Module walking using an SH3-like cell-wall-binding domain leads to a new GH184 family of muramidases.

Muramidases (also known as lysozymes) hydrolyse the peptidoglycan component of the bacterial cell wall and are found in many glycoside hydrolase (GH) families. Similar to other glycoside hydrolases, muramidases sometimes have noncatalytic domains that facilitate their interaction with the substrate. Here, the identification, characterization and X-ray structure of a novel fungal GH24 muramidase from Trichophaea saccata is first described, in which an SH3-like cell-wall-binding domain (CWBD) was identified by structure comparison in addition to its catalytic domain. Further, a complex between a triglycine peptide and the CWBD from T. saccata is presented that shows a possible anchor point of the peptidoglycan on the CWBD. A `domain-walking' approach, searching for other sequences with a domain of unknown function appended to the CWBD, was then used to identify a group of fungal muramidases that also contain homologous SH3-like cell-wall-binding modules, the catalytic domains of which define a new GH family. The properties of some representative members of this family are described as well as X-ray structures of the independent catalytic and SH3-like domains of the Kionochaeta sp., Thermothielavioides terrestris and Penicillium virgatum enzymes. This work confirms the power of the module-walking approach, extends the library of known GH families and adds a new noncatalytic module to the muramidase arsenal.

Fungal lysozyme leverages the gut microbiota to curb DSS-induced colitis.

Colitis is characterized by colonic inflammation and impaired gut health. Both features aggravate obesity and insulin resistance. Host defense peptides (HDPs) are key regulators of gut homeostasis and generally malfunctioning in above-mentioned conditions. We aimed here to improve bowel function in diet-induced obesity and chemically induced colitis through daily oral administration of lysozyme, a well-characterized HDP, derived from Acremonium alcalophilum.C57BL6/J mice were fed either low-fat reference diet or HFD ± daily gavage of lysozyme for 12 weeks, followed by metabolic assessment and evaluation of colonic microbiota encroachment. To further evaluate the efficacy of intestinal inflammation, we next supplemented chow-fed BALB/c mice with lysozyme during Dextran Sulfate Sodium (DSS)-induced colitis in either conventional or microbiota-depleted mice. We assessed longitudinal microbiome alterations by 16S amplicon sequencing in both models.Lysozyme dose-dependently alleviated intestinal inflammation in DSS-challenged mice and further protected against HFD-induced microbiota encroachment and fasting hyperinsulinemia. Observed improvements of intestinal health relied on a complex gut flora, with the observation that microbiota depletion abrogated lysozyme's capacity to mitigate DSS-induced colitis.Akkermansia muciniphila associated with impaired gut health in both models, a trajectory that was mitigated by lysozyme administration. In agreement with this notion, PICRUSt2 analysis revealed specific pathways consistently affected by lysozyme administration, independent of vivarium, disease model and mouse strain.Taking together, lysozyme leveraged the gut microbiota to curb DSS-induced inflammation, alleviated HFD-induced gastrointestinal disturbances and lowered fasting insulin levels in obese mice. Collectively, these data present A. alcalophilum-derived lysozyme as a promising candidate to enhance gut health.

A muramidase from Acremonium alcalophilum hydrolyse peptidoglycan found in the gastrointestinal tract of broiler chickens.

This study evaluates peptidoglycan hydrolysis by a microbial muramidase from the fungus Acremonium alcalophilum in vitro and in the gastrointestinal tract of broiler chickens. Peptidoglycan used for in vitro studies was derived from 5 gram-positive chicken gut isolate type strains. In vitro peptidoglycan hydrolysis was studied by three approaches: (a) helium ion microscopy to identify visual phenotypes of hydrolysis, (b) reducing end assay to quantify solubilization of peptidoglycan fragments, and (c) mass spectroscopy to estimate relative abundances of soluble substrates and reaction products. Visual effects of peptidoglycan hydrolysis could be observed by helium ion microscopy and the increase in abundance of soluble peptidoglycan due to hydrolysis was quantified by a reducing end assay. Mass spectroscopy confirmed the release of hydrolysis products and identified muropeptides from the five different peptidoglycan sources. Peptidoglycan hydrolysis in chicken crop, jejunum, and caecum samples was measured by quantifying the total and soluble muramic acid content. A significant increase in the proportion of the soluble muramic acid was observed in all three segments upon inclusion of the microbial muramidase in the diet.

