Einfluss immunevasiver Strategien des humanen Cytomegalovirus auf die MHC-Klasse-I-Pr��sentation der viralen Antigene pp65 und IE1

Das Humane Cytomegalovirus (HCMV) stellt eine gro��e Bedrohung f��r Patienten mit geschw��chtem oder unausgereiftem Immunsystem dar. Bei immunkompetenten Personen hingegen werden schwere Erkrankungen insbesondere durch die Wirkung antiviraler zytotoxischer CD8+-T-Lymphozyten (CTL) weitgehend verhindert. Aus Zellkultur-Systemen war bekannt, dass virale Glykoproteine, welche in der US2-US11-Region des HCMV-Genoms kodiert werden, inhibitorisch in den MHC-Klasse-I-Pr��sentationsweg eingreifen und somit die entsprechende Pr��sentation durch infizierte Zellen behindern. ��ber die Bedeutung dieser US2-US11-vermittelten Immunevasion f��r die Pr��sentation viraler Antigene im Kontext der Virusinfektion war jedoch nichts bekannt. Im Rahmen der vorliegenden Arbeit sollte daher der Einfluss der Immunevasion auf die MHC-Klasse-I-Pr��sentation der beiden wichtigsten CTL-Zielstrukturen von HCMV, dem Tegumentprotein pp65 und dem regulatorischen immediate early Protein IE1, untersucht werden. In Erg��nzung dazu sollte das immunevasive Potential eines durch HCMV kodierten Homologs des immunmodulatorischen Zytokins Interleukin-10 (cmvIL-10) analysiert werden. Hierzu wurden ��ber Peptidimmunisierung HLA-A2-transgener M��use CTL-Klone hergestellt, welche ausgesuchte Peptide aus pp65 und IE1 in Assoziation mit HLA-A2 mit hoher Spezifit��t und Sensitivit��t erkannten. Auf diese Weise konnte eine direkte Beeinflussung der MHC-Klasse-I-Pr��sentation durch cmvIL-10 falsifiziert und somit der Hypothese, dass das von infizierten Zellen freigesetzte Zytokin die MHC-Klasse-I-Pr��sentation nicht infizierter Nachbarzellen beeinflussen k��nnte, widersprochen werden. Mit Hilfe einer US2-US11-Deletionsmutante des Virus konnte zum ersten Mal gezeigt werden, dass die Pr��sentation von sowohl pp65 als auch IE1 durch die Immunevasion beeintr��chtigt wird. Dabei war die Pr��sentation des IE1-Peptids zu jedem untersuchten Zeitpunkt nach Infektion vollst��ndig unterdr��ckt. Die Pr��sentation des pp65-Peptids hingegen war noch bis zu 72 Stunden nach Infektion detektierbar. Diese anhaltende Pr��sentation wurde dabei durch MHC-Klasse-I-Komplexe hervorgerufen, die trotz der Expression der US2-US11-Region an die Zelloberfl��che transportiert wurden. Anhand des pp65 konnte somit erstmals gezeigt werden, dass die Immunevasion von HCMV Bildung und Transport bestimmter MHC-Klasse-I-Peptid-Komplexe zwar beeintr��chtigen, jedoch nicht vollst��ndig blockieren kann. Weitere Untersuchungen ergaben, dass die Pr��sentation von IE1-Peptiden durch das Vorhandensein des pp65-Proteins nicht beeinflusst wurde. Damit konnten aus der Literatur bekannte Daten anderer widerlegt werden. Mit Hilfe einer weiteren Virusmutante konnte schlie��lich gezeigt werden, das die Expression eines der Immunevasine, des gpUS11, hinreichend ist, die IE1-Pr��sentation vollst��ndig zu unterdr��cken, jedoch keinerlei messbaren Einfluss auf die Pr��sentation von pp65 aus��bt. Die vorliegende Arbeit hat wichtige Erkenntnisse erbracht, die die Grundlage f��r weiterf��hrende Untersuchungen zur Aufkl��rung der Bedeutung der einzelnen Immunevasionsgene f��r die Pr��sentation viraler Antigene im Rahmen der Virusinfektion darstellen.

