Wolfgang Holtmeier

γδ T Cells Link Innate and Adaptive Immune Responses

While most T cells use a CD3-associated alpha/beta T cell receptor as antigen recognition structure, a second population of T cells expresses the alternative gamma/delta T cell receptor. gamma/delta T cells are a minor population in the peripheral blood but constitute a major population among intestinal intraepithelial lymphocytes. Most gamma/delta T cells recognize ligands which are fundamentally different from the short peptides that are seen by alpha/beta T cells in the context of MHC class I or class II molecules. Thus, human Vdelta2 T cells recognize small bacterial phosphoantigens, alkylamines and synthetic aminobisphosphonates, whereas Vdelta1 T cells recognize stress-inducible MHC-related molecules MICA/B as well as several other ligands. At the functional level, gamma/delta T cells rapidly produce a variety of cytokines and usually exert potent cytotoxic activity, also towards many tumor cells. In this article, we discuss the role of gamma/delta T cells as a bridge between the innate and the adaptive immune system, based on the interpretation that gamma/delta T cells use their T cell receptor as a pattern recognition receptor. Our increasing understanding of the ligand recognition and activation mechanisms of gamma/delta T cells also opens new perspectives for the development of gamma/delta T cell-based immunotherapies.

Randomized, placebo‐controlled, double‐blind trial of Boswellia serrata in maintaining remission of Crohn's disease: Good safety profile but lack of efficacy

Background: Complementary therapies are frequently used by patients with inflammatory bowel disease (IBD). The aim of this study was to evaluate the efficacy and safety of long‐term therapy with a new Boswellia serrata extract (Boswelan, PS0201Bo) in maintaining remission in patients with Crohns disease (CD). Methods: In 22 German centers a double‐blind, placebo‐controlled, randomized, parallel study was performed. In all, 108 outpatients with CD in clinical remission were included. Patients were randomized to Boswelan (3×2 capsules/day; 400 mg each) or placebo for 52 weeks. The primary endpoint was the proportion of patients in whom remission was maintained throughout the 52 weeks. Secondary endpoints were time to relapse, changes of Crohns Disease Activity Index (CDAI), and IBD Questionnaire (IBDQ) scores. Results: The trial was prematurely terminated due to insufficient discrimination of drug and placebo with regard to the primary efficacy endpoint. A total of 82 patients were randomized to Boswelan (n = 42) or placebo (n = 40). Sixty‐six patients could be analyzed for efficacy. 59.9% of the actively treated patients and 55.3% of the placebo group stayed in remission (P = 0.85). The mean time to diagnosis of relapse was 171 days for the active group and 185 days for the placebo group (P = 0.69). With respect to CDAI, IBDQ, and laboratory measurements of inflammation, no advantages in favor of active treatment were detected. Regarding safety concerns, no disadvantages of taking the drug compared to placebo were observed. Conclusions: The trial confirmed good tolerability of a new Boswellia serrata extract, Boswelan, in long‐term treatment of CD. However, superiority versus placebo in maintenance therapy of remission could not be demonstrated. (Inflamm Bowel Dis 2011)

Development of γδ thymocyte subsets during prenatal and postnatal ontogeny

In this report, we describe 12 subpopulations of porcine γδ thymocytes based on their expression of CD1, CD2, CD4, CD8‐isoforms and CD45RC. Our data suggest that γδ thymocytes can be divided into two major families: (a) one large family of CD4–γδ thymocytes that could be further subdivided according to the CD2/CD8αα phenotype and (b) a small family of CD4+γδ thymocytes bearing CD8αβ and possessing certain unusual features in comparison with other γδ thymocytes. Maturation of γδ thymocytes within the CD4– family begins with proliferation of the CD2+ CD8– CD1+ CD45RC–γδ common precursor. This developmental stage is followed by diversification into the CD2+ CD8αα+, CD2+ CD8– and CD2– CD8– subsets. Their further maturation is accompanied by a loss of expression of CD1 and by increased expression of CD45RC. Therefore, individual subsets develop from CD1+ CD45RC– through CD1– CD45RC– into CD1– CD45RC+ cells. On the other hand, γδ thymocytes within the CD4+ family bear exclusively CD8αβ, always express CD1, but may coexpress CD45RC. These cells have no counterpart in the periphery. Our observations suggest that all peripheral CD8+γδ T cells express CD8αα and that two subsets of these cells differing in major histocompatibility complex II expression, occur. We propose that one subset acquires CD8αα in the thymus while the second acquires CD8αα as a result of stimulation in the periphery.

Identification of human cathepsin G as a functional target of boswellic acids from the anti-inflammatory remedy frankincense.

