In situ analysis of FOXP3+ regulatory T cells in human colorectal cancer

The immune system spontaneously responds to tumor-associated antigens in peripheral blood of colorectal cancer (CRC) patients. Regulatory T cells (Treg) are suspected of influencing the interaction between the tumor and immune system and thus the course of malignant diseases. However, the function of Tregs in the development of T cell responses and on the clinical course of CRC is not clear. We analyzed Treg infiltration (FOXP3 staining) in situ in 40 CRC patients and investigated whether there is a correlation to disease stage, systemic T cell response, and survival. Treg infiltration was significantly higher in CRC than in healthy colon. Stromal Treg infiltration was significantly higher than epithelial infiltration in CRC. Furthermore, Treg infiltration in the tumor was significantly higher in limited disease than in metastatic CRC. The average Treg infiltration rate in the tumor was non-significantly higher in patients without systemic TAA-specific T cell response. Survival did not differ between patients with high Treg infiltration and those with low Treg infiltration. In conclusion, a direct link between Treg infiltration in the tumor and the development of a systemic T cell response in CRC cannot be proven. However, local Treg infiltration was significantly higher in limited disease, in which a systemic TAA-directed T cell responses is less frequently observed

Ex vivo screening for immunodominant viral epitopes by quantitative real time polymerase chain reaction (qRT-PCR)

The identification and characterization of viral epitopes across the Human Leukocyte Antigen (HLA) polymorphism is critical for the development of actives-specific or adoptive immunotherapy of virally-mediated diseases. This work investigates whether cytokine mRNA transcripts could be used to identify epitope-specific HLA-restricted memory T lymphocytes reactivity directly in fresh peripheral blood mononuclear cells (PBMCs) from viral-seropositive individuals in response to ex vivo antigen recall. PBMCs from HLA-A*0201 healthy donors, seropositive for Cytomegalovirus (CMV) and Influenza (Flu), were exposed for different periods and at different cell concentrations to the HLA-A*0201-restricted viral FluM1(58–66 )and CMVpp65(495–503 )peptides. Quantitative real time PCR (qRT-PCR) was employed to evaluate memory T lymphocyte immune reactivation by measuring the production of mRNA encoding four cytokines: Interferon-γ (IFN-γ), Interleukin-2 (IL-2), Interleukin-4 (IL-4), and Interleukin-10 (IL-10). We could characterize cytokine expression kinetics that illustrated how cytokine mRNA levels could be used as ex vivo indicators of T cell reactivity. Particularly, IFN-γ mRNA transcripts could be consistently detected within 3 to 12 hours of short-term stimulation in levels sufficient to screen for HLA-restricted viral immune responses in seropositive subjects. This strategy will enhance the efficiency of the identification of viral epitopes independently of the individual HLA phenotype and could be used to follow the intensity of immune responses during disease progression or in response to in vivo antigen-specific immunization

Comparison of T-cell receptor repertoire restriction in blood and tumor tissue of colorectal cancer patients

Several immunotherapeutic approaches rely on antigen-specific T-cells. Restrictions in the T-cell receptor (TCR) repertoire were reported as indicator of anti-tumor cytotoxic T-lymphocyte (CTL) response in various tumor entities. It is unclear yet whether a TCR restriction in peripheral blood mirrors the tumor compartment. We compared the expression of TCR Vβ-families for the quantification of TCR repertoire alterations in blood and tissue samples from patients with colorectal carcinoma. Blood samples from patients with colorectal carcinoma and healthy volunteers and tissue samples of normal colonic mucosa and colorectal carcinoma were analyzed. Relative Vβ-family quantification was performed based on quantitative reverse transcribed PCR. Standard deviation and average mean of the single families were determined. Two variables describing the degree of Vβ-repertoire restriction were defined. Forty-eight blood samples and 37 tissue samples were analyzed. TCR repertoire restriction was higher in blood of tumor patients than in blood of healthy controls (p < 0.05). No difference in the degree of TCR repertoire restriction was found between carcinoma and unaffected colon tissue. We found no corresponding elevated TCR families among the different compartments blood, normal colon, and carcinoma tissue of the same patient. In conclusion, we observed a repertoire restriction in peripheral blood as well as in tumor tissue of cancer patients. However, in tumor tissue, repertoire alterations were comparable to normal mucosa, suggesting compartment-specific TCR distribution rather than alterations due to tumor-T-cell interaction questioning the presence of highly restricted clonal T-cell expansions in colorectal cancer as they have been described in other, assumingly more immunogenic tumor entities

