TY - JOUR A1 - Langenhorst, Daniela A1 - Tabares, Paula A1 - Gulde, Tobias A1 - Becklund, Bryan R. A1 - Berr, Susanne A1 - Surh, Charles D. A1 - Beyersdorf, Niklas A1 - Hünig, Thomas T1 - Self-recognition sensitizes mouse and human regulatory T cells to low-dose CD28 superagonist stimulation JF - Frontiers in Immunology N2 - In rodents, low doses of CD28-specific superagonistic monoclonal antibodies (CD28 superagonists, CD28SA) selectively activate regulatory T cells (Treg). This observation has recently been extended to humans, suggesting an option for the treatment of autoimmune and inflammatory diseases. However, a mechanistic explanation for this phenomenon is still lacking. Given that CD28SA amplify T cell receptor (TCR) signals, we tested the hypothesis that the weak tonic TCR signals received by conventional CD4\(^{+}\) T cells (Tconv) in the absence of cognate antigen require more CD28 signaling input for full activation than the stronger TCR signals received by self-reactive Treg. We report that in vitro, the response of mouse Treg and Tconv to CD28SA strongly depends on MHC class II expression by antigen-presenting cells. To separate the effect of tonic TCR signals from self-peptide recognition, we compared the response of wild-type Treg and Tconv to low and high CD28SA doses upon transfer into wild-type or H-2M knockout mice, which lack a self-peptide repertoire. We found that the superior response of Treg to low CD28SA doses was lost in the absence of self-peptide presentation. We also tested if potentially pathogenic autoreactive Tconv would benefit from self-recognition-induced sensitivity to CD28SA stimulation by transferring TCR transgenic OVA-specific Tconv into OVA-expressing mice and found that low-dose CD28SA application inhibited, rather than supported, their expansion, presumably due to the massive concomitant activation of Treg. Finally, we report that also in the in vitro response of human peripheral blood mononuclear cells to CD28SA, HLA II blockade interferes with the expansion of Treg by low-dose CD28SA stimulation. These results provide a rational basis for the further development of low-dose CD28SA therapy for the improvement of Treg activity. KW - D665 KW - regulatory T cells KW - self-reactivity KW - autoimmunity KW - CD28 superagonists KW - TGN1412 KW - TAB08 Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-159387 VL - 8 IS - 1985 ER - TY - JOUR A1 - Langenhorst, Daniela A1 - Haack, Stephanie A1 - Göb, Selina A1 - Uri, Anna A1 - Lühder, Fred A1 - Vanhove, Bernhard A1 - Hünig, Thomas A1 - Beyersdorf, Niklas T1 - CD28 costimulation of T helper 1 cells enhances cytokine release in vivo JF - Frontiers in Immunology N2 - Compared to naive T cells, differentiated T cells are thought to be less dependent on CD28 costimulation for full activation. To revisit the role of CD28 costimulation in mouse T cell recall responses, we adoptively transferred in vitro generated OT-II T helper (Th) 1 cells into C57BL/6 mice (Thy1.2\(^{+}\)) and then either blocked CD28–ligand interactions with Fab fragments of the anti-CD28 monoclonal antibody (mAb) E18 or deleted CD28 expression using inducible CD28 knock-out OT-II mice as T cell donors. After injection of ovalbumin protein in adjuvant into the recipient mice we observed that systemic interferon (IFN)γ release strongly depended on CD28 costimulation of the Th1 cells, while secondary clonal expansion was not reduced in the absence of CD28 costimulation. For human memory CD4\(^{+}\) T cell responses we also noted that cytokine release was reduced upon inhibition of CD28 costimulation. Together, our data highlight the so far underestimated role of CD28 costimulation for the reactivation of fully differentiated CD4\(^{+}\) T cells. KW - CD4\(^{+}\) T helper cells KW - T helper 1 cells KW - antigenic recall KW - CD28 costimulation KW - cytokine secretion KW - mouse KW - human Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-176726 VL - 9 IS - 1060 ER - TY - JOUR A1 - Langenhorst, Daniela A1 - Gogishvili, Tea A1 - Ribechini, Eliana A1 - Kneitz, Susanne A1 - McPherson, Kirsty A1 - Lutz, Manfred B. A1 - Hünig, Thomas T1 - Sequential induction of effector function, tissue migration and cell death during polyclonal activation of mouse regulatory T-cells N2 - The ability of CD4+Foxp3+ regulatory T-cells (Treg) to produce interleukin (IL)-10 is important for the limitation of inflammation at environmental interfaces like colon or lung. Under steady state conditions, however, few Tregs produce IL-10 ex vivo. To investigate the origin and fate of IL-10 producing Tregs we used a superagonistic mouse anti-mouse CD28 mAb (CD28SA) for polyclonal in vivo stimulation of Tregs, which not only led to their numeric expansion but also to a dramatic increase in IL-10 production. IL-10 secreting Tregs strongly upregulated surface receptors associated with suppressive