Frankincense preparations, used in folk medicine to cure inflammatory diseases, showed anti-inflammatory effectiveness in animal models and clinical trials. Boswellic acids (BAs) constitute major pharmacological principles of frankincense, but their targets and the underlying molecular modes of action are still unclear. Using a BA-affinity Sepharose matrix, a 26-kDa protein was selectively precipitated from human neutrophils and identified as the lysosomal protease cathepsin G (catG) by mass spectrometry (MALDI-TOF) and by immunological analysis. In rigid automated molecular docking experiments BAs tightly bound to the active center of catG, occupying the same part of the binding site as the synthetic catG inhibitor JNJ-10311795 (2-[3-{methyl[1-(2-naphthoyl)piperidin-4-yl]amino}carbonyl)-2-naphthyl]-1-(1-naphthyl)-2-oxoethylphosphonic acid). BAs potently suppressed the proteolytic activity of catG (IC50 of ∼600 nM) in a competitive and reversible manner. Related serine proteases were significantly less sensitive against BAs (leukocyte elastase, chymotrypsin, proteinase-3) or not affected (tryptase, chymase). BAs inhibited chemoinvasion but not chemotaxis of challenged neutrophils, and they suppressed Ca2+ mobilization in human platelets induced by isolated catG or by catG released from activated neutrophils. Finally, oral administration of defined frankincense extracts significantly reduced catG activities in human blood ex vivo vs placebo. In conclusion, we show that catG is a functional and pharmacologically relevant target of BAs, and interference with catG could explain some of the anti-inflammatory properties of frankincense.

Epithelial Defence by γδ T Cells

γδ T cells constitute a separate lineage of T lymphocytes which differ from conventional αβ T cells with regard to T cell receptor (TCR) repertoire and tissue localization. In murine skin, γδ T cells expressing a canonical Vγ5 TCR are abundant and contribute as so-called dendritic epidermal T cells to local immune surveillance. In humans, major subsets of γδ T cells are recognized on the basis of their TCR Vδ usage. While Vδ2 cells dominate in the peripheral blood, Vδ1 cells are preferentially localized in mucosal tissue including the intestinal epithelia. In this article we summarize basic features of intraepithelial γδ T cells and discuss their possible role in epithelial defence.

Proliferating intestinal γ/δ T cells recirculate rapidly and are a major source of the γ/δ T cell pool in the peripheral blood

The proliferation, recirculation and repertoire of gut‐derived γ/δ T cells were studied in pigs in vivo. Proliferating γ/δ T cells (detected by BrdU labeling) are present in all intestinal compartments. In the gut lymph ∼0.5% of all γ/δ T cells were proliferating. These gut‐derived BrdU+ γ/δ T cells re‐enter the intestinal tissues, and re‐appear in the intestinal lymph far more often than other cells: about 22% of i.v.‐injected BrdU+ γ/δ T cells were recovered again from the intestinal lymph within 72 h (compare with BrdU+ B cells 2%, and other BrdU+ T cells 10%). The contribution of the gut to the migrating γ/δ T cell pool in the blood became obvious: the proportion of BrdU+ γ/δ T cells was three‐times larger in control versus cannulated pigs. In 9‐month‐old pigs, clonally expanded T cells were identified in the intestine by complementarity‐determining region 3 spectratyping of TCR‐δ transcripts. Such expansions were not visible in the blood or intestinal lymph. The distribution of γ/δ T cells within the intestinal tract is likely to depend to a large degree on the proliferation and the migratory properties of these cells which are different to those of α/β T cells and B lymphocytes.

Development and Compartmentalization of the Porcine TCR δ Repertoire at Mucosal and Extraintestinal Sites: The Pig as a Model for Analyzing the Effects of Age and Microbial Factors

γδ T cells are an important component of the mucosal immune system. Previously, we have shown that the TCR δ repertoire in human intestine is polyclonal at birth and becomes increasingly restricted with age. In this study, we expand those studies to the pig which allows more extensive experiments including several organs. Tissues from different mucosal sites like the stomach, duodenum, ileum, Peyer’s patches, jejunum, and colon, and also extraintestinal sites like the lung, spleen, thymus and mesenteric lymph nodes, were obtained from conventionally reared pigs aged 2 wk to 5.5 years. In addition, tissues were also obtained from 10-wk-old specified pathogen- and germ-free pigs. TCRDV1-DV5 transcripts were amplified by RT-PCR after which complementarity-determining region 3 spectratyping was performed. Individual bands were excised from the gels and directly sequenced. The intestinal TCR δ repertoire showed increasing restriction with age and was highly oligoclonal in the adult 2- to 5.5-year-old pigs. In old pigs, we observed a striking compartmentalization. Different TCR δ repertoires were present between the lungs and the intestinal mucosa but also within different parts of the gastrointestinal tract. However, occasionally we observed identical TCR δ transcripts in the intestine and the lungs and shared clones could be detected also along the entire gastrointestinal tract. Thus, subsets of γδ T cells are likely to transport immunological information between different compartments of the immune system. Furthermore, these data support the hypothesis that in each mucosal site, different Ags are responsible for selecting and maintaining the γδ TCR over time.