Fungal GH25 muramidases: New family members with applications in animal nutrition and a crystal structure at 0.78Å resolution.

Muramidases/lysozymes hydrolyse the peptidoglycan component of the bacterial cell wall. They are found in many of the glycoside hydrolase (GH) families. Family GH25 contains muramidases/lysozymes, known as CH type lysozymes, as they were initially discovered in the Chalaropsis species of fungus. The characterized enzymes from GH25 exhibit both ß-1,4-N-acetyl- and ß-1,4-N,6-O-diacetylmuramidase activities, cleaving the ß-1,4-glycosidic bond between N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) moieties in the carbohydrate backbone of bacterial peptidoglycan. Here, a set of fungal GH25 muramidases were identified from a sequence search, cloned and expressed and screened for their ability to digest bacterial peptidoglycan, to be used in a commercial application in chicken feed. The screen identified the enzyme from Acremonium alcalophilum JCM 736 as a suitable candidate for this purpose and its relevant biochemical and biophysical and properties are described. We report the crystal structure of the A. alcalophilum enzyme at atomic, 0.78 Å resolution, together with that of its homologue from Trichobolus zukalii at 1.4 Å, and compare these with the structures of homologues. GH25 enzymes offer a new solution in animal feed applications such as for processing bacterial debris in the animal gut.

Glucagon-like Peptide 1 Conjugated to Recombinant Human Serum Albumin Variants with Modified Neonatal Fc Receptor Binding Properties. Impact on Molecular Structure and Half-Life.

Glucagon-like peptide 1 (GLP-1) is a small incretin hormone stimulated by food intake, resulting in an amplification of the insulin response. Though GLP-1 is interesting as a drug candidate for the treatment of type 2 diabetes mellitus, its short plasma half-life of <3 min limits its clinical use. A strategy for extending the half-life of GLP-1 utilizes the long half-life of human serum albumin (HSA) by combining the two via chemical conjugation or genetic fusion. HSA has a plasma half-life of around 21 days because of its interaction with the neonatal Fc receptor (FcRn) expressed in endothelial cells of blood vessels, which rescues circulating HSA from lysosomal degradation. We have conjugated GLP-1 to C34 of native sequence recombinant HSA (rHSA) and two rHSA variants, one with increased and one with decreased binding affinity for human FcRn. We have investigated the impact of conjugation on FcRn binding affinities, GLP-1 potency, and pharmacokinetics, combined with the solution structure of the rHSA variants and GLP-1-albumin conjugates. The solution structures, determined by small-angle X-ray scattering, show the GLP-1 pointing away from the surface of rHSA. Combining the solution structures with the available structural information about the FcRn and GLP-1 receptor obtained from X-ray crystallography, we can explain the observed in vitro and in vivo behavior. We conclude that the conjugation of GLP-1 to rHSA does not affect the interaction between rHSA and FcRn, while the observed decrease in the potency of GLP-1 can be explained by a steric hindrance of binding of GLP-1 to its receptor.

Direct demonstration of a neonatal Fc receptor (FcRn)-driven endosomal sorting pathway for cellular recycling of albumin.

