Fusion of Bacterial Flagellin to a Dendritic Cell-Targeting αCD40 Antibody Construct Coupled With Viral or Leukemia-Specific Antigens Enhances Dendritic Cell Maturation and Activates Peptide-Responsive T Cells

Conventional dendritic cell (DC) vaccine strategies, in which DCs are loaded with antigens ex vivo, suffer biological issues such as impaired DC migration capacity and laborious GMP production procedures. In a promising alternative, antigens are targeted to DC-associated endocytic receptors in vivo with antibody–antigen conjugates co-administered with toll-like receptor (TLR) agonists as adjuvants. To combine the potential advantages of in vivo targeting of DCs with those of conjugated TLR agonists, we generated a multifunctional antibody construct integrating the DC-specific delivery of viral- or tumor-associated antigens and DC activation by TLR ligation in one molecule. We validated its functionality in vitro and determined if TLR ligation might improve the efficacy of such a molecule. In proof-of-principle studies, an αCD40 antibody containing a CMV pp65-derived peptide as an antigen domain (αCD40CMV) was genetically fused to the TLR5-binding D0/D1 domain of bacterial flagellin (αCD40.FlgCMV). The analysis of surface maturation markers on immature DCs revealed that fusion of flagellin to αCD40CMV highly increased DC maturation (3.4-fold elevation of CD80 expression compared to αCD40CMV alone) by specifically interacting with TLR5. Immature DCs loaded with αCD40.FlgCMV induced significantly higher CMVNLV-specific T cell activation and proliferation compared to αCD40CMV in co-culture experiments with allogeneic and autologous T cells (1.8-fold increase in % IFN-γ/TNF-α+ CD8+ T cells and 3.9-fold increase in % CMVNLV-specific dextramer+ CD8+ T cells). More importantly, we confirmed the beneficial effects of flagellin-dependent DC stimulation using a tumor-specific neoantigen as the antigen domain. Specifically, the acute myeloid leukemia (AML)-specific mutated NPM1 (mNPM1)-derived neoantigen CLAVEEVSL was delivered to DCs in the form of αCD40mNPM1 and αCD40.FlgmNPM1 antibody constructs, making this study the first to investigate mNPM1 in a DC vaccination context. Again, αCD40.FlgmNPM1-loaded DCs more potently activated allogeneic mNPM1CLA-specific T cells compared to αCD40mNPM1. These in vitro results confirmed the functionality of our multifunctional antibody construct and demonstrated that TLR5 ligation improved the efficacy of the molecule. Future mouse studies are required to examine the T cell-activating potential of αCD40.FlgmNPM1 after targeting of dendritic cells in vivo using AML xenograft models

Interferon-γ Receptor Signaling in Dendritic Cells Restrains Spontaneous Proliferation of CD4+ T Cells in Chronic Lymphopenic Mice

In lymphopenic mice, T cells become activated and undergo lymphopenia-induced proliferation (LIP). However, not all T cells are equally sensitive to lymphopenia. Several lymphopenia-insensitive T cell clones were described and their non-responsiveness was mainly attributed to clone-specific properties. Here, we provide evidence for an additional, host-dependent mechanism restraining LIP of lymphopenia-insensitive CD4+ T cells. We show that such cells undergo LIP in lymphopenic mice lacking IFN-γ receptor (IFN-γR) expression, a process, which is promoted by the autocrine action of T cell-derived IFN-γ. Additionally, LIP of lymphopenia-insensitive CD4+ T cells requires an intact microflora and is accompanied by the massive accumulation of IL-6 and dendritic cells (DCs). Consistent with these results, IL-6 neutralization and the DC-specific restoration of IFN-γR expression are both sufficient to restrict LIP. Hence, the insensitivity of CD4+ T cells to lymphopenia relies on cell-intrinsic properties and a complex interplay between the commensal microflora, IL-6, IFN-γR+ DCs, and T cell-derived IFN-γ

Interleukin-7 Links T Lymphocyte and Intestinal Epithelial Cell Homeostasis

Interleukin-7 (IL-7) is a major survival factor for mature T cells. Therefore, the degree of IL-7 availability determines the size of the peripheral T cell pool and regulates T cell homeostasis. Here we provide evidence that IL-7 also regulates the homeostasis of intestinal epithelial cells (IEC), colon function and the composition of the commensal microflora. In the colon of T cell-deficient, lymphopenic mice, IL-7-producing IEC accumulate. IEC hyperplasia can be blocked by IL-7-consuming T cells or the inactivation of the IL-7/IL-7R signaling pathway. However, the blockade of the IL-7/IL-7R signaling pathway renders T cell-deficient mice more sensitive to chemically-induced IEC damage and subsequent colitis. In summary, our data demonstrate that IL-7 promotes IEC hyperplasia under lymphopenic conditions. Under non-lymphopenic conditions, however, T cells consume IL-7 thereby limiting IEC expansion and survival. Hence, the degree of IL-7 availability regulates both, T cell and IEC homeostasis