Identifizierung von T-Zell-erkannten Nierenzellkarzinom-Antigenen

Eine Voraussetzung f��r die Entwicklung neuer immunmodulatorischer Therapieverfahren ist die Kenntnis immunogener Tumorantigene, die von tumorreaktiven T-Zellen erkannt werden. In der vorliegenden Arbeit wurden tumorreaktive CD8+ zytotoxische T-Lymphozyten (CTL, cytotoxic T-lymphocytes) aus dem Blut eines HLA (human leukocyte antigen)-kompatiblen Fremdspenders generiert. Methodisch wurden hierzu CD8-selektionierte periphere Blutlymphozyten repetitiv mit der klarzelligen Nierenzellkarzinomlinie MZ1851-RCC (RCC, renal cell carcinoma) in einer allogenen gemischten Lymphozyten-Tumorzell Kultur (MLTC, mixed lymphocyte tumor cell culture) stimuliert. Aus den Responderlymphozyten wurden mit Hilfe des Grenzverd��nnungsverfahrens klonale zytotoxische T-Zellen generiert und expandiert. Die CTL-Klone wurden anschlie��end ph��notypisch mittels Durchflu��zytometrie sowie funktionell mittels HLA-Antik��rper-Blockadeexperimenten und Kreuzreaktivit��tstests detailliert charakterisiert. Dabei konnte gezeigt werden, da�� aus dem Blut eines allogenen gesunden Spenders CD8+ T-Zellen isoliert werden k��nnen, welche Reaktivit��t gegen Nierenzellkarzinome (NZK) aufweisen und ��ber verschiedene HLA-Klasse-I-Allele restringiert sind. Die von den einzelnen CTL-Klonen erkannten Zielstrukturen zeigten entweder ubiquit��re (z.B. HLA-Cw*0704-reaktiver CTL-Klon E77) oder eine tumorspezifische (z.B. HLA-B*0702-restringierter CTL-Klon A4) Gewebeexpression. Zur Identifizierung der nat��rlich prozessierten Peptidliganden wurden die HLA-B/C-Allele unter Verwendung des monoklonalen Antik��rpers B123.2 aus einem zuvor hergestellten Detergenslysat der Nierenzellkarzinomlinie MZ1851-RCC immunchromatographisch aufgereinigt. Aus den so isolierten HLA-Peptid-Komplexen wurden die tumorassoziierten Peptidliganden nach S��ureeluation und Filtration abgespalten und ��ber eine ���reverse phase���-HPLC (high performance liquid chromatography) fraktioniert. Die ��berpr��fung der einzelnen HPLC-Fraktionen auf Bioaktivit��t erfolgte mit den korrespondierenden CTL-Klonen in 51Cr-Zytotoxizit��tstests. Dabei wurde eine HPLC-Fraktion identifiziert, die die lytische Funktion des HLA-B*0702-restringierten CTL-Klons A4 ausl��sen konnte. Die bioaktive HPLC-Fraktion wurde dazu durch eine zweite (second dimension) Kapillar-Fl��ssigkeitschromatographie (Cap-LC, capillar liquid chromatography) in Subfraktionen geringerer Komplexit��t aufgetrennt und die darin enthaltenen Peptidepitope durch das MALDI-TOF/TOF (matrix assisted laser desorption/ionization- time of flight/time of flight)-Analyseverfahren sequenziert. Innerhalb dieser HPLC-Fraktion wurden eine Vielzahl von HLA-B/C-assoziierten Peptidliganden erfolgreich sequenziert, was die Effektivit��t dieser Verfahrenstechnik zur Identifizierung nat��rlich prozessierter HLA-Klasse-I-bindender Peptide unter Beweis stellt. Leider war es mit dieser Methode bisher nicht m��glich, das von CTL-Klon A4 detektierte Peptidepitop zu sequenzieren. Dies liegt m��glicherweise in der unzureichenden Konzentration des Peptidepitops in der bioaktiven HPLC-Fraktion begr��ndet. In Folgearbeiten soll nun mit erh��hter Probenmenge beziehungsweise verbesserter Analytik der erneute Versuch unternommen werden, das Zielantigen des CTL-Klons A4 zu identifizieren. Die Kenntnis von Antigenen, die tumorspezifisch exprimiert und von CD8+ CTL aus gesunden Spendern erkannt werden, er��ffnet neue therapeutische M��glichkeiten, das spezifische Immunsystem des Stammzellspenders nach allogener Blutstammzelltransplantation gezielt zur Steigerung von Tumorabsto��ungsreaktionen (z.B. durch Vakzinierung oder adoptivem T-Zelltransfer) zu nutzen.

Die Identifizierung und Charakterisierung von HLA-Klasse-I-restringierten Minorhistokompatibilit��tsantigenen und Leuk��mie-assoziierten Antigenen mittels cDNA-Expressionsklonierung

Die allogene h��matopoetische Stammzelltransplantation (allo-HSCT) bietet bei einem hohen Anteil akuter Leuk��mien die einzige kurative Behandlungsm��glichkeit. Um die mit ihr assoziierte Morbidit��t und Mortalit��t zu senken und ihre Effektivit��t zu steigern, soll die GvL (graft-versus-leukemia)-Reaktion als eigentliches Therapieziel gegen��ber der unerw��nschten GvHD (graft-versus-host disease) m��glichst selektiv verst��rkt werden. Wesentliche Mediatoren beider Effekte sind alloreaktive T-Zellen. Bei HLA-��bereinstimmung zwischen Spender und Empf��nger sind so genannte Minorhistokompatibilit��tsantigene (mHAgs) und Leuk��mie-assoziierte Antigene (LAA) die mutma��lichen Zielstrukturen beider Reaktionen. Im Rahmen der vorliegenden Arbeit wurden in dem Leuk��mie-Modell der Patientin MZ201 [akute myeloische Leuk��mie (AML) vom Subtyp FAB M5] mittels T-Zell-basierter cDNA-Expressionsklonierung zwei neue Antigene identifiziert, die von allogenen, AML-reaktiven CD8+ T-Lymphozyten aus Blut eines HLA-passenden gesunden Spenders erkannt wurden. Es handelt sich zum einen um das HLA-B*5601-restringierte mHAg PLAUR-317P, das aus einem Polymorphismus des Gens PLAUR (plasminogen activator, urokinase receptor) resultiert. Das von den T-Zellen am Besten erkannte Peptid enth��lt die Aminos��uren 316 - 327. PLAUR wird in lymphoh��matopoetischen Zellen und in verschiedenen Malignomen ��berexprimiert und ist dabei mit schlechterer Prognose und vermehrter Gewebeinvasivit��t assoziiert. Etwa 30% getesteter Individuen tragen das Allel PLAUR-317P. Zum anderen handelt es sich um ein Epitop aus der Signalregion des Chemokins CXCL3 [chemokine (C-X-C motif) ligand 3], das von CD8+ T-Zellen des gleichen Spenders auf Leuk��miezellen der Patientin MZ201 in Assoziation mit HLA-A*0201 erkannt wurde. Auch CXCL3 wird vorwiegend in Zellen der Myelopoese exprimiert. Aufgrund ihres Expressionsmusters sind beide Antigene potentielle Zielstrukturen f��r die Elimination der Empf��nger-H��matopoese unter Einschluss der Leuk��mieblasten im Rahmen der allo-HSCT. Weiterf��hrende Untersuchungen m��ssen zeigen, ob diese Antigene tats��chlich in vivo GvL-Reaktionen hervorrufen. Die Kenntnis eines repr��sentativen Spektrums solcher Antigene w��rde verbesserte Spenderselektionen erlauben und neue Wege des adoptiven T-Zelltransfers erschlie��en helfen.