Albumin is the most abundant plasma protein involved in the transport of many compounds, such as fatty acids, bilirubin, and heme. The endothelial cellular neonatal Fc receptor (FcRn) has been suggested to play a central role in maintaining high albumin plasma levels through a cellular recycling pathway. However, direct mapping of this process is still lacking. This work presents the use of wild-type and engineered recombinant albumins with either decreased or increased FcRn affinity in combination with a low or high FcRn-expressing endothelium cell line to clearly define the FcRn involvement, intracellular pathway, and kinetics of albumin trafficking by flow cytometry, quantitative confocal microscopy, and an albumin-recycling assay. We found that cellular albumin internalization was proportional to FcRn expression and albumin-binding affinity. Albumin accumulation in early endosomes was independent of FcRn-binding affinity, but differences in FcRn-binding affinities significantly affected the albumin distribution between late endosomes and lysosomes. Unlike albumin with low FcRn-binding affinity, albumin with high FcRn-binding affinity was directed less to the lysosomes, suggestive of FcRn-directed albumin salvage from lysosomal degradation. Furthermore, the amount of recycled albumin in cell culture media corresponded to FcRn-binding affinity, with a ∼3.3-fold increase after 1 h for the high FcRn-binding albumin variant compared with wild-type albumin. Together, these findings uncover an FcRn-dependent endosomal cellular-sorting pathway that has great importance in describing fundamental mechanisms of intracellular albumin recycling and the possibility to tune albumin-based therapeutic effects by FcRn-binding affinity.

Secretion, blood levels and cutaneous expression of TL1A in psoriasis patients.

TL1A is a TNF-like cytokine which has been shown to co-stimulate TH1 and TH17 responses during chronic inflammation. The expression of this novel cytokine has been investigated in inflammatory disorders like rheumatoid arthritis and inflammatory bowel disease, but little is known about expression and induction in psoriasis. Indeed, the pathogenesis in psoriasis is still not fully understood and it is speculated that cytokines other than TNF-α are important in subsets of patients. Also, for patients with severe disease that are treated with systemic anti-TNF-α blockade, novel candidates to be used as disease and response biomarkers are of high interest. Here, we demonstrate TL1A expression in biopsies from psoriatic lesions. Also, we investigated spontaneous and induced TL1A secretion from PBMCs and blood levels from a cohort of psoriasis patients. Here, increased spontaneous secretion from PBMCs was observed as compared to healthy controls and a small subset of patients had highly elevated TL1A in the blood. Interestingly, activation of PBMCs with various cytokines showed a decreased sensitivity for TL1A activation in psoriasis patients compared to healthy controls.TL1A levels in blood and biopsies could not be correlated with disease activity with this patient cohort. Thus, additional large-scale studies are warranted to investigate TL1A as a biomarker.

Enteroantigen (eAg)-binding B lymphocytes in the mouse - phenotype, distribution, function and eAg-specific antibody secretion.

Studies reporting beneficial effects of B lymphocytes in autoimmune diseases have been accumulating and a regulatory role for certain B cell subsets is hence getting more and more recognition. Recently, B cells were shown to exhibit a regulatory effect in a T cell transfer model of colitis. Here, B cells exposed to enteroantigen (eAg) ex vivo abrogated the colitogenic effect if co-transplanted with Treg-depleted (CD4+CD25-) T cells into severe combined immune deficiency (SCID) mice. These data may imply a role for B cells that bind eAg (eAg+ B cells) in the immunopathology of colitis. Here, we report the detection of a subset of eAg+ B cells, including both B2 and B1 lineages, and show that these cells are present in all peripheral lymphoid organs of the mouse including the peritoneal cavity. eAg+ B cells are far more efficient as eAg-presenting cells than unfractionated splenocytes or eAg- B cells in causing proliferation of eAg-specific T cells. In comparison with eAg- B cells, eAg+ B cells secrete a significant amount of IL-10 in vitro, suggesting an anti-inflammatory potential. Compared with wild-type B cells, B cell receptor (BCR) transgenic, hen egg lysozyme-specific B cells show inferior eAg binding and T cell stimulatory activity suggesting involvement of the BCR in eAg binding and processing. After activation of CD19(+) B cells by eAg and hybridization with hypoxanthine-aminopterin-thymidine (HAT) sensitive ×63 lymphoma cells followed by cloning at limiting dilution conditions, around 10% of the hybridoma cells secrete eAg-specific antibodies.

B7-H4-Ig treatment of normal mice changes lymphocyte homeostasis and increases the potential of regulatory T cells.

