Modulation of intestinal inflammation via antigen-targeting to DEC-205 on dendritic cells

Morbus Crohn und Kolitis ulcerosa sind zwei die Hauptformen chronisch-entzündlicher Darmerkrankungen (CED) im Menschen. Für die Behandlung von CED sind derzeit nur wenige Therapieansätze verfügbar. Die wirksamste Therapieform stellt die Behandlung mit anti-TNF-α Antikörpern dar, allerdings geht dieser Ansatz mit starken Nebenwirkungen einher und führt bei einem Großteil der Patienten nicht zur Verbesserung des klinischen Bildes. Aus diesen Gründen ist es notwendig, neue und spezifischere Therapieansätze für CED zu entwickeln. Der immunologische Toleranzverlust gegenüber Nahrungsantigenen und / oder Antigenen kommensaler Bakterien scheint ein Auslöser für die Entwicklung von CED zu sein. Daher ist ein vielversprechender Ansatz für die Unterbindung dieser Entzündungsprozesse die Induktion immunologischer Toleranz im Darm. In früheren Studien konnte gezeigt werden, dass die spezifische Antigenbeladung unreifer dendritischer Zellen (DCs) über den Oberflächenrezeptor DEC-205 zur Toleranzvermittlung u.a. über die Induktion Foxp3+ regulatorischer T-Zellen (Treg) und damit zur Unterbindung von Entzündungsprozessen in verschiedenen Mausmodellen führt. Ob dieser Ansatz auch für die Toleranzinduktion im Darm genutzt werden kann, ist unklar. In der vorliegenden Arbeit konnte erstmals gezeigt werden, dass das Antigen Targeting über DEC-205 zur effizienten Konversion naiver, antigen-spezifischer CD4+ T-Zellen in Foxp3+ Treg im Darm führt. Bemerkenswerterweise konnte über diesen Ansatz die Induktion intestinaler Entzündung in einem neu etablierten Mausmodell unterdrückt werden. Darüber hinaus zeigen die Ergebnisse dieser Arbeit, dass die DEC-205 vermittelte Antigenbeladung zur Modulation der Migration differenzierter, antigen-spezifischer TH1-Zellen führt, die eine wichtige Rolle bei humanen CED spielen. Dieser Ansatz führte in einem TH1-Zell vermittelten Darmentzündungsmodell durch die reduzierte Migration der Effektorzellen in den Darm zu einem deutlichen Schutz vor der Entzündung. Interessanterweise konnte durch das DEC-205 vermittelte Antigen Targeting eine verminderte Expression des Chemokinrezeptors CXCR3 auf den TH1 Zellen beobachtet werden, was zu einer reduzierten Migration der Zellen in den Darm führte. In weiteren Analysen, in denen der Mechanismus hinter diesen Beobachtungen untersucht wurde, konnte gezeigt werden, dass es nach Antigen Targeting über DEC-205 zu einer verstärkten IL-10-Sekretion durch unreife DCs kommt. Bemerkenswerterweise führte die erhöhte IL-10-Expression zur direkten Einschränkung der TH1 Zellmigration in den Darm, da die in vivo Neutralisierung von IL 10 die Aufhebung des DEC-205 vermittelten Schutzes im untersuchten Entzündungsmodell zur Folge hatte. Die Analyse von humanen CED Proben ergab, dass der prozentuale Anteil von CD4+CXCR3+ T-Zellen im Blut der Patienten reduziert, in der entzündeten Darmmukosa allerdings stark erhöht ist. Dies deutet auf eine verstärkte Migration von CD4+CXCR3+ T-Zellen während eines akuten Entzündungsschubes aus der Peripherie in die intestinale Mukosa hin. Interessanterweise wird der systemische Block der Effektorzellmigration derzeit in klinischen Studien als therapeutischer Ansatz für die Behandlung humaner CED untersucht. Zusammengefasst zeigen die Ergebnisse dieser Arbeit deutlich, dass das Antigen Targeting über DEC-205 zu einem deutlichen Schutz vor intestinaler Entzündung führt. Dieser Schutz wird durch die Modulation der CXCR3-abhängigen Migration von TH1-Zellen in den Darm vermittelt, so dass ein neuer, hochspezifischer Ansatz für die Behandlung von CED identifiziert werden konnte. In diesem Zusammenhang wurde erstmals gezeigt, dass IL-10 in der Lage ist das Migrationsverhalten von Effektorzellen in Entzündungsherde zu beeinflussen und u.a. auf diese Weise anti inflammatorische Funktionen ausübt.Crohn´s disease and ulcerative colitis are the two major types of inflammatory bowel disease (IBD) in humans. Therapeutic options for IBD to date are rare with anti TNF α medication being most efficacious. However, accompanied by side effects and a significant fraction of non-responders there is need for new, more specific therapies for IBD. The loss of tolerance towards commensal gut bacteria and / or food antigens seems to be an initial event for the manifestation of disease. Therefore, a promising approach for the prevention of IBD is the induction of tolerance in the gut. Antigen targeting to DEC-205 on immature dendritic cells (DCs) was reported to induce tolerance in different experimental disease settings, i.e. by the induction of Foxp3+ regulatory T cells (Treg). However, whether this approach is sufficient to also induce tolerance in the intestine is currently unknown. In the present study it was demonstrated for the first time that antigen-targeting to DEC 205 leads to the efficient conversion of naive, antigen-specific CD4+ T cells into Foxp3+ Treg in the gut. Remarkably, this approach resulted in a significant amelioration of disease in a newly established mouse model of intestinal inflammation. More important, this study provides evidence that targeting antigens to DEC-205 leads to the interference with migratory properties of fully differentiated, antigen specific TH1 effector cells, some of the key mediators of human IBD. In a TH1-cell-mediated mouse model of intestinal inflammation DEC-205-mediated antigen-targeting led to the prevention of inflammation via the reduced migration of TH1 cells into intestinal compartments. Further experiments revealed that the chemokine receptor CXCR3 was down-regulated on TH1 cells with this approach, thereby limiting recruitment of the effector cells into the gut. Further investigations on the mechanism behind these observations showed that antigen-targeting to DEC-205 induces high secretion of anti-inflammatory IL-10 by immature DCs. Strikingly, IL-10 directly interfered with migratory properties of TH1 cells as the in vivo neutralization of IL-10 abolished the protective effect of antigen-targeting to DEC-205 in the examined inflammation model by the significant reduction of TH1 cell migration to the gut. Moreover, analysis of human IBD samples revealed a reduced percentage of CD4+CXCR3+ T cells in peripheral blood but increased numbers of these cells in the inflamed mucosa suggesting a preferential migration of CD4+CXCR3+ T cells from the periphery to the intestinal mucosa during acute inflammation. Interestingly, the systemic block of effector cell migration to the gut is currently under investigation in clinical trials as therapeutic approach for IBD. In summary, the results from the present study clearly show that antigen-targeting to DEC-205 significantly reduces inflammation of the gut in different inflammatory settings. Protection from inflammation was achieved through the impairment of CXCR3 dependent TH1 effector cell migration to the gut, providing a novel, highly specific therapeutic approach for IBD. Importantly, it could be demonstrated for the first time that IL-10 is capable of interfering with migratory abilities of effector T cells to sites of inflammation, thereby mediating its anti-inflammatory properties