function as compared to non-producing Tregs. Furthermore, polyclonally expanding Tregs shifted their migration receptor pattern after activation from a CCR7+CCR52 lymph node-seeking to a CCR72CCR5+ inflammationseeking phenotype, explaining the preferential recruitment of IL-10 producers to sites of ongoing immune responses. Finally, we observed that IL-10 producing Tregs from CD28SA stimulated mice were more apoptosis-prone in vitro than their IL-10 negative counterparts. These findings support a model where prolonged activation of Tregs results in terminal differentiation towards an IL-10 producing effector phenotype associated with a limited lifespan, implicating built-in termination of immunosuppression. KW - Medizin Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-76009 ER - TY - THES A1 - Langenhorst, Daniela T1 - Induktion und Aktivierung regulatorischer T-Zellen durch superagonistische Stimulation des CD28 Moleküls T1 - Induction and activation of regulatory T-cells by superagonistic Stimulation of the CD28 molecule N2 - Regulatorische T-Zellen (Tregs) spielen eine ntscheidende Rolle beim Erhalt der Immunhomöostase und bei der Kontrolle überschießender Immunantworten. Sie können anhand ihres Entstehungsortes in im Thymus generierte natürliche Tregs (nTregs) und in der Peripherie generierte induzierte Tregs (iTregs) unterteilt werden. Ihr Phänotyp wie auch ihre Funktion werden zu einem großen Teil durch den transkriptionellen Masterregulator Foxp3 kontrolliert. Das kostimulatorische Molekül CD28 wird von nTregs für die Differenzierung benötigt und von Tregs und konventionellen T-Zellen (Tkons) für ihre Aktivierung. Superagonistische CD28 spezifische monoklonale Antikörper (CD28SA) aktivieren T-Zellen im Gegensatz zu konventionellen anti-CD28 Antikörpern ohne zusätzliche Ligation des T-Zellrezeptors. Die in vivo Applikation des CD28SA bewirkt eine starke Aktivierung der Tregs und eine präferentielle Expansion der Tregs gegenüber Tkons. Dies erklärt die präventive und therapeutische Wirkung der CD28SA Behandlung in verschiedenen Krankheitsmodellen bei Nagern. Die erste Anwendung des humanisierten CD28SA TGN1412 führte in den Testpersonen jedoch zu einem unerwarteten „Cytokine-Release Syndrom“. Daher wurde hier am Mausmodell der Zusammenhang zwischen Treg Aktivierung und systemischer Zytokinausschüttung näher untersucht. Es konnte gezeigt werden, dass die CD28SA vermittelte Proliferation der T-Zellen abhängig vom CD28 Signal und von parakrinem Interleukin (IL)-2 ist. Durch die in vivo Depletion der Tregs vor der CD28SA Injektion wurde deutlich, dass es auch in Mäusen nach CD28SA Stimulation zu einer systemischen Ausschüttung pro-inflammatorischer Zytokine kommt, die jedoch, im Gegensatz zum humanen System, von Tregs effektiv kontrolliert werden kann. Um die usschüttung pro-inflammatorischer Zytokine zu verhindern, wäre eine zusätzliche prophylaktische Behandlung mit Corticosteroiden möglich, da diese auch in hohen Dosen die CD28SA vermittelte Aktivierung und Expansion der Tregs nicht beeinflussen. Neben der Expansion wird durch die Stimulation mit CD28SA auch die Produktion des anti-inflammatorischen Zytokins IL-10 in Tregs induziert und so eine genauere Untersuchung des Ursprungs und des Schicksals IL-10 produzierender Tregs ermöglicht. Diese Tregs exprimieren im Vergleich zu IL-10 negativen Tregs ein höheres Niveau an Molekülen, die mit einer supprimierenden Aktivität verbunden sind. Zudem werden IL-10 Produzenten aufgrund der Veränderung im Expressionsmuster der Migrationsrezeptoren nach der Stimulation von einem lymphknotensuchenden CCR7+CCR5-CCR6- zu einem entzündungssuchenden CCR7-CCR5+CCR6+ Phänotyp verstärkt in Bereiche mit stattfindender Immunantwort rekrutiert. Schließlich sind IL-10 produzierende Tregs von CD28SA stimu2 lierten Mäusen in vitro stärker apoptoseanfällig als die IL-10 negativen Tregs. Die Aktivierung der Tregs scheint somit die terminale Differenzierung zu einem IL-10 produzierenden Effektorphänotyp mit begrenzter Lebensdauer zu induzieren. Dies führt auch zur Beendigung der Immunsuppression. Die Kombination aus schwachem TZR und starkem CD28 Signal, die die CD28SA Stimulation in naiven T-Zellen auslöst, induziert zumindest in vitro abhängig von IL-2 und TGFβ effizient die Expression von Foxp3. Die so generierten iTregs haben, ähnlich wie konventionell in vitro erzeugte iTregs, in Bezug auf die Expression von Oberflächenmolekülen und den Methylierungsstatus bestimmter Regionen des Foxp3 Gens einen Phänotyp, der zwischen dem von Tkons und Tregs liegt. Da auch die supprimierende Aktivität der iTregs geringer ist als die der ex vivo Tregs bedarf es einer weiteren Optimierung des Stimulationsprotokolls, um diese Zellen für therapeutische Zwecke verwenden zu können. Zusammenfassend zeigt diese Arbeit, dass die superagonistische Stimulation des CD28 Moleküls ein vielseitig einsetzbares