Enteroantigens (eAgs) drive tolerogenic and inflammatory immune responses in the gut and are of importance for sustained immune homeostasis in colonic mucosa. Decline of regulatory activity in the gut mucosa might result in chronic colitis. B7-H4 is a co-inhibitory receptor expressed by professional antigen-presenting cells. By delivering signal 2 during T cell activation, it inhibits T cell proliferation and inflammation. In this study, we have used a newly developed B7-H4-Ig fusion protein and evaluated its effect on eAg-activated effector and regulatory T cells (Treg) in vitro and in vivo. T cells were recovered from the mesenteric lymph nodes (MLNs) of untreated or B7-H4-Ig-treated BALB/c mice. Treatment of cells in vitro did neither affect the proliferation of effector T cells nor the function of Tregs. In vivo, B7-H4 treatment increased the total number of MLN-derived CD4⁺ and CD8⁺ T cell subsets as well as the functional activity of MLN-derived Tregs, whereas the proliferative activity of eAg or alloantigen specific effector T cells was not influenced, although treatment resulted in less secretion of inflammatory cytokines and chemokines from these cells. B7-H4-Ig treatment of severe combined immune-deficient (SCID) mice undergoing T cell transfer colitis did not influence the course of disease probably reflecting the lack of Tregs in this model of chronic colitis. In conclusion, we show that treatment with B7-H4-Ig in vivo changes lymphocyte homeostasis and increases the regulatory potential in normal mice, but does not affect the course of disease development in SCID mice undergoing T cell transfer colitis.

TH17 cell induction and effects of IL-17A and IL-17F blockade in experimental colitis.

BACKGROUND: T helper (TH) 17 cells are believed to play a pivotal role in development of inflammatory bowel disease, and their contribution to intestinal inflammation has been studied in various models of colitis. TH17 cells produce a range of cytokines, some of which are potential targets for immunotherapy. However, blockade of IL-17A alone with secukinumab was not effective in Crohn's disease. In this regard, the pathogenic impact of IL-17A versus IL-17F during intestinal inflammation is still unresolved. METHODS: Development of IFN-γ-producing, IL-17A-producing, and IL-17F-producing CD4 T cells was analyzed in the CD4CD25 T-cell transfer model of colitis at varying degrees of colitis. The pathogenic roles of IL-17A and IL-17F were investigated by treating colitic mice with neutralizing antibodies against these 2 cytokines. RESULTS: We found that colitis development was associated with an increase in IL-17A-producing TH17 cells in spleen, mesenteric lymph nodes, and lamina propria. In contrast, the relative abundance of IFN-γ-producing TH1 cell was stable in all 3 organs during progression of colitis, and the frequency of IFN-γIL-17A double-positive cells declined in spleen and mesenteric lymph node but not in lamina propria. IL-17F was coexpressed in TH17 cells and IFN-γIL-17A double positive but not in TH1 cells and its expression inversely correlated with colitis development. In vivo neutralization of both IL-17A and IL-17F ameliorated colitis in particular at early administration, whereas neutralization of IL-17A or IL-17F alone was inefficient. CONCLUSIONS: TH17 cell development correlates with colitis progression, and concurrent neutralization of their cytokine products IL-17A and IL-17F ameliorates intestinal inflammation. These findings suggest combined IL-17A and IL-17F blockade as a potential strategy in inflammatory bowel disease therapy.

B cells exposed to enterobacterial components suppress development of experimental colitis.

BACKGROUND: B cells positively contribute to immunity by antigen presentation to CD4(+) T cells, cytokine production, and differentiation into antibody secreting plasma cells. Accumulating evidence implies that B cells also possess immunoregulatory functions closely linked to their capability of IL-10 secretion. METHODS: Colitis development was followed in CD4(+) CD25(-) T cell transplanted SCID mice co-transferred with B cells exposed to an enterobacterial extract (ebx-B cells). B and T cell cytokine expression was measured by flow cytometry and enzyme-linked immunosorbent assay (ELISA). RESULTS: We demonstrate that splenic B cells exposed to ebx produce large amounts of IL-10 in vitro and express CD1d and CD5 previously known to be associated with regulatory B cells. In SCID mice transplanted with colitogenic CD4(+) CD25(-) T cells, co-transfer of ebx-B cells significantly suppressed development of colitis. Suppression was dependent on B cell-derived IL-10, as co-transfer of IL-10 knockout ebx-B cells failed to suppress colitis. Ebx-B cell-mediated suppression of colitis was associated with a decrease in interferon gamma (IFN-γ)-producing T(H) 1 cells and increased frequencies of Foxp3-expressing T cells. CONCLUSIONS: These data demonstrate that splenic B cells exposed to enterobacterial components acquire immunosuppressive functions by which they can suppress development of experimental T cell-mediated colitis in an IL-10-dependent way.