Impact of IL-7 signaling on adoptive T cell therapy

Das Zytokin Interleukin-7 (IL-7) ist für die Entstehung und das Überleben reifer T Zellen von zentraler Bedeutung. Die Gabe von IL-7 führt sowohl in der Maus als auch im Menschen zu erhöhten T Zellzahlen und einem veränderten T Zellphänotyp. Folglich könnte sich die therapeutische Gabe von IL-7 bei Patienten mit geschwächtem Immunsystem positiv auswirken. Diese Hypothese wird derzeit in mehreren klinischen Studien untersucht. Bisher wurde allerdings nur die Wirkung von IL-7 auf T-Zellen studiert. Zu dessen Wirkung auf andere Immun- oder Stromazellen sowie deren IL-7-abhängigen Beitrag zur Regulation der T-Zellhomöostase ist nur wenig bekannt. Daher war es Ziel der Arbeit, den Einfluss einer therapeutischen Gabe von IL-7 auf adoptiv-transferierte T-Zellen in IL-7-Rezeptor (IL-7R)-kompetenten und defizienten lymphopenischen Mäusen zu studieren. Die Untersuchungen bestätigen, dass die Gabe von IL-7 T-Zellantworten unterstützt, zeigen jedoch auch, daß viele dieser Effekte von IL-7R-exprimierenden Wirtszellen abhängig sind. Dies weist darauf hin, dass IL-7R-vermittelte Signale in Wirtszellen indirekt T-Zellantworten beeinflussen. Zudem zeigte sich, dass effiziente anti-Tumor-T Zellantworten von IL 7R-vermittelten Signalen in Wirtszellen abhängen. Vor allem nicht-hämatopoetische Wirtszellen fungieren hier als Regulatoren der IL-7-Therapie-vermittelten T Zelldifferenzierung. Unsere Ergebnisse bestätigen außerdem, dass Stromazellen in verschiedenen Organen il-7 exprimieren und zeigen darüber hinaus, dass diese Zellen durch die Gabe von IL-7 beeinflusst werden. Wir folgern daraus, dass die Effekte der IL-7-Therapie auf T Zellhomöostase teilweise indirekt über il-7-exprimierende Stromazellen vermittelt werden. Um diese Zellen genauer identifizieren und untersuchen zu können, haben wir ein neues transgenes Mausmodell charakterisiert, was es erleichtern wird, die beteiligten molekularen Signalwege zu analysieren und den Erfolg der adoptiven T Zelltherapie zu verbessern.Interleukin-7 (IL-7) is an essential cytokine required for the development and maintenance of mature T cell. Its availability is limited under normal conditions, but rises during lymphopenia, leading to increased T cell proliferation. The administration of recombinant IL-7 to normal or lymphopenic mice and humans results in increased T cell numbers and altered T cell phenotype. Hence, IL-7 administration could mediate therapeutic benefits in immunocompromised patients and is currently tested in several clinical trials. However, besides its well-studied effects on T cells little is known about the effect of IL-7 on other immune and non-immune cells and their influence on T cell homeostasis. Therefore, we evaluated the effect of IL-7 therapy on adoptively transferred T cells in IL-7 receptor (IL-7R)-competent and IL-7R-deficient lymphopenic mice. We confirm the benefits of IL-7 therapy on T cell responses but additionally show that many of these effects are dependent on IL-7R expression by host cells, indicating that IL-7R signaling in host cells modulates T cell responses. We show that efficient T cell responses against cancer are dependent on host IL-7R signaling. Based on studies in bone-marrow chimeric mice, we identify non-hematopoietic host cells as main regulators of IL-7 therapy-modulated T cell differentiation. We conclude from these data that IL-7 therapy affects non-hematopoietic stromal cells that modulate the success of adoptive T cell therapy. Our results confirm that stromal cells in various organs express il-7 and show that these cells are targeted by IL-7 therapy in vivo. Hence, we propose that il-7-expressing cells regulate IL-7 therapy-modulated T cell homeostasis. To identify and study these il-7 expressing stromal cells in more detail, we characterized a new transgenic mouse model that will facilitate determining the molecular pathways to improve the success of adoptive T cell therapy