In vitro characterization of human AML-reactive CTL clones generated from the naive subset of healthy donors and adoptive transfer into leukemia-engrafted NSG mice

Acute myeloid leukemia (AML) is a very aggressive cancer of the hematopoietic system. Chemotherapy and immunotherapeutical approaches including hematopoietic stem cell transplantation (HSCT) and donor lymphocyte infusion (DLI) are the only curative options available. The beneficial graft-versus-leukemia (GVL) effect of cellular immunotherapy is mostly mediated by donor-derived CD8+ T lymphocytes that recognize minor histocompatibility antigens (mHags) and leukemia-associated antigens (LAAs) presented on the surface of AML blasts (Falkenburg et al. 2008; Kolb 2008). A main complication is graft-versus-host disease (GVHD) that can be induced when cytotoxic T lymphocytes (CTLs) recognize broadly expressed antigens. To reduce the risk of GVHD, specific allogeneic T-cell therapy inducing selective GVL responses could be an option (Barrett & Le Blanc 2010; Parmar et al. 2011; Smits et al. 2011). This requires efficient in vitro strategies to generate AML-reactive T cells with an early differentiation phenotype as well as vigorous effector functions and humanized mouse models to analyze the anti-leukemic potential of adoptively transferred T cells in vivo. In this study, AML-reactive CTL clones and oligoclonal T-cell lines could be reliably generated from the naive subset of healthy HLA-class I-identical donors by stimulation with primary AML blasts in mini-mixed-lymphocyte / leukemia cultures (MLLCs) in eight different patient / donor pairs. These CTLs were promising candidates for cellular immunotherapy because of their relatively early differentiation phenotype and strong proliferative and lytic capabilities. The addition of the common ��-chain cytokine IL-21 to the stimulation protocol enabled more precursors to develop into potent leukemia-reactive CTLs, presumably by its beneficial effects on cell survival and antigen-specific proliferation during the first weeks of cultures. It also strengthened the early-stage phenotype. Three long-term cultured CTLs exemplarily transferred into leukemia-engrafted immunodeficient NSG mice mediated a significant reduction of the leukemic burden after a single transfusion. These results demonstrate that CTL clones with reactivity to patient-derived AML blasts can be isolated from the naive compartment of healthy donors and show potent anti-leukemic effects in vivo. The herein described allo-MLLC approach with in vitro ���programmed��� naive CTL precursors independent of a HSCT setting is a valuable alternative to the conventional method of isolating in vivo primed donor CTLs out of patients after transplantation (Kloosterboer et al. 2004; Warren et al. 2010). This would make leukemia-reactive CTLs already available at the time point of HSCT, when residual leukemia disease is minimal and the chances for complete leukemia eradication are high. Furthermore, leukemia-reactive CTLs effectively expanded by this in vitro protocol can be used as screening populations to identify novel candidate LAAs and mHags for antigen-specific immunotherapy.