Consumption of probiotics increases the effect of regulatory T cells in transfer colitis.

BACKGROUND: Probiotics may alter immune regulation. Recently, we showed that the probiotic bacteria Lactobacillus acidophilus NCFM™ influenced the activity of regulatory T cells (Tregs) in vitro. The aim of the present work was to demonstrate if L. acidophilus NCFM™ also affects the function of Tregs in vivo. METHODS: Development of colitis after transfer of CD4+CD25- T cells and protection from colitis by Tregs was studied in immunodeficient SCID mice which were simultaneously tube-fed with L. acidophilus NCFM™ or L. salivarius Ls-33 for 5 weeks. RESULTS: Probiotic-fed SCID mice transplanted with low numbers of Tregs in addition to the disease-inducing T cells were completely protected from colitis. This was in contrast to the control group, which showed intermediate levels of inflammation. In addition, feeding with probiotics lowered serum levels of inflammatory cytokines in both colitic mice and in mice protected from colitis by Tregs. Gene expression patterns of rectum samples of protected mice that receive either one of the probiotics showed a closer resemblance to naïve SCID mice than did patterns of the control group. The mechanism of action of the probiotics appears to be an indirect effect by inducing a Treg-favorable environment rather than a direct effect on the Tregs. CONCLUSIONS: L. acidophilus NCFM™ and L. salivarius Ls-33 feeding of SCID mice increases the in vivo effect of Tregs, resulting in a gene expression pattern in the rectum resembling that of the naïve SCID mouse.

Enteroantigen-presenting B cells efficiently stimulate CD4(+) T cells in vitro.

BACKGROUND: Presentation of enterobacterial antigens by antigen-presenting cells and activation of enteroantigen-specific CD4(+) T cells are considered crucial steps in inflammatory bowel disease (IBD) pathology. The detrimental effects of such CD4(+) T cells have been thoroughly demonstrated in models of colitis. Also, we have previously established an in vitro assay where murine enteroantigen-specific colitogenic CD4(+) CD25(-) T cells are activated by splenocytes pulsed with an enterobacterial extract. METHODS: CD4(+) CD25(-) T cells were stimulated in vitro with various kinds of enterobacterial extract-pulsed antigen-presenting cells. T-helper phenotypes were detected by flow cytometry. RESULTS: We found that enteroantigen-pulsed splenic B cells possess a significantly higher and more sustained T cell stimulatory capacity than similarly pulsed splenic dendritic cells (DCs) measured by the level of enteroantigen-specific CD4(+) CD25(-) T cell proliferation. In support of this, we observed upregulation of classic maturation markers in B cells following incubation with enterobacterial antigens. Peritoneal and mesenteric lymph node-derived B cells were equally effective as enteroantigen-presenting stimulator cells. B cells greatly expanded the number of stimulated CD4(+) T cells, which acquired a T(H) 2 phenotype. Interestingly, regulatory T cells were primarily activated by enteroantigen-pulsed B cells but not by similarly pulsed DCs. CONCLUSIONS: We conclude that B cells are superior stimulators of enteroantigen-specific CD4(+) T cells in vitro, favoring T(H) 2 polarization. Thus, enteroantigen-processing and -presentation by B cells instead of by DCs might have opposing consequences for IBD development.

Antigen-presenting cells exposed to Lactobacillus acidophilus NCFM, Bifidobacterium bifidum BI-98, and BI-504 reduce regulatory T cell activity.