T cell responses against tumor associated antigens and prognosis in colorectal cancer patients

INTRODUCTION: Spontaneous T cell responses against specific tumor-associated antigens (TAA) are frequently detected in peripheral blood of tumor patients of various histiotypes. However, little is known about whether these circulating, spontaneously occurring, TAA-reactive T cells influence the clinical course of disease. METHODS: Fifty-four HLA-A2 positive colorectal cancer patients had been analyzed for the presence of T cell responses against epitopes derived from the TAA Ep-CAM, her-2/neu, and CEA either by ELISPOT assay or by intracellular cytokine staining. Then, Kaplan-Meier survival analysis was performed comparing T-cell-responders and T-cell-non-responders. For comparison, a group of T-cell-non-responders was compiled stringently matched to T-cell-responders based on clinical criteria and also analyzed for survival. RESULTS: Sixteen out of 54 patients had a detectable T cell response against at least one of the three tested TAA. Two out of 21 patients (9.5%) with limited stage of disease (UICC I and II) and 14 out of 33 patients (42.4%) with advanced disease (UICC III and IV) were T cell response positive. Comparing all T-cell-responders (n = 16) and all T-cell-non-responders (n = 38), no survival difference was found. In an attempt to reduce the influence of confounding clinical factors, we then compared 16 responders and 16 non-responders in a matched group survival analysis; and again no survival difference was found (p = 0.7). CONCLUSION: In summary, we found no evidence that spontaneous peripheral T cell responses against HLA-A2-binding epitopes of CEA, her-2/neu and Ep-CAM are a strong prognostic factor for survival

Delayed polarization of mononuclear phagocyte transcriptional program by type I interferon isoforms

BACKGROUND: Interferon (IFN)-α is considered a key modulator of immunopathological processes through a signature-specific activation of mononuclear phagocytes (MPs). This study utilized global transcript analysis to characterize the effects of the entire type I IFN family in comparison to a broad panel of other cytokines on MP previously exposed to Lipopolysaccharide (LPS) stimulation in vitro. RESULTS: Immature peripheral blood CD14+ MPs were stimulated with LPS and 1 hour later with 42 separate soluble factors including cytokines, chemokines, interleukins, growth factors and IFNs. Gene expression profiling of MPs was analyzed 4 and 9 hours after cytokine stimulation. Four hours after stimulation, the transcriptional analysis of MPs revealed two main classes of cytokines: one associated with the alternative and the other with the classical pathway of MP activation without a clear polarization of type I IFNs effects. In contrast, after 9 hours of stimulation most type I IFN isoforms induced a characteristic and unique transcriptional pattern separate from other cytokines. These "signature" IFNs included; IFN-β, IFN-α2b/α2, IFN-αI, IFN-α2, IFN-αC, IFN-αJ1, IFN-αH2, and INF-α4B and induced the over-expression of 44 genes, all of which had known functional relationships with IFN such as myxovirus resistance (Mx)-1, Mx-2, and interferon-induced hepatitis C-associated microtubular aggregation protein. A second group of type I IFNs segregated separately and in closer association with the type II IFN-γ. The phylogenetic relationship of amino acid sequences among type I IFNs did not explain their sub-classification, although differences at positions 94 through 109 and 175 through 189 were present between the signature and other IFNs. CONCLUSION: Seven IFN-α isoforms and IFN-β participate in the late phase polarization of MPs conditioned by LPS. This information broadens the previous view of the central role played by IFN-α in autoimmunity and tumor rejection by including and/or excluding an array of related factors likely to be heterogeneously expressed by distinct sub-populations of individuals in sickness or in response to biological therapy

Selection and validation of endogenous reference genes using a high throughput approach