Instrument ist. Einerseits können durch die CD28SA Stimulation Tregs polyklonal aktiviert und für therapeutische Zwecke mobilisiert werden und andererseits kann die besondere Art der T-Zellstimulation auch dazu genutzt werden, neue Aspekte von nTregs und iTregs zu untersuchen. N2 - Regulatory T-cells (Tregs) are important to maintain immune homeostasis and to control overshooting immune responses. Depending on the place where they are generated Tregs can be divided into thymus-derived natural Tregs (nTregs) and peripheral-derived induced Tregs (iTregs). Their phenotype and function is controlled by the transcriptional masterregulator Foxp3. The costimulatory molecule CD28 is required for nTreg differentiation and for the activation of Tregs and conventional T-cells (Tcons). Superagonistic CD28 specific monoclonal antibodies (CD28SA) activate T-cells without additional T-cell receptor ligation in contrast to conventional anti-CD28 antibodies. In vivo CD28SA treatment induces strong Treg activation and preferential expansion of Tregs over Tcons. This explains the preventive and therapeutic effects of CD28SA in various rodent disease models. In contrast, a first-in-man trail of the human CD28SA TGN1412 resulted in an unexpected cytokine release syndrome. Thus, using the mouse system the relationship between Treg activation and systemic cytokine release was reinvestigated. It could be shown that CD28SA induced T-cell proliferation is dependent on the CD28 signal and on paracrine interleukin (IL)-2. In vivo depletion of Tregs prior CD28SA injection showed that in mice, too, CD28SA stimulation induces a systemic release of pro-inflammatory cytokines, but in contrast to humans, this can be effectively controlled by Tregs. To prevent the pro-inflammatory cytokine release an additional prophylactic treatment with corticosteroids might be feasible since even high doses of corticosteroids have no effect on CD28SA driven Treg activation and expansion. Beside expansion, CD28SA stimulation also induces the production of anti-inflammatory IL-10 in Tregs. This allows for examination of the origin and fate of IL-10 producing Tregs. In comparison to IL-10 negative Tregs, these cells express higher levels of molecules associated with suppressive activity. In addition IL-10 producers are preferentially recruited to sites of ongoing immune responses, as they shift their expression pattern of migration receptors after stimulation from a CCR7+CCR5-CCR6- lymph node-seeking to a CCR7-CCR5+CCR6+ inflammation-seeking phenotype. Finally, IL-10 producing Tregs of CD28SA stimulated mice are more apoptosis-prone in vitro than their IL-10 negative counterparts. Thus, activation of Tregs induces terminal differentiation towards an IL-10 producing effector phenotype with a limited life span. This also terminates the immune suppression. The special combination of weak TCR and strong CD28 signals following CD28SA stimulation of naïve T-cells can, at least in vitro and dependent on IL-2 and TGFβ, efficiently induce Foxp3 expression. The phenotype regarding expression of certain surface molecules and the methylation level of particular regulatory regions of the Foxp3 gene is similar in CD28SA and anti-CD3/ anti-CD28 (+ IL-2/TGFβ) induced iTregs, and shows properties of both Tcons and Tregs. Since the suppressive activity of these iTregs is also lower than that of ex vivo Tregs, further improvement of the stimulation protocol is needed before these cells could be used for therapeutic purposes. In conclusion, this study shows that superagonistic stimulation of CD28 molecules is a versatile tool. On the one hand CD28SA stimulation polyclonally activates Tregs and can be used as a therapeutic, on the other hand the special way of T-cell stimulation can also be utilized to investigate new aspects of nTregs and iTregs. KW - Antigen CD28 KW - regulatorische T-Zellen KW - CD28 Molekül KW - T-Zellaktivierung KW - CD28 Superagonist KW - T-Lymphozyt KW - Immunreaktion KW - Immunologie Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-96700 ER - TY - JOUR A1 - Haack, Stephanie A1 - Baiker, Sarah A1 - Schlegel, Jan A1 - Sauer, Markus A1 - Sparwasser, Tim A1 - Langenhorst, Daniela A1 - Beyersdorf, Niklas T1 - Superagonistic CD28 stimulation induces IFN‐γ release from mouse T helper 1 cells in vitro and in vivo JF - European Journal of Immunology N2 - Like human Th1 cells, mouse Th1 cells also secrete IFN‐γ upon stimulation with a superagonistic anti‐CD28 monoclonal antibody (CD28‐SA). Crosslinking of the CD28‐SA via FcR and CD40‐CD40L interactions greatly increased IFN‐γ release. Our data stress the utility of the mouse as a model organism for immune responses in humans. KW - CD28 KW - Th1 cells KW - cytokine release KW - interferon γ KW - Superagonistic antibody Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-239028 VL - 51 IS - 3 SP - 738 EP - 741 ER -