BACKGROUND: The effect in vitro of six different probiotic strains including Lactobacillus acidophilus NCFM, Lactobacillus salivarius Ls-33, Lactobacillus paracasei subsp. paracasei YS8866441, Lactobacillus plantarum Lp-115, Bifidobacterium bifidum BI-504 and BI-98 was studied on splenic enteroantigen-presenting cells (APC) and CD4(+)CD25(+) T-regulatory cells (Tregs) in splenocyte-T cell proliferation assays. METHODS: Splenocytes exposed to enteroantigen +/- probiotics were used to stimulate cultured CD4(+)CD25(-) T cells to which titrated numbers of Tregs were added. Cytokine assays were performed by use of neutralizing antibodies and ELISA. RESULTS: Exposure of APCs to enteroantigens and the series of probiotic strains mentioned above did not influence the stimulatory capacity of APCs on proliferative enteroantigen-specific T cells. However, exposure to B. bifidum BI-98, BI-504 and L. acidophilus NCFM consistently reduced the suppressive activity of Tregs. The suppressive activity was analyzed using fractionated components of the probiotics, and showed that a component of the cell wall is responsible for the decreased Treg activity in the system. The probiotic-induced suppression of Treg function is not mediated by changes in APC-secretion of the inflammatory cytokines IL-6 or IL-1b. CONCLUSION: We conclude that certain probiotic strains can modify APCs to cause reduced Treg activity. This effect apparently depends on a direct APC-to-Treg cell contact. The APC-mediated suppressive effect on Treg function of certain probiotic strains may constrain the anti-inflammatory activity, which is often desired from probiotic therapy. This unexpected function of certain probiotic strains should be taken into consideration when designing adjuvant therapies with these bacteria, or when probiotic strains are selected for improvement of gut-associated inflammation like IBD.

Peptide specific expansion of CD8(+) T cells by recombinant plate bound MHC/peptide complexes.

Development of methods for efficient in vitro stimulation and expansion of peptide specific CD8(+) T cells is compelling not only with respect to adoptive T cell therapy but also regarding analysis of T cell responses and search for new immunogenic peptides. In the present study, a new approach to in vitro T cell stimulation was investigated. By use of an antigenic peptide derived from the cytomegalovirus (CMVp) we tested the stimulatory efficacy of recombinant plate bound MHC molecules (PB-MHC), being immobilized in culture plates. A single stimulation of non-adherent peripheral blood mononuclear cells (NA-PBMCs) with PB-MHC/CMVp resulted in significant expansion of CMVp specific CD8(+) T cells, which was comparable to that achieved by CMVp pulsed mature dendritic cells (DCs). By repeated exposure of NA-PBMCs to PB-MHC/CMVp more than 60% CMVp specific CD8(+) T cells, representing a 240-fold expansion, were reached after only two stimulations. Although stimulation with PB-MHC/CMVp clearly demonstrated efficient peptide specific expansion of CD8(+) T cells, there was a tendency to proliferative exhaustion of the cells after 3-4 stimulations. Thus, it will be of interest to examine the effect of new stimulatory cocktails, e.g. cytokines and co-stimulatory molecules, by use of the present rapid and easy-to-use method of expanding peptide specific T cells.

Dexamethasone/1alpha-25-dihydroxyvitamin D3-treated dendritic cells suppress colitis in the SCID T-cell transfer model.

Autoantigen-presenting immunomodulatory dendritic cells (DCs) that are used for adoptive transfer have been shown to be a promising therapy for a number of autoimmune diseases. We have previously demonstrated that enteroantigen-pulsed DCs treated with interleukin-10 (IL-10) can partly protect severe combined immunodeficient (SCID) mice adoptively transferred with CD4+ CD25(-) T cells from the development of wasting disease and colitis. We therefore established an in vitro test that could predict the in vivo function of DCs and improve strategies for the preparation of immunomodulatory DCs in this model. Based on these in vitro findings, we here evaluate three methods for DC generation including short-term and long-term IL-10 exposure or DC exposure to dexamethasone in combination with vitamin D3 (Dex/D3). All DCs resulted in lower CD4+ CD25(-) T-cell enteroantigen-specific responses in vitro, but Dex/D3 DCs had the most prominent effect on T-cell cytokine secretion. In vivo, Dex/D3 DCs most efficiently prevented weight loss and gut pathology upon CD4+ CD25(-) T-cell transfer in SCID mice, although the effect on gut pathology was antigen independent. Our data in the SCID T-cell transfer model illustrate some correlation between in vitro and in vivo DC function and document that prevention of experimental inflammatory bowel disease by transfer of immunosuppressive DCs is possible.