Cutting Edge: Innate Lymphoid Cells Suppress Homeostatic T Cell Expansion in Neonatal Mice

In adult mice, lymphopenia-induced proliferation (LIP) leads to T cell activation, memory differentiation, tissue destruction, and a loss of TCR diversity. Neonatal mice are lymphopenic within the first week of life. This enables some recent thymic emigrants to undergo LIP and convert into long-lived memory T cells. Surprisingly, however, most neonatal T cells do not undergo LIP. We therefore asked whether neonate-specific mechanisms prevent lymphopenia-driven T cell activation. In this study, we show that IL-7R-dependent innate lymphoid cells (ILCs) block LIP of CD8(+) T cells in neonatal but not adult mice. Importantly, CD8(+) T cell responses against a foreign Ag are not inhibited by neonatal ILCs. This ILC-based inhibition of LIP ensures the generation of a diverse naive T cell pool in lymphopenic neonates that is mandatory for the maintenance of T cell homeostasis and immunological self-tolerance later in life

Host IL-7R signaling is required for CD8⁺ T cell-mediated tumor rejection in response to IL-7 treatment.

<p>(A-C) Rag^-/- and Rag^-/-IL-7R^-/- mice were reconstituted with 7–10 x 10⁵ CD8⁺CD90.1⁺ OT-I T cells or were left untreated (+/- OT-I). OT-I-reconstituted mice received rIL-7 (+ IL-7) or PBS (- IL-7) every 3–4 days for 18 days starting one day before T cell transfer. (A, B) 22–23 days after T cell transfer, some mice were challenged s.c. with 1 x 10⁶ ovalbumin-expressing EG7 lymphoma cells. Mice with tumors >250 mm³ were scored as tumor positive. Pooled data from 2 independent experiments with a total of 10–12 mice per group are shown. Statistical significance was calculated using the log-rank test. (C, D) Some recipients were not challenged with EG7 but analyzed for OT-I expansion and phenotype. (C) Splenic CD8⁺CD90.1⁺ OT-I T cells were quantified 21–25 days after adoptive transfer. Pooled data (±SEM) from 2 independent experiments with a total of 7–9 mice/group are shown. (D) CD127 expression of splenic OT-I T cells was determined 5 days after adoptive transfer. Data (±SEM) for 3–4 mice/group are shown.</p

The combination of rIL-7 therapy and peptide vaccination impairs T cell-dependent tumor rejection in Rag^-/- mice.

<p>(A) Rag^-/- and (B) Rag^-/-IL-7R^-/- mice were reconstituted with 1 x 10⁶ CD8⁺CD90.1⁺ OT-I T cells or were left untreated (+/- OT-I). One day later, OT-I-reconstituted mice were either vaccinated with 50 μg SIINFEKL or received PBS (+/- Pep). rIL-7 or PBS (+/- IL-7) were injected every 3–4 days for 19 days starting one day before T cell transfer. Mice were challenged s.c. with 1 x 10⁶ EG7 tumor cells three weeks after T cell transfer. Mice with tumors larger than 250 mm³ were scored as tumor positive. Shown are pooled data from 2 independent experiments with a total of 12–13 T cell reconstituted mice. Primary tumor growth was analyzed in untreated Rag^-/- and Rag^-/-IL-7R^-/- mice (n = 3). Statistical significance was calculated using the log-rank test. (C) The numbers of splenic DCs were determined in tumor-bearing mice 28–37 days after tumor challenge. Pooled data (±SEM) from 2 independent experiments with a total of 6–11 mice/group are shown. Statistical significance was calculated using the Mann-Whitney test.</p

Host IL-7R signaling is required for granulocyte and DC expansion in response to rIL-7.

<p>Rag^-/- and Rag^-/-IL-7R^-/- mice were treated with rIL-7 or PBS (+/- IL-7) every 3–4 days and spleens were analyzed by flow cytometry after 10–24 days. (A-E) Shown are numbers of (A) splenocytes (Splen.), (B) CD11b⁺Gr1⁺ granulocytes (Gran.), (C) CD11c⁺MHC-II⁺ dendritic cells (DCs), (D) CD8⁺ and (E) CD8^- DCs. Shown are pooled data (±SEM) from 2 independent experiments with a total of 7–8