Negative Selection by an Endogenous Retrovirus Promotes a Higher-Avidity CD4+ T Cell Response to Retroviral Infection

Effective T cell responses can decisively influence the outcome of retroviral infection. However, what constitutes protective T cell responses or determines the ability of the host to mount such responses is incompletely understood. Here we studied the requirements for development and induction of CD4+ T cells that were essential for immunity to Friend virus (FV) infection of mice, according to their TCR avidity for an FV-derived epitope. We showed that a self peptide, encoded by an endogenous retrovirus, negatively selected a significant fraction of polyclonal FV-specific CD4+ T cells and diminished the response to FV infection. Surprisingly, however, CD4+ T cell-mediated antiviral activity was fully preserved. Detailed repertoire analysis revealed that clones with low avidity for FV-derived peptides were more cross-reactive with self peptides and were consequently preferentially deleted. Negative selection of low-avidity FV-reactive CD4+ T cells was responsible for the dominance of high-avidity clones in the response to FV infection, suggesting that protection against the primary infecting virus was mediated exclusively by high-avidity CD4+ T cells. Thus, although negative selection reduced the size and cross-reactivity of the available FV-reactive naïve CD4+ T cell repertoire, it increased the overall avidity of the repertoire that responded to infection. These findings demonstrate that self proteins expressed by replication-defective endogenous retroviruses can heavily influence the formation of the TCR repertoire reactive with exogenous retroviruses and determine the avidity of the response to retroviral infection. Given the overabundance of endogenous retroviruses in the human genome, these findings also suggest that endogenous retroviral proteins, presented by products of highly polymorphic HLA alleles, may shape the human TCR repertoire that reacts with exogenous retroviruses or other infecting pathogens, leading to interindividual heterogeneity