One-pot, mix-and-read peptide-MHC tetramers.

BACKGROUND: Cytotoxic T Lymphocytes (CTL) recognize complexes of peptide ligands and Major Histocompatibility Complex (MHC) class I molecules presented at the surface of Antigen Presenting Cells (APC). Detection and isolation of CTL's are of importance for research on CTL immunity, and development of vaccines and adoptive immune therapy. Peptide-MHC tetramers have become important reagents for detection and enumeration of specific CTL's. Conventional peptide-MHC-tetramer production involves recombinant MHC production, in vitro refolding, biotinylation and tetramerization; each step followed by various biochemical steps such as chromatographic purification, concentration etc. Such cumbersome production protocols have limited dissemination and restricted availability of peptide-MHC tetramers effectively precluding large-scale screening strategies involving many different peptide-MHC tetramers. METHODOLOGY/PRINCIPAL FINDINGS: We have developed an approach whereby any given tetramer specificity can be produced within 2 days with very limited effort and hands-on time. The strategy is based on the isolation of correctly oxidized, in vivo biotinylated recombinant MHC I heavy chain (HC). Such biotinylated MHC I HC molecules can be refolded in vitro, tetramerized with streptavidin, and used for specific T cell staining-all in a one-pot reaction without any intervening purification steps. CONCLUSIONS/SIGNIFICANCE: We have developed an efficient "one-pot, mix-and-read" strategy for peptide-MHC tetramer generation, and demonstrated specific T cell straining comparable to a commercially available MHC-tetramer. Here, seven peptide-MHC tetramers representing four different human MHC (HLA) class I proteins have been generated. The technique should be readily extendable to any binding peptide and pre-biotinylated MHC (at this time we have over 40 different pre-biotinylated HLA proteins). It is simple, robust, and versatile technique with a very broad application potential as it can be adapted both to small- and large-scale production of one or many different peptide-MHC tetramers for T cell isolation, or epitope screening.

Identification of a new hTERT-derived HLA-A*0201 restricted, naturally processed CTL epitope.

By the use of a neural network capable of performing quantitative predictions of peptides binding to HLA-A*0201 molecules, we identified a number of nonamer peptides derived from the catalytic subunit of telomerase, human telomerase reverse transcriptase (hTERT). Five nonimmunogenic peptides with measured binding affinities for HLA-A*0201 ranging from 155 to 1,298 nM were modified at the P1, P2 and P9 positions, respectively, to achieve stronger HLA-A*0201 binding. One peptide, mp30-38 (mp30), with an L to V substitution at position 9 was subsequently found to be immunogenic in mp30 immunized HLA-A*0201/H2K(b) or HHD transgenic mice. The T cell reactivity obtained was directed against both the mp30 and against the unmodified p30. Anti-mp30 specific T cells generated in HLA-A*0201 transgenic mice were dependent on TCR-CD8/MHC-I alpha3 binding and therefore not capable of recognizing mp30-pulsed human HLA-A*0201(+) cells or murine HLA-A*0201 transfectants. In order to show reactivity against naturally processed peptide in human tumor cells, an hTERT positive HLA-A*0201 negative colon carcinoma cell line (CCL220) was transfected with an HLA-A*0201/H2K(b) cDNA construct and used as target in ELISPOT and cytotoxicity assays. The data show that T cells from mp30 immunized HHD transgenic mice react specifically against the CCL220 transfectant indicating that p30 is naturally processed. In conclusion, we have identified a new CTL HLA-A*0201 restricted hTERT epitope, which is now, included in an ongoing phase 2 vaccine trial of patients with disseminated cancer.