Generation of FLT3-ITD-reactive T-cell populations from different sources

Approximately 25% of acute myeloid leukemias (AMLs) carry internal tandem duplications (ITD) of various lengths within the gene encoding the FMS-like tyrosine kinase receptor 3 (FLT3). Although varying duplication sites exist, most of these length mutations affect the protein��s juxtamembrane domain. FLT3-ITDs support leukemic transformation by constitutive phosphorylation resulting in uncontrolled activation, and their presence is associated with worse prognosis. As known form previous work, they represent leukemia- and patient-specific neoantigens that can be recognized by autologous AML-reactive CD8+ T cells (Graf et al., 2007; Graf et al., unpublished). Herein, in patient FL, diagnosed with FLT3-ITD+ AML and in first complete remission after induction chemotherapy, T cells against her leukemia��s individual FLT3-ITD were detected at a frequency up to 1.7x10-3 among peripheral blood CD8+ T lymphocytes. This rather high frequency suggested, that FLT3-ITD-reactive T cells had been expanded in vivo due to the induction of an anti-leukemia response.rnrnCell material from AML patients is limited, and the patients�� anti-leukemia T-cell repertoire might be skewed, e.g. due to complex previous leukemia-host interactions and chemotherapy. Therefore, allogeneic sources, i.e. buffy coats (BCs) from health donors and umbilical cord blood (UCB) donations, were exploited for the presence and the expansion of FLT3-ITD-reactive T-cell populations. BC- and UCB-derived CD8+ T cells, were distributed at 105 cells per well on microtiter plates and, were stimulated with antigen-presenting cells (APCs) transfected with in vitro-transcribed mRNA (IVT-mRNA) encoding selected FTL3-ITDs. APCs were autologous CD8- blood mononuclear cells, monocytes or FastDCs.rnrnBuffy coat lymphocytes from 19 healthy individuals were analyzed for CD8+ T-cell reactivity against three immunogenic FLT3-ITDs previously identified in patients VE, IN and QQ and designated as VE_, IN_ and QQ_FLT3-ITD, respectively. These healthy donors carried at least one of the HLA I alleles known to present an ITD-derived peptide from one of these FLT3-ITDs. Reactivities against single ITDs were observed in 8/19 donors. In 4 donors the frequencies of ITD-reactive T cells were determined and were estimated to be in the range of 1.25x10-6 to 2.83x10-7 CD8+ T cells. These frequencies were 1,000- to 10,000-fold lower than the frequency of autologous FLT3-ITD-reactive T cells observed in patient FL. Restricting HLA I molecules were identified in two donors. In one of them, the recognition of VE_FLT3-ITD was found to be restricted by HLA-C*07:02, which is different from the HLA allele restricting the anti-ITD T cells of patient VE. In another donor, the recognition of IN_FLT3-ITD was restricted by HLA-B*35:01, which also had been observed in patient IN (Graf et al., unpublished). By gradual 3��-fragmentation of the IN_FLT3-ITD cDNA, the 10-mer peptide CPSDNEYFYV was identified as the target of allogeneic T cells against IN_FLT3-ITD. rnLymphocytes in umbilical cord blood predominantly exhibit a na��ve phenotype. Seven UCB donations were analyzed for T-cell responses against the FLT3-ITDs of patients VE, IN, QQ, JC and FL irrespective of their HLA phenotype. ITD-reactive responses against all stimulatory FLT3-ITDs were observed in 5/7 UCB donations. The frequencies of T cells against single FLT3-ITDs in CD8+ lymphocytes were estimated to be in the range of 1.8x10-5 to 3.6x10-6, which is nearly 15-fold higher than the frequencies observed in BCs. Restricting HLA I molecules were identified in 4 of these 5 positive UCB donations. They were mostly different from those observed in the respective patients. But in one UCB donation T cells against the JC_FLT3-ITD had exactly the same peptide specificity and HLA restriction as seen before in patient JC (Graf et al., 2007). Analyses of UCB responder lymphocytes led to the identification of the 10-mer peptide YESDNEYFYV, encoded by FL_FLT3-ITD, that was recognized in association with the frequent allele HLA-A*02:01. This peptide was able to stimulate and enrich ITD-reactive T cells from UCB lymphocytes in vitro. Peptide responders not only recognized the peptide, but also COS-7 cells co-transfected with FL_FLT3-ITD and HLA-A*02:01.rnrnIn conclusion, T cells against AML- and individual-specific FLT3-ITDs were successfully generated not only from patient-derived blood, but also from allogeneic sources. Thereby, ITD-reactive T cells were detected more readily and at higher frequencies in umbilical cord blood than in buffy coat lymphocytes. It occurred that peptide specificity and HLA restriction of allogeneic, ITD-reactive T cells were identical to autologous patient-derived T cells. As shown herein, allogeneic, FLT3-ITD-reactive T cells can be used for the identification of FLT3-ITD-encoded peptides, e.g. for future therapeutic vaccination studies. In addition, these T cells or their receptors can be applied to adoptive transfer.

Identifizierung und Charakterisierung von Zielantigenen alloreaktiver zytotoxischer T-Zellen mittels cDNA-Bank-Expressionsklonierung in akuten myeloischen Leuk��mien

