malignancy, is suggested to arise from the nephrogenic blastema that undergoes partial differentiation and as such is an attractive model to study renal stem cells leading to cancer initiation and maintenance. Previously we have made use of blastema‐enriched WT stem‐like xenografts propagated in vivo to define a ‘WT‐stem’ signature set, which includes cell surface markers convenient for cell isolation (frizzled homolog 2 [Drosophila] – FZD2, FZD7, G‐protein coupled receptor 39, activin receptor type 2B, neural cell adhesion molecule – NCAM). We show by fluorescence‐activated cell sorting analysis of sphere‐forming heterogeneous primary WT cultures that most of these markers and other stem cell surface antigens (haematopoietic, CD133, CD34, c‐Kit; mesenchymal, CD105, CD90, CD44; cancer, CD133, MDR1; hESC, CD24 and putative renal, cadherin 11), are expressed in WT cell sub‐populations in varying levels. Of all markers, NCAM, CD133 and FZD7 were constantly detected in low‐to‐moderate portions likely to contain the stem cell fraction. Sorting according to FZD7 resulted in extensive cell death, while sorted NCAM and CD133 cell fractions were subjected to clonogenicity assays and quantitative RT‐PCR analysis, exclusively demonstrating the NCAM+ fraction as highly clonogenic, overexpressing the WT ‘stemness’ genes and topoisomerase2A (TOP2A), a bad prognostic marker for WT. Moreover, treatment of WT cells with the topoisomerase inhibitors, Etoposide and Irinotecan resulted in down‐regulation of TOP2A along with NCAM and WT1. Thus, we suggest NCAM as a marker for the WT progenitor cell population. These findings provide novel insights into the cellular hierarchy of WT, having possible implications for future therapeutic options.

Early preservation of effector functions followed by eventual T cell memory depletion: a model for the delayed onset of the effect of thiopurines

Objective: The onset of the effect of thiopurines is delayed for several months. The aim of this study was to investigate immune mechanisms for this delay. Methods: The effects of thiopurines on human peripheral blood T cells and on lamina propria lymphocytes were investigated for apoptosis induction by Annexin V/propidium iodide (PI) and for cytokine secretion by intracellular staining and ELISA assays. To investigate the mechanism of the effect of thiopurines in vivo, Balb/C mice were co-immunised with HEL/OVA (hen egg lysozyme/ovalbumin) antigens, and then repeatedly challenged by HEL only, while being treated by mercaptopurine or vehicle alone for either 4 or 20 weeks. The memory response of CD4+ splenocytes towards HEL/OVA was then determined by CFSE (carboxyfluorescein succinimidyl ester) dilution. Results: Thiopurines arrested the proliferation of stimulated T cells but did not enhance the apoptosis of either resting T cells or activated T cells until day 5 poststimulation. Despite the proliferation arrest, stimulated T cells successfully differentiated into effector cells, as evidenced by their capacity for proinflammatory cytokine secretion, potent adhesion and cytotoxicity. Prolonged mercaptopurine treatment of mice for 20 weeks selectively reduced the CD4+ memory response to a repeatedly encountered HEL antigen, but did not affect the T cell memory pool to the previously presented OVA antigen. A shorter, 4 weeks, treatment with mercaptopurine did not inhibit the memory response to either antigen. Conclusions: T cells arrested from cycling by thiopurines can still differentiate into potent effector cells capable of propagating the inflammatory process. Thiopurine treatment results in depletion of antigen-specific memory T cells, but this effect is dependent upon repeated encounters with the antigen over a prolonged time course.

Reduced central tolerance in Omenn syndrome leads to immature self-reactive oligoclonal T cells.