BACKGROUND: Endogenous reference genes are commonly used to normalize expression levels of other genes with the assumption that the expression of the former is constant in different tissues and in different physiopathological conditions. Whether this assumption is correct it is, however, still matter of debate. In this study, we searched for stably expressed genes in 384 cDNA array hybridization experiments encompassing different tissues and cell lines. RESULTS: Several genes were identified whose expression was highly stable across all samples studied. The usefulness of 8 genes among them was tested by normalizing the relative gene expression against test genes whose expression pattern was known. The range of accuracy of individual endogenous reference genes was wide whereas consistent information could be obtained when information pooled from different endogenous reference genes was used. CONCLUSIONS: This study suggests that even when the most stably expressed genes in array experiments are used as endogenous reference, significant variation in test gene expression estimates may occur and the best normalization is achieved when data from several endogenous reference genes are pooled together to minimize minimal but significant variation among samples. We are presently optimizing strategies for the preparation of endogenous reference gene mixtures that could yield information comparable to that of data pooled from individual endogenous reference gene normalizations

Tumor-infiltrating macrophages and dendritic cells in human colorectal cancer: relation to local regulatory T cells, systemic T-cell response against tumor-associated antigens and survival

<p>Abstract</p> <p>Introduction</p> <p>Although systemic T-cell responses against tumor antigens and tumor infiltration by T cells have been investigated in colorectal cancer (CRC), the initiation of spontaneous immune responses <it>in situ </it>is not well understood. Macrophages and dendritic cells (DC) play an important role as a link between innate and adaptive immune response. The aim of the present study was to analyze macrophage and DC infiltration in CRC and to investigate whether there is a correlation to systemic T-cell response, regulatory T cell (Treg) infiltration, and survival.</p> <p>Methods</p> <p>Immunohistological staining was performed with nine markers for macrophages and DC (CD68, CD163, S100, CD11c, CD208, CD209, CD123, CD1a, Langerin) in 40 colorectal cancer samples from patients, in whom the state of systemic T-cell responses against tumor-associated antigens (TAA) and Treg infiltration had previously been determined.</p> <p>Results</p> <p>All specimens contained cells positive for CD68, CD163, S100 and CD1a in epithelial tumor tissue and tumor stroma. Only a very few (less than median 3/HPF) CD123+, CD1a+, CD11c+, CD 208+, CD209+, or Langerin+ cells were detected in the specimens. Overall, we found a trend towards increased infiltration by S100-positive DC and a significantly increased number of stromal S100-positive DC in patients without T-cell response. There was an increase of stromal S100 DC and CD163 macrophages in limited disease (S100: 11.1/HPF vs. 7.3/HPF, p = 0.046; CD163: 11.0/HPF vs. 8.1/HPF, p = 0.06). We found a significant, positive correlation between S100-positive DC and FOXP3-positive Tregs. Survival in patients with high DC infiltration was significantly better than that in those with low DC infiltration (p < 0.05). Furthermore, we found a trend towards better survival for increased infiltration with CD163-positive macrophages (p = 0.07).</p> <p>Conclusion</p> <p>The present <it>in situ </it>study adds new data to the discussion on the interaction between the innate and adoptive immune system. Our data strongly support the hypothesis that tumor-infiltrating DC are a key factor at the interface between innate and adaptive immune response in malignant disease. Tumor infiltrating S100-positive DC show an inverse relationship with the systemic antigen-specific T-cell response, a positive correlation with regulatory T cells, and a positive association with survival in CRC. These data put tumor-infiltrating DC at the center of the relevant immune response in CRC.</p

Antigen-specific T cell immunity and antigen-presenting cells in malignant diseases