Allogene h��matopoetische Stammzelltransplantationen (HSZTs) werden insbesondere zur Behandlung von Patienten mit Hochrisiko-Leuk��mien durchgef��hrt. Dabei bewirken T-Zellreaktionen gegen Minorhistokompatibilit��tsantigene (mHAgs) sowohl den therapeutisch erw��nschten graft-versus-leukemia (GvL)-Effekt als auch die sch��digende graft-versus-host (GvH)-Erkrankung. F��r die Identifizierung neuer mHAgs mittels des T-Zell-basierten cDNA-Expressionsscreenings waren leuk��miereaktive T-Zellpopulationen durch Stimulation na��ver CD8+-T-Lymphozyten gesunder HLA-Klasse I-identischer Buffy Coat-Spender mit Leuk��miezellen von Patienten mit akuter myeloischer Leuk��mie (AML) generiert worden (Albrecht et al., Cancer Immunol. Immunother. 60:235, 2011). Im Rahmen der vorliegenden Arbeit wurde mit diesen im AML-Modell des Patienten MZ529 das mHAg CYBA-72Y identifiziert. Es resultiert aus einem bekannten Einzelnukleotidpolymorphismus (rs4673: CYBA-242T/C) des Gens CYBA (kodierend f��r Cytochrom b-245 ��-Polypeptid; syn.: p22phox), der zu einem Austausch von Tyrosin (Y) zu Histidin (H) an Aminos��ureposition 72 f��hrt. Das mHAg wurde von T-Lymphozyten sowohl in Assoziation mit HLA-B*15:01 als auch mit HLA-B*15:07 erkannt. Eine allogene T-Zellantwort gegen CYBA-72Y wurde in einem weiteren AML-Modell (MZ987) beobachtet, die ebenso wie in dem AML-Modell MZ529 polyklonal war. Insgesamt konnte bei drei von f��nf getesteten HLA-B*15:01-positiven Buffy Coat-Spendern, die homozygot f��r CYBA-72H (H/H) waren, eine CYBA-72Y-spezifische T-Zellantwort generiert werden. Das von den T-Lymphozyten ��bereinstimmend in niedrigster Konzentration erkannte Peptid umfasste die Aminos��uren 69 - 77, wobei das homologe Peptid aus CYBA-72H auch in hohen Konzentrationen keine Reaktivit��t ausl��ste. Eine reziproke Immunogenit��t des mHAg ist bislang nicht belegt. T-Lymphozyten gegen CYBA-72Y erkannten Leuk��miezellen bei acht von zw��lf HLA-B*15:01-positiven Patienten (FAB-Subtypen: M1, M2, M4, M5). Da das Gen CYBA f��r eine Komponente des mikrobiziden Oxidasesystems von phagozytierenden Zellen kodiert, ist es ��berwiegend in Zellen des h��matopoetischen Systems exprimiert. Von Leukozytensubtypen, aufgereinigt aus HLA-B*15:01-positiven Buffy Coat-Spendern mit CYBA-242T-Allel, wurden Monozyten und daraus abgeleitete dendritische Zellen durch CYBA-72Y-reaktive T-Lymphozyten sehr stark, untransformierte B-Zellen in weit geringerem Ma��e und Granulozyten sowie T-Lymphozyten nicht erkannt. Das f��r CYBA-72Y kodierende Allel CYBA-242T wurde bei 56% aller getesteten gesunden Spender und Malignompatienten (n=481) nachgewiesen. Unter Ber��cksichtigung der H��ufigkeit des pr��sentierenden HLA-Allels ist davon auszugehen, dass etwa 4,5% der Kaukasier das mHAg CYBA-72Y zusammen mit HLA-B*15:01 tragen. Nach bisherigen Beobachtungen f��hrt ein immunogener CYBA-72Y-Mismatch bei allogenen HSZTs nicht notwendigerweise zu einer schweren GvH-Erkrankung. Das hier beschriebene mHAg CYBA-72Y erscheint potenziell geeignet, im Rahmen einer allogenen HSZT die pr��ferenzielle Elimination der Empf��nger-H��matopoese unter Einschluss von myeloischen Leuk��miezellen zu bewirken. Jedoch sind weiterf��hrende Untersuchungen erforderlich, um die therapeutische Relevanz des Antigens zu belegen.

Comprehensive analysis of CpG island methylator phenotype (CIMP)-high, -low, and -negative colorectal cancers based on protein marker expression and molecular features

CpG island methylator phenotype (CIMP) is being investigated for its role in the molecular and prognostic classification of colorectal cancer patients but is also emerging as a factor with the potential to influence clinical decision-making. We report a comprehensive analysis of clinico-pathological and molecular features (KRAS, BRAF and microsatellite instability, MSI) as well as of selected tumour- and host-related protein markers characterizing CIMP-high (CIMP-H), -low, and -negative colorectal cancers. Immunohistochemical analysis for 48 protein markers and molecular analysis of CIMP (CIMP-H: ? 4/5 methylated genes), MSI (MSI-H: ? 2 instable genes), KRAS, and BRAF were performed on 337 colorectal cancers. Simple and multiple regression analysis and receiver operating characteristic (ROC) curve analysis were performed. CIMP-H was found in 24 cases (7.1%) and linked (p < 0.0001) to more proximal tumour location, BRAF mutation, MSI-H, MGMT methylation (p = 0.022), advanced pT classification (p = 0.03), mucinous histology (p = 0.069), and less frequent KRAS mutation (p = 0.067) compared to CIMP-low or -negative cases. Of the 48 protein markers, decreased levels of RKIP (p = 0.0056), EphB2 (p = 0.0045), CK20 (p = 0.002), and Cdx2 (p < 0.0001) and increased numbers of CD8+ intra-epithelial lymphocytes (p < 0.0001) were related to CIMP-H, independently of MSI status. In addition to the expected clinico-pathological and molecular associations, CIMP-H colorectal cancers are characterized by a loss of protein markers associated with differentiation, and metastasis suppression, and have increased CD8+ T-lymphocytes regardless of MSI status. In particular, Cdx2 loss seems to strongly predict CIMP-H in both microsatellite-stable (MSS) and MSI-H colorectal cancers. Cdx2 is proposed as a surrogate marker for CIMP-H.