BACKGROUND Omenn syndrome (OS) is characterized by a peculiar severe T-cell immune deficiency associated with autoimmunelike manifestations. Dysregulations of the central and peripheral immune tolerance, mediated by the protein autoimmune regulator (AIRE) and regulatory T cells, respectively, were proposed as possible mechanisms of this aberrant inflammatory process. OBJECTIVE We studied mechanisms of central and peripheral tolerance in patients with OS and also examined the gene expression profile associated with OS features. METHODS T-cell receptor diversity, DNA rearrangement, and the expression of AIRE and forkhead box P3 mRNA as well as the expression of regulatory T cells in cells obtained from patients with OS were studied. Characterization of gene expression in these cells was carried out by using the TaqMan Low-Density Array. RESULTS Transcript expression of peripheral blood AIRE but not forkhead box P3 was reduced in patients with OS. The expression of natural killer T and regulatory T cells was normal, although the latter showed an abnormal CD4-negative population. Patients with OS have oligoclonal T cells with limited DNA recombination activity, including the presence of early but not late T-cell maturation events, regardless of the genetic defect underlying the syndrome. The transcriptional profile associated with OS features reveals significant changes in 25.5% of the tested genes compared with normal control. CONCLUSION Our findings suggest that T-cell oligoclonal expansion in OS emanates from an incomplete block before the maturation stage of negative selection, which may explain escape of autoreactive T cells from the thymus. Dysregulated genes in patients with OS are closely involved with self-tolerance and autoimmunity.

α1β1 Integrin+ and Regulatory Foxp3+ T Cells Constitute Two Functionally Distinct Human CD4+ T Cell Subsets Oppositely Modulated by TNFα Blockade

The expression of the collagen receptor α1β1 integrin (VLA-1) on CD4+ T cells is largely restricted to CCR7−CD45RO+ cells that localize to inflamed tissues. Moreover, neutralizing α1 integrin, in vivo, has been shown to compromise cell-mediated immunity. Our current study shows that the expression of VLA-1 on human CD4+ T cells is restricted to conventional effectors. In contrast, Foxp3+ T regulatory cells (Tregs) do not express this receptor. Moreover, Foxp3 or VLA-1 expression remained a mutually exclusive event in CD4+ T cells even upon polyclonal anti-CD3-induced activation. Because TNFα blockade ameliorates certain T cell-dependent autoimmune disorders in humans, we investigated, in vitro, whether neutralizing TNFα affected the balance between the proinflammatory VLA-1+ effectors and the counteracting Tregs. We found that anti-CD3 stimulation of freshly isolated PBL from healthy individuals, coupled with continuous TNFα blockade, inhibited the typical activation-dependent generation of CD4+VLA-1+ Th1 cells. In contrast, it augmented the outgrowth of VLA-1neg/dimCD25high and Foxp3+CD4+ T cells. Indeed, repeated anti-CD3 stimulation coupled with TNFα blockade generated CD4+ T cell lines enriched for VLA-1−Foxp3+ Tregs. Importantly, these CD4+ T cells displayed potent suppressive functions toward autologous CD4+ PBL, including the suppression of the activation-dependent induction of VLA-1+ effectors. Thus, we propose a novel mechanism by which anti-TNFα therapy may restore self-tolerance, by shifting the balance between VLA-1+ effectors and Foxp3+ Tregs, during immune activation, in favor of the latter suppressor cell population.

Expression of the α1β1 integrin, VLA-1, marks a distinct subset of human CD4 + memory T cells

The α1β1 integrin, very late antigen-1 (VLA-1), is a collagen receptor expressed in many CD4+ T cells localizing to inflamed tissues. Here we show that the expression of VLA-1 is a stable marker of a distinct subset of CD4+ memory T cells. Thus, in human peripheral blood lymphocytes (PBLs), approximately 1–4% of the CD4+ T cells express VLA-1, and following T cell receptor activation ex vivo, the percentage of VLA-1+ cells increases within the CD45RO+ population. Importantly, the activated VLA-1+ and VLA-1– cells can be isolated and maintained in culture as phenotypically stable subsets. Functionally, CD4+ memory T cells, operationally defined as the cells that divide rapidly following stimulation with a recall antigen, are highly enriched for VLA-1+ cells. Moreover, depletion of the small fraction of VLA-1+ cells present in CD4+ PBLs prior to stimulation significantly abrogated the proliferative response to recall antigens. Notably, the VLA-1+ cells in fresh CD4+ PBLs are composed of resting CD45RO+/RA–, CCR7–, CD62L+, CD25–, and VLA-4hi cells. Interestingly, this VLA-1+ subset is enriched for Th1-type cells, and Th1-polarizing conditions during T cell activation favor the emergence of VLA-1+ cells. Thus, VLA-1 expression is a stable marker of a unique subset of human memory CD4+ T cells that predominantly differentiates into Th1 cells.