IL10-Deficiency in CD4+ T Cells Exacerbates the IFNγ and IL17 Response During Bacteria Induced Colitis

Background/Aims: IL10 is a key inhibitor of effector T cell activation and a mediator of intestinal homeostasis. In addition, IL10 has emerged as a key immunoregulator during infection with various pathogens, ameliorating the excessive T-cell responses that are responsible for much of the immunopathology associated with the infection. Because IL10 plays an important role in both intestinal homeostasis and infection, we studied the function of IL10 in infection-associated intestinal inflammation. Methods: Wildtype mice and mice deficient in CD4+ T cell-derived or regulatory T cells-derived IL10 were infected with the enteric pathogen Citrobacter (C.) rodentium and analyzed for the specific immune response and pathogloy in the colon. Results: We found that IL10 expression is upregulated in colonic tissue after infection with C. rodentium, especially in CD4+ T cells, macrophages and dendritic cells. Whereas the deletion of IL10 in regulatory T cells had no effect on C. rodentium induced colitis, infection of mice deficient in CD4+ T cell-derived IL10 exhibited faster clearance of the bacterial burden but worse colitis, crypt hyperplasia, and pathology than did WT mice. In addition, the depletion of CD4+ T cell-derived IL10 in infected animals was accompanied by an accelerated IFNγ and IL17 response in the colon. Conclusion: Thus, we conclude that CD4+ T cell-derived IL10 is strongly involved in the control of C. rodentium-induced colitis. Interference with this network could have implications for the treatment of infection-associated intestinal inflammation

IL10-Deficiency in CD4+ T Cells Exacerbates the IFNγ and IL17 Response During Bacteria Induced Colitis

Background/Aims: IL10 is a key inhibitor of effector T cell activation and a mediator of intestinal homeostasis. In addition, IL10 has emerged as a key immunoregulator during infection with various pathogens, ameliorating the excessive T-cell responses that are responsible for much of the immunopathology associated with the infection. Because IL10 plays an important role in both intestinal homeostasis and infection, we studied the function of IL10 in infection-associated intestinal inflammation. Methods: Wildtype mice and mice deficient in CD4+ T cell-derived or regulatory T cells-derived IL10 were infected with the enteric pathogen Citrobacter (C.) rodentium and analyzed for the specific immune response and pathogloy in the colon. Results: We found that IL10 expression is upregulated in colonic tissue after infection with C. rodentium, especially in CD4+ T cells, macrophages and dendritic cells. Whereas the deletion of IL10 in regulatory T cells had no effect on C. rodentium induced colitis, infection of mice deficient in CD4+ T cell-derived IL10 exhibited faster clearance of the bacterial burden but worse colitis, crypt hyperplasia, and pathology than did WT mice. In addition, the depletion of CD4+ T cell-derived IL10 in infected animals was accompanied by an accelerated IFNγ and IL17 response in the colon. Conclusion: Thus, we conclude that CD4+ T cell-derived IL10 is strongly involved in the control of C. rodentium-induced colitis. Interference with this network could have implications for the treatment of infection-associated intestinal inflammation

Detection of env-specific CD4⁺ T cells by A^b-env_123-141 tetramer eclipsed by antigen-induced TCR downregulation.

<p>(A) A^b-hCLIP (control) or A^b-env_123-141 tetramer staining in total CD4⁺ T cells isolated from the spleen of wild-type B6 mice 7 days post FV infection. Plots are representative of 7 mice. (B) Frequency of Vα2 cells in either bulk naïve (CD44^lo), bulk memory (CD44^hi) or A^b- env_123-141 tetramer⁺ CD4⁺ T cells from the same FV infected mice. Horizontal short lines in naïve and memory subsets denote the mean frequency of Vα2 cells in the same populations from uninfected mice. Each symbol represents an individual mouse. (C–F) CD45.1⁺ EF4.1 CD4⁺ T cells were adoptively transferred into wild-type B6 recipients that were infected with FV the same day. (C) A^b-env_123-141 tetramer staining in host (CD45.1⁻) or donor (CD45.1⁺) CD4⁺ T cells according to TCRα or TCRβ staining. Gates in donor CD4⁺ T cells are set around the median TCRα and TCRβ staining, respectively. (D) Percentage of A^b-env_123-141 tetramer⁺ cells in donor CD4⁺ T cells with TCRβ (<i>left</i>) or TCRα (<i>right</i>) staining below or above the median. (E) A^b-hCLIP or A^b-env_123-141 tetramer staining in host or donor CD4⁺ T cells from the same recipients assessed directly <i>ex vivo</i> (<i>top</i>) or following 3-day <i>in vitro</i> culture in the absence of antigenic stimulation (<i>bottom</i>). (F) Percentage of A^b-env_123-141 tetramer⁺ cells in donor CD4⁺ T cells before and after <i>in vitro</i> culture. In (D) and (F) each symbol represents an individual mouse from one of two experiments.</p