T cell-mediated immunoregulation in the gastrointestinal tract

In the intestinal tract, only a single layer of epithelial cells separates innate and adaptive immune effector cells from a vast amount of antigens. Here, the immune system faces a considerable challenge in tolerating commensal flora and dietary antigens while preventing the dissemination of potential pathogens. Failure to tightly control immune reactions may result in detrimental inflammation. In this respect, 'conventional' regulatory CD4(+) T cells, including naturally occurring and adaptive CD4(+) CD25(+) Foxp3(+) T cells, Th3 and Tr1 cells, have recently been the focus of considerable attention. However, regulatory mechanisms in the intestinal mucosa are highly complex, including adaptations of nonhaematopoietic cells and innate immune cells as well as the presence of unconventional T cells with regulatory properties such as resident TCRgammadelta or TCRalphabeta CD8(+) intraepithelial lymphocytes. This review aims to summarize the currently available knowledge on conventional and unconventional regulatory T cell subsets (Tregs), with special emphasis on clinical data and the potential role or malfunctioning of Tregs in four major human gastrointestinal diseases, i.e. inflammatory bowel diseases, coeliac disease, food allergy and colorectal cancer. We conclude that the clinical data confirms some but not all of the findings derived from experimental animal models.

Glucocorticoid alterations in the human type-1/type-2 cytokine balance and the role of CD28/B7 costimulation

We have recently reported that psychological stress is associated with a shift in the human type-1/type-2 cytokine balance toward a type-2 cytokine response. The mechanisms of these cytokine alterations are unknown, but likely involve glucocorticoid (GC) modulation of cytokine production. Therefore we sought to characterize the effects of GC on the in vitro human type-1/type-2 cytokine balance. We hypothesized that GC induce a type-2 cytokine shift through modulation of critical regulatory cytokines and alterations in the CD28/B7 costimulatory pathway. ^ We first sought to characterize the effect of the GC, dexamethasone (DEX), on type-1 (IFN-��, IL-12) and type-2 (IL-4, IL-10) cytokine production by human peripheral blood mononuclear blood cells (pBMC) stimulated with a variety of T-lymphocyte and monocyte stimuli. DEX, at concentrations mimicking stress and supraphysiologic levels of cortisol, decreased IFN-�� and IL-12 production and increased IL-4 and IL-10 production, indicating a shift in the type-1/type-2 cytokine balance toward a type-2 response. Furthermore, both CD4+ and CD8+ T-lymphocytes were susceptible to the cytokine modulating effects of DEX. Furthermore, in the absence of the monocyte, the DEX-induced alterations in T-lymphocyte cytokine production were reduced, indicating that the interaction between the monocyte and T-lymphocyte plays a significant role. ^ We next determined the role of regulatory cytokines, known to modulate the type-1/type-2 cytokine balance, in the DEX-induced cytokine alterations. The addition of the recombinant IL-12p70 and IFN-��, but not the neutralization of IL-4, IL-10 or IL-13 using monoclonal antibodies, attenuated the DEX-induced type-1/type-2 cytokine alterations. These data suggest that the DEX-induced cytokine alterations are mediated, at least in part, through the initial inhibition type-1 cytokines. Lastly, we investigated the role of the CD28/B7 costimulatory pathway in these cytokine alterations. DEX decreased the expression of CD80 and CD86 on THP-1 cells, a monocyte cell line, and the expression of CD28 and CTLA-4 on PHA-stimulated pBMC. The DEX-induced decrease in CD28 and CTLA-4 expression was attenuated by rhIL-12. Finally, CD28 activation attenuated the DEX-induced decrease in IFN-�� production, suggesting that modulation of the CD28/B7 costimulatory pathway may contribute to the DEX-induced type-1/type-2 cytokine alterations. ^

Spontaneous inflammatory demyelinating disease in transgenic mice showing central nervous system-specific expression of tumor necrosis factor alpha.

Cytokines are now recognized to play important roles in the physiology of the central nervous system (CNS) during health and disease. Tumor necrosis factor alpha (TNF-alpha) has been implicated in the pathogenesis of several human CNS disorders including multiple sclerosis, AIDS dementia, and cerebral malaria. We have generated transgenic mice that constitutively express a murine TNF-alpha transgene, under the control of its own promoter, specifically in their CNS and that spontaneously develop a chronic inflammatory demyelinating disease with 100% penetrance from around 3-8 weeks of age. High-level expression of the transgene was seen in neurons distributed throughout the brain. Disease is manifested by ataxia, seizures, and paresis and leads to early death. Histopathological analysis revealed infiltration of the meninges and CNS parenchyma by CD4+ and CD8+ T lymphocytes, widespread reactive astrocytosis and microgliosis, and focal demyelination. The direct action of TNF-alpha in the pathogenesis of this disease was confirmed by peripheral administration of a neutralizing anti-murine TNF-alpha antibody. This treatment completely prevented the development of neurological symptoms, T-cell infiltration into the CNS parenchyma, astrocytosis, and demyelination, and greatly reduced the severity of reactive microgliosis. These results demonstrate that overexpression of TNF-alpha in the CNS can cause abnormalities in nervous system structure and function. The disease induced in TNF-alpha transgenic mice shows clinical and histopathological features characteristic of inflammatory demyelinating CNS disorders in humans, and these mice represent a relevant in vivo model for their further study.