TITEL UND INHALTSVERZEICHNIS 1\. EINLEITUNG 2\. EIGENE ARBEITEN 3\. DISKUSSION 4\. ZUSAMMENFASSUNG 5\. LITERATURVERZEICHNISDie Auseinandersetzung des Immunsystems mit neoplastischen Zellen ist seit Jahrzehnten Gegenstand wissenschaftlicher Debatten. Im Wesentlichen gibt es zwei Hauptannahmen zu dieser Interaktion. Die eine Theorie besagt, dass das Immunsystem durch Entzündungsreize, wie zum Beispiel Zytokine, zu Proliferation und Metastasierung eines Tumors beitragen kann (Immunstimulation); die andere Theorie hingegen geht davon aus, dass das Immunsystem einen protektiven Effekt hat und das Tumorwachstum überwachen und, wenn vielleicht nicht komplett verhindern, so doch zumindest eingrenzen kann (Immunosurveillance). In der vorliegenden Habilitationsschrift wurden zwei wesentliche Elemente der Interaktion zwischen Tumor und Immunsystems näher untersucht: Antigen (Ag)-spezifische T-Zellen und Ag-präsentierende Zellen. Diese Zellpopulationen spielen sowohl für die spontane Entwicklung Tumor- gerichteter Immunantworten als auch für Entwicklung immuntherapeutischer Strategien gegen Tumoren eine wichtige Rolle. Bis vor einigen Jahren war es unklar, ob T-Zellen, die sich spezifisch gegen Tumor-assoziierte Antigene (TAA) richten, im Blut von Tumorpatienten ohne vorherige Immuntherapie vorhanden sind. Wir konnten nachweisen, dass solche spezifischen T-Zellen im peripheren Blut von Kolorektalkarzinom (CRC)-Patienten existieren. Es konnte außerdem gezeigt werden, dass sich im peripheren Blut von CRC-Patienten deutlich häufiger TAA-spezifische T-Zellen finden lassen als im Blut von Mammakarzinompatientinnen. Die Ursache für diese Beobachtung bleibt unklar, könnte jedoch im unterschiedlichen Tumor-Mikroenvironment oder in einem unterschiedlichen Homing-Verhalten der T-Zellen liegen. Die gegen die TAA CEA, EpCAM und her-2/neu gerichteten T-Zellen beim CRC konnten einem zytotoxischen Zelltyp zugeordnet werden. Es war zudem besonders auffällig, dass diese T-Zellen signifikant häufiger im metastasierten Stadium des CRC auftraten. Lymphknotenkontakt des Tumors als Voraussetzung für die Entwicklung einer T -Zell-Antwort und Tumor Escape Mechanismen sind potentielle Ursachen für diesen Befund. Ein Überlebensvorteil für die Patienten mit spontanen TAA- spezifischen T-Zellen im peripheren Blut konnte nicht gezeigt werden. Ebenso konnte kein direkter Zusammenhang der systemischen, TAA-spezifischen T-Zell- Antwort mit einer lokalen Infiltration durch regulatorische T-Zellen (Treg) nachgewiesen werden. Interessanterweise war die Treg-Infiltration im Tumor signifikant erhöht im limitierten Stadium des CRC, in dem wiederum auch weniger häufig systemische, TAA-gerichtete T-Zell-Antworten beschrieben sind. Dies könnte ein Hinweis auf einen indirekten Zusammenhang zwischen den beiden Zell-Populationen sein. Eine der mittlerweile gängigsten Methoden zur Analyse TAA-spezifischer T-Zellen ist die Tetramer-Färbung. In einer Methodenarbeit haben wir herausgearbeitet, dass CD20 und CD4 als Marker für eine negative Selektion zur Erhöhung der Spezifität des Assays verwendet werden können. In der vorliegenden Arbeit wurden verschiedene Aspekte Ag-präsentierender Zellen (APC) mit besonderem Bezug zu tumorimmuntherapeutischen Überlegungen untersucht. Im Rahmen von Vakzinierungen gegen maligne Erkrankungen werden häufig reife DC verwendet, um eine besonders starke Immunantwort zu induzieren. Wir haben das Zytokin-Repertoire, das während der Reifung der DC sezerniert wird, mittels Protein-Profiling Assays untersucht und dabei Zytokine und Chemokine nachgewiesen, die die Ag-Aufnahme und die Aktivierung des unspezifischen Immunsystems vermitteln (TNF[alpha], IL-10, IL-12, IFN[gamma]), verschiedene Immunzellpopulationen anlocken (MIP1[alpha], MIP1[beta], IL-8, MCP-1, MDC, RANTES, MIG, IP-10, Eotaxin) und schließlich T-Zellen aktivieren (IL-2, IL-6, IL-7). Es ist anzunehmen, dass sich die DC während einer in vitro Reifung in Bezug auf ihre Zytokinausschüttung erschöpfen. APC selbst sind in vivo auch einer Vielzahl von Zytokinen und Chemokinen im Rahmen eines Entzündungsprozesses oder einer Tumor-Immunsystem- Interaktion ausgesetzt. In einer Genexpressionsanalyse haben wir gezeigt, dass LPS-aktivierte mononukleäre Phagozyten (MP) durch eine große Bandbreite von Zytokinen und Chemokinen stark polarisiert stimuliert werden. Zwei Zytokinhauptklassen wurden identifiziert, die entweder den klassischen (IFN[gamma], M1) oder den alternativen Weg (IL-4, M2) der MP Aktivierung induzierten. Unsere Befunde legen zudem eine zentrale Rolle des NF[kappa]B in der Steuerung der MP-Aktivierung/Differenzierung nahe. Neben der Zytokin-APC- Interaktion ist auch eine effektive Epitoppräsentation für die Ausprägung einer Immunantwort wichtig. Wir haben ein rekombinantes Vacciniavirus als Vektor verwendet, um das TAA gp100 in DC zu expremieren. Das Ag ließ sich in reifen und unreifen DC vergleichbar gut expremieren. Die Reifungsmarker der DC wurden nach der Infektion nur wenig herunterreguliert. Die spezifische T-Zell- Stimulation war mit dem nativen gp100 Epitop g209 jedoch sehr gering, konnte allerdings durch Verwendung des modifizierten, stärker HLA-bindenden gp100 Epitops g209-2M wiederhergestellt werden. Die direkte T-Zell-Stimulation durch reife und unreife DC war dabei gleich. Eine rVV-Infektion zur Expression von Ag kann also durchaus nach der DC Reifung erfolgen. In einer weiteren Analyse fanden wir, dass die erhöhte HLA-Affinität des modifizierten gp100 Epitops sogar eine geringere proteasomale Spaltung kompensieren kann. Die HLA- Affinität des Epitops scheint einer der zentralen Punkte für die Aktivierung von T-Zellen zu sein. Schließlich haben wir in einer Übersichtsarbeit, die über 500 Patienten mit metastasiertem CRC einschloss, die klinische und immunologische Wirksamkeit einer aktiven spezifischen Immunisierung evaluiert. Während sich humorale und zelluläre Immunantworten bei etwa 50% der Patient zeigten, fand sich nur bei etwa 1% der Patienten ein komplette oder partielle Remission des CRC. Die Debatte um Immunstimulation und Immunosurveillance von Tumoren wird nicht so bald beendet sein. Wahrscheinlich treten im zeitlichen Verlauf einer Tumorerkrankung gegensätzliche tumortoxische wie proliferogene - Funktionen des Immunsystems auf. Hier wurde gezeigt, dass das CRC grundsätzlich ein immunogener Tumor ist, gegen dessen Antigene sich eine spontane T-Zell-Antwort entwickeln kann. Es scheinen bisher jedoch weder die spontanen noch die Vakzine-induzierten T-Zell-Antworten im peripheren Blut einen wesentlichen Einfluss auf den Verlauf der Erkrankung zu haben. Es wird aber auch klar, dass das Immunsystem ein großes Potential für die Erkennung und damit die Behandlung von Tumoren hat. Die vorgelegten Arbeiten leisten wichtige Beiträge nicht nur zur Analyse TAA-spezifischer T-Zellen bei Tumoren sondern auch zum Verständnis der Rolle der Zytokine bei der Aktivierung Ag- präsentierender Zellen und zu Mechanismen der Epitop-Präsentation durch DC. Diese Informationen stellen Grundlagen für die rationale, dringend notwendige Weiterentwicklung immuntherapeutischer Ansätze bei malignen Erkrankungen dar.The interaction between immune system and malignant cells has been a matter of scientific debate for decades. There are basically two major theories regarding this interaction. One assumption is that the immune system may contribute to tumor proliferation and spread by inflammational stimuli, e.g. cytokines (immunostimulation); the other theory is based on the assumption that the immune system has got a protective effect and is able to control tumor growth (immunosurveillance). Two main elements of the interaction between tumor and immune system were analyzed in-depth in the present work ( Habilitationsschrift ): antigen (Ag)-specific T cells and Ag-presenting cells. These two cell populations play an important role for spontaneous development of tumor-directed responses as well as for rational development of immunotherapeutic strategies against tumors. For a long time it has been unknown whether T cells specifically directed against tumor-associated antigens (TAA), exist in peripheral blood of tumor patients without prior immunotherapy. We have shown that TAA-specific T cells exist in peripheral blood of patients with colorectal cancer (CRC). Additionally we found that TAA-specific T cells were significantly more often found in colorectal cancer patients than in breast cancer patients. The reason for this observation remains unknown. Different tumor microenvironments or different T cell homing behavior could be possible explanations. T cell responses directed against TAA CEA, EpCAM, and her-2/neu were identified as cytotoxic phenotype. Furthermore, it was very interesting to see that these T cell responses were significantly more frequent in patients with metastatic CRC