Genetic contribution to a high-avidity env-reactive CD4⁺ T cell repertoire.

<p>(A) Frequency of env_124-138L- reactive cells in Vα2 or non-Vα2 primary CD4⁺ T cells isolated from either B6 (B6-EF4.1) or 129S8 (129S8-EF4.1) EF4.1 mice. (B) Functional avidity of env_124-138L-reactive Vα2 or non-Vα2 primary CD4⁺ T cells from the same donors in A. (C) Frequency of Vα2 cells in env_124-138L-reactive CD4⁺ T cells from the same donors in A as a function of peptide concentration. (D) Frequency of env_124-138L- reactive cells in Vα2 or non-Vα2 primary CD4⁺ T cells isolated from either B6×129S8-EF4.1 F₁, B6-<i>Emv2</i>^−/−×129S8-EF4.1 F₁ or B6-<i>Tcra</i>^−/−×129S8-EF4.1 F₁, EF4.1 mice. (E) Functional avidity of env_124-138L-reactive Vα2 or non-Vα2 primary CD4⁺ T cells from the same donors in D. (F) Frequency of Vα2 cells in env_124-138L-reactive CD4⁺ T cells from the same donors in D as a function of peptide concentration. Numbers in (B) and (E) represent the ED₅₀. In (C) and (F) the CD4⁺ T cell response elicited by the last peptide dose (10⁻⁸ M) was too small to allow accurate measurement of the frequency of Va2 cells and was therefore omitted. Data in (A–F) are the means ± SEM (<i>n</i> = 4–8) of 18-hr stimulations from 3 experiments.</p

<i>Emv2</i> preferentially selects against non-Vα2 env-specific CD4⁺ T cells.

<p>(A) Dose-response to env_124-138L stimulation of CD4⁺ T cells isolated from either B6 (B6-EF4.1) or <i>Emv2</i>-deficient B6 (B6-EF4.1 <i>Emv2</i>^−/−) EF4.1 mice. (B) Frequency of env_124-138L-specific cells in Vα2 or non-Vα2 primary CD4⁺ T cells from the same donors. (C) Functional avidity of <i>Emv2</i>-selected (B6-EF4.1) or -nonselected (B6-EF4.1 <i>Emv2</i>^−/−) EF4.1 CD4⁺ T cells for env_124-138L. (D) Frequency of Vα2 cells in env_124-138L-specific CD4⁺ T cells from the same donors as a function of peptide concentration. (E) Frequency of env_124-138Y-specific cells in Vα2 or non-Vα2 primary CD4⁺ T cells from the same donors. (F) Functional avidity of <i>Emv2</i>-selected (B6-EF4.1) or -nonselected (B6-EF4.1 <i>Emv2</i>^−/−) EF4.1 CD4⁺ T cells for env_124-138Y. Numbers in (C) and (F) represent the ED₅₀. Data in (A–F) are the means ± SEM (<i>n</i> = 9–12) of 18-hr stimulations from 3 experiments.</p

<i>Emv2</i>-selected CD4⁺ T cells mount a predominantly high-avidity response.

<p>(A–C) CD45.2⁺ (<i>Ptprc</i>^2/2) CD4⁺ T cells isolated from either B6 (B6-EF4.1) or <i>Emv2</i>-deficient B6 (B6-EF4.1 <i>Emv2</i>^−/−) EF4.1 donor mice were adoptively transferred into <i>Ptprc</i>^1/2 B6 recipients that were infected with FV the same day and analyzed 7 days later. (A) Absolute number of total, Vα2 or non-Vα2 FV-responding (CD44^hi) donor (CD45.2⁺CD45.1⁻) CD4⁺ T cells isolated from the spleens of recipient mice according to donor type. (B) Flow cytometric example and (C) frequency of high-avidity Vα2 cells in responding CD4⁺ T cells according to donor type. In (A) and (C) each symbol is an individual mouse.</p