Passive immunotherapy for retroviral disease: influence of major histocompatibility complex type and T-cell responsiveness.

Administration of virus-specific antibodies is known to be an effective early treatment for some viral infections. Such immunotherapy probably acts by antibody-mediated neutralization of viral infectivity and is often thought to function independently of T-cell-mediated immune responses. In the present experiments, we studied passive antibody therapy using Friend murine leukemia virus complex as a model for an immunosuppressive retroviral disease in adult mice. The results showed that antibody therapy could induce recovery from a well-established retroviral infection. However, the success of therapy was dependent on the presence of both CD4+ and CD8+ T lymphocytes. Thus, cell-mediated responses were required for recovery from infection even in the presence of therapeutic levels of antibody. The major histocompatibility type of the mice was also an important factor determining the relative success of antibody therapy in this system, but it was less critical for low-dose than for high-dose infections. Our results imply that limited T-cell responsiveness as dictated by major histocompatibility genes and/or stage of disease may have contributed to previous immunotherapy failures in AIDS patients. Possible strategies to improve the efficacy of future therapies are discussed.

Deletion of high-avidity T cells by thymic epithelium.

Tolerance induction by thymic epithelium induces a state of so-called "split tolerance," characterized in vivo by tolerance and in vitro by reactivity to a given thymically expressed antigen. Using a model major histocompatibility complex class I antigen, H-2Kb (Kb), three mechanisms of thymic epithelium-induced tolerance were tested: induction of tolerance of tissue-specific antigens exclusively, selective inactivation of T helper cell-independent cytotoxic T lymphocytes, and deletion of high-avidity T cells. To this end, thymic anlagen from Kb-transgenic embryonic day 10 mouse embryos, taken before colonization by cells of hemopoietic origin, were grafted to nude mice. Tolerance by thymic epithelium was not tissue-specific, since Kb-bearing skin and spleen grafts were maintained indefinitely. Only strong priming in vivo could partially overcome the tolerant state and induce rejection of some skin grafts overexpressing transgenic Kb. Furthermore, the hypothesis that thymic epithelium selectively inactivates those T cells that reject skin grafts in a T helper-independent fashion could not be supported. Thus, when T-cell help was provided by a second skin graft bearing an additional major histocompatibility complex class II disparity, tolerance to the Kb skin graft was not broken. Finally, direct evidence could be obtained for the avidity model of thymic epithelium-induced negative selection, using Kb-specific T-cell receptor (TCR) transgenic mice. Thymic epithelium-grafted TCR transgenic mice showed a selective deletion of those CD8+ T cells with the highest density of the clonotypic TCR. These cells presumably represent the T cells with the highest avidity for Kb. We conclude that split tolerance induced by thymic epithelium was mediated by the deletion of those CD8+ T lymphocytes that have the highest avidity for antigen.

Combination gene therapy for liver metastasis of colon carcinoma in vivo.

The efficacy of combination therapy with a "suicide gene" and a cytokine gene to treat metastatic colon carcinoma in the liver was investigated. Tumor in the liver was generated by intrahepatic injection of a colon carcinoma cell line (MCA-26) in syngeneic BALB/c mice. Recombinant adenoviral vectors containing various control and therapeutic genes were injected directly into the solid tumors, followed by treatment with ganciclovir. While the tumors continued to grow in all animals treated with a control vector or a mouse interleukin 2 vector, those treated with a herpes simplex virus thymidine kinase vector, with or without the coadministration of the mouse interleukin 2 vector, exhibited dramatic necrosis and regression. However, only animals treated with both vectors developed an effective systemic antitumoral immunity against challenges of tumorigenic doses of parental tumor cells inoculated at distant sites. The antitumoral immunity was associated with the presence of MCA-26 tumor-specific cytolytic CD8+ T lymphocytes. The results suggest that combination suicide and cytokine gene therapy in vivo can be a powerful approach for treatment of metastatic colon carcinoma in the liver.

An��lise fenot��pica de c��lulas dendr��ticas e de linf��citos T efetores, polifuncionais e reguladores no l��quen plano