than in limited CRC. Lymph node involvement as prerequisite for the development of specific T cell responses and tumor escape mechanisms are potential reasons for this observation. A survival benefit for patients with systemic TAA-directed T cell responses was not shown. Furthermore, no direct correlation was found between systemic TAA- specific T cell response and local infiltration by regulatory T cells (Treg). Interestingly, Treg infiltration in the tumor was significantly increased in limited stage of CRC, in which in turn TAA-directed T cell responses are less frequently described. This might indicate an indirect interrelation between these two T cell populations. One well-established method for analyzing TAA- specific T cells is tetramer staining. We have described that CD20 and CD4 may be used as markers for negative selection to increase the specificity of this assay. In the present work various aspects of Ag-presenting cells (APC) were analyzed with specific regard to immunotherapeutic considerations. Mature DC are often used for immunization against malignant diseases to induce strong immune responses. We analyzed the cytokine repertoire, which is secreted during DC maturation, by protein profiling assays. At this, we found cytokines and chemokines, which mediate Ag-uptake and activation of the innate immune system (TNF[alpha], IL-10, IL-12, IFN[gamma]), which attract various immune cell populations (MIP1[alpha], MIP1[beta], IL-8, MCP-1, MDC, RANTES, MIG, IP-10, Eotaxin) and, eventually, activate T cells (IL-2, IL-6, IL-7). It can be assumed that DC exhaust during in vitro maturation with regard to their cytokine secretion. APC are in vivo confronted with numerous cytokines and chemokines during an inflammation or during a tumor/immune system interaction. In a gene expression analysis, we have shown that LPS-activated mononuclear phagocytes (MP) are stimulated and polarized by a broad range of cytokines and chemokines Two major cytokine classes were identified, which either induced the classical (IFN[gamma], M1) or the alternative pathway (IL-4, M2) of MP activation. Our findings additionally suggest a central role of NF[kappa]B in controlling MP-activation/differentiation. Besides cytokine-APC-interaction, also an effective epitope presentation is important for the occurrence of an immune response. We used a recombinant vaccinia virus as a vector to express TAA gp100 in DC. The Ag was expressed at similar levels in both mature and immature DC. DC maturation markers were only minimally down regulated after infection. Specific T cell stimulation was very low with the native gp100 epitope g209; it was, however, restored by using the modified, gp100 epitope g209-2M, which has a stronger HLA-binding. Direct T cell stimulation by mature and immature DC was similar at this point. An rVV infection to express Ag may, thus, follow the DC maturation. In a further analysis, we found that increased HLA affinity of the modified gp100 epitope may even compensate for less proteasomal cleavage. HLA-affinity of epitopes seems to stand at the center of T cell activation. Finally, we evaluated clinical and immunological responses after active specific immunization in a review, which included more than 500 patients with metastatic CRC. While 50% of patients showed a humoral or cellular immunological response only 1% of patients had a complete or partial clinical remission after vaccination. The debate on immunostimulation and immunosurveillance of tumors will not be resolved in a near future. It seems probable that various, partly opposing tumor toxic as well as tumor growth supporting immunological functions appear during the time course of a malignant disease. It was shown that CRC is an immunogenic tumor and that spontaneous T cell responses may develop against its Ags. So far, neither spontaneous nor vaccine-induced T cell responses in peripheral blood seem to substantially influence the course of disease. However, it is clear that the immune system has got a great potential for detection and, thus, treatment of tumors. The presented scientific works contribute to the analysis of TAA- specific T cells in tumors and to the understanding of the role of cytokines in the activation of Ag-presenting cells, and mechanisms of epitope presentation by DC. This knowledge represents a basis for the rational development of immunotherapeutic approaches of malignant diseases