Two Groups of Porcine TCRγδ+ Thymocytes Behave and Diverge Differently

Developmental pathways of γδ T cells are still unknown, largely because of the absence of recognized lineage-specific surface markers other than the TCR. We have shown that porcine γδ thymocytes can be divided into 12 subsets of the following two major groups: 1) CD4− γδ thymocytes that can be further subdivided according to their CD2/CD8αα phenotype, and 2) CD4+ γδ thymocytes that are always CD1+CD2+CD8αβ+ and have no counterpart in the periphery. In this study, we have analyzed γδ thymocyte subsets with respect to behavior during cultivation, cell cycle status, and lymphocyte-specific transcripts. The group of CD4− γδ thymocytes gives rise to all γδ T cells found in the periphery. Proliferating CD2+CD8−CD1+CD45RC− γδ thymocytes are a common precursor of this group. These precursors differentiate into CD2+CD8αα+, CD2+CD8−, and CD2−CD8− γδ T cell subsets, which subsequently mature by loss of CD1 and by eventual gain of CD45RC expression. In contrast, the group of CD4+ γδ thymocytes represents transient and independent subsets that are never exported from thymus as TCRγδ+ T cells. In accordance with the following findings, we propose that CD4+CD8αβ+ γδ thymocytes extinguish their TCRγδ expression and differentiate along the αβ T cell lineage program: 1) CD4+ γδ thymocytes are actively dividing; 2) CD4+ γδ thymocytes do not die, although their numbers decreased with prolonged cultivation; 3) CD4+ γδ thymocytes express transcripts for RAG-1, TdT, and TCRβ; and 4) CD4+ γδ thymocytes are able to alter their phenotype to TCRαβ+ thymocytes under appropriate culture conditions.

Increased Bioavailability of 11-Keto-β-Boswellic Acid Following Single Oral Dose Frankincense Extract Administration After a Standardized Meal in Healthy Male Volunteers: Modeling and Simulation Considerations for Evaluating Drug Exposures

T use of gum resin extracts from the Boswellia serrata tree (frankincense) for the treatment of inflammatory conditions originates from traditional Ayurvedic medicine in India. In vitro studies suggests that 11-keto-β-boswellic acid (KBA) and 3-acetyl-11-keto-β-boswellic acid (AKBA) are the 2 lead boswellic acids with inhibition most pronounced in vitro for 5-lipoxygenase, thus reducing leukotrienes as inflammatory lipid mediators. Therapeutic effects of frankincense extracts were shown in laboratory animal disease models as well as in clinical trials studying inflammatory bowel diseases, arthritis, asthma, and cancer. However, valid recommendations regarding clinical use are scarce—perhaps not surprising, as natural products such as frankincense vary in their pharmaceutical quality and are available as different formulations with varying ratios of boswellic acids, and wellcontrolled clinical trials with unequivocal evidence of efficacy are missing. This suggests a cautious use of frankincense; however, clinical practice suggests the opposite. Frankincense extracts have been used within cancer support groups, supported by German import statistics for Gufic H 15, a Boswellia serrata gum resin extract. In addition, 36% of surveyed German patients have treated their inflammatory bowel disease with B serrata extracts.

Regional variation of the αβ T cell repertoire in the colon of healthy individuals and patients with Crohn's disease

Clonally expanded T cells might be involved in the pathogenesis of Crohns disease (CD). To test the impact of CD on the regional distribution of expanded T cells, this study analyzed the T cell receptor beta (TCRB) repertoire within colonic biopsy specimens from 12 CD patients and 6 noninflammatory controls by TCR spectratyping. Migration characteristics of dominant CDR3 bands from different sites of the normal mucosa suggested focal, segmental, or ubiquitous spreading of individual expanded clones. Similar patterns were observed when inflamed and noninflamed areas of the colon of CD patients were compared, suggesting that regional expansion of T cells was more closely related to anatomic proximity than to local inflammatory activity. CDR3-sequence analysis of TCRBV12+ T cells, which were selectively expanded in the inflamed colon of 3 CD patients, failed to reveal a public CDR3 motif. Our data indicate the existence of distinct patterns of regional T cell expansions in the normal gut mucosa, which are not significantly disrupted by chronic intestinal inflammation. This does not exclude a pathogenic role of expanded T cells in CD through more subtle changes, but emphasizes the need to distinguish them from a discontinuous distribution of clonally expanded T cells in normal colon.