INTRODU����O: L��quen plano (LP) �� uma doen��a mucocut��nea de natureza inflamat��ria cr��nica de etiologia ainda desconhecida. A estimula����o da imunidade inata via os receptores Toll-like (TLRs) podem influenciar as c��lulas dendr��ticas e direcionar a resposta de c��lulas T CD4+ e CD8+ efetoras, assim como tamb��m favorecer o estado inflamat��rio do LP. OBJETIVOS: Avaliar o perfil fenot��pico de c��lulas dendr��ticas miel��ides (mDCs) e plasmocit��ides (pDCs) e de linf��citos T CD4+ e CD8+ ap��s est��mulo com agonistas de TLRs no sangue perif��rico de pacientes com LP. Al��m disto, avaliar a frequ��ncia, perfil de matura����o e os subtipos de c��lulas T CD4+ e TCD8+ reguladores. M��TODOS: Foram selecionados 18 pacientes com LP (15 mulheres, 3 homens), com 41,57 �� 4,73 anos de idade e um grupo controle com 22 indiv��duos sadios (18 mulheres, 4 homens), com 43,92 �� 7,83 anos de idade. As c��lulas mononucleares (CMNs) de sangue perif��rico foram avaliadas por citometria de fluxo quanto ��: 1) Produ����o de TNF-? em mDCs e de IFN-? em pDCs em CMNs ativadas por agonistas de TLR 4, 7, 7/8 e 9; 2) An��lise de c��lulas T CD4+ e CD8+ monofuncionais e polifuncionais ap��s est��mulo com agonistas de TLR 4, 7/8, 9 e enterotoxina B de Staphylococcus aureus (SEB); 3) Avalia����o de c��lulas Th17 e Th22/Tc22 em CMNs ap��s est��mulo com SEB; 4) Frequ��ncia, perfil de matura����o e subtipos de c��lulas T CD4+ e CD8+ reguladoras. RESULTADOS: 1) Nos pacientes com LP foi demonstrado um aumento na frequ��ncia de mDCs TNF-alfa+ ap��s est��mulo com agonistas de TLR4/LPS e TLR7-8/CL097, mas com imiquimode/TLR7 houve diminui����o da express��o de CD83. J�� nas pDCs do grupo LP, o imiquimode foi capaz de diminuir a express��o de CD80 e o CpG/TLR9 diminuiu a express��o de CD83 no LP. 2) As c��lulas T CD4+ secretoras de IL-10 mostraram aumento da frequ��ncia nos n��veis basais, que diminuiu ap��s est��mulo com LPS e SEB. Em contraste, a produ����o de IFN-y aumentou em resposta ao LPS enquanto diminuiu para CpG. As c��lulas T CD4+ polifuncionais, secretoras de 5 citocinas simult��neas (CD4+IL-17+IL-22+TNF+IL-10+IFN-y+) diminu��ram no LP ap��s est��mulo com CL097 e CpG. Entretanto, na aus��ncia de IL-10, houve aumento da frequ��ncia de c��lulas CD4+IL-17+IL-22+TNF+IFN-y+ em resposta ao LPS. Um aumento na polifuncionalidade foi observado em c��lulas TCD4+ que expressam CD38, marcador de ativa����o cr��nica e na aus��ncia de IL-10. Similarmente, ��s TCD4+, uma diminui����o de c��lulas T CD8+ IFN-y+ e TNF+ foram detectadas ap��s est��mulo com CpG. 3) As c��lulas Th22/Tc22 nos n��veis basais e ap��s est��mulo com SEB se mostraram aumentadas. As c��lulas Th17 n��o mostraram diferen��as entre os grupos. 4) A frequ��ncia das c��lulas T CD4+ e CD8+ reg totais (CD25+Foxp3+CD127low/-) est�� elevada no LP. Quanto aos perfis de matura����o, h�� aumento na frequ��ncia de c��lulas TCD4+ de mem��ria efetora enquanto que para as c��lulas T CD8+ h�� predom��nio das c��lulas de mem��ria central. Quanto aos subtipos, h�� aumento nas c��lulas T CD4+ regs perif��ricas (pT reg). CONCLUS��ES: O estado de ativa����o das mDCs ap��s ativa����o das vias de TLRs 4 e 7/8 pode influenciar na gera����o de resposta T efetoras no LP. O perfil de resposta monofuncional e polifuncional aos est��mulos TLRs reflete a ativa����o destas c��lulas no sangue perif��rico. Al��m disso, o aumento de Th22/Tc22 e das c��lulas T regs indicam uma rela����o entre regula����o e c��lulas efetoras no sangue perif��rico evidenciando que existem altera����es extracut��neas no LP

Single-cell derived clones from human adipose stem cells present different immunomodulatory properties

Human adipose mesenchymal stem cells are a heterogeneous population, where cell cultures derived from single cell-expanded clones present varying degrees of differential plasticity. This work focuses on the immunomodulatory/anti-inflammatory properties of these cells. To this end, 5 single cell clones were isolated (generally called 1.X and 3.X) from 2 volunteers. Regarding the expression level of the lineage-characteristic surface antigens, clones 1.10 and 1.22 expressed the lowest amounts, while clones 3.10 and 3.5 expressed more CD105 than the rest and clone 1.7 expressed higher amounts of CD73 and CD44. Regarding cytokine secretion, all clones were capable of spontaneously releasing high levels of IL-6 and low to moderate levels of IL-8. These differences can be explained in part by the distinct methylation profile exhibited by the clones. Furthermore and after lipopolysaccharide stimulation, clone 3.X produced the highest amounts of pro-inflammatory cytokines such as IL-1��, while clones 1.10 and 1.22 highly expressed IL-4 and IL-5. In co-culture experiments, clones 1.X are altogether more potent inhibitors than clones 3.X for proliferation of total, CD3+T, CD4+T and CD8+T lymphocytes and NK cells. The results of this work indicates that adipose stem cell population is heterogeneous in cytokine production profile, and that isolation, characterization and selection of the appropriate cell clone is a more exact method for the possible treatment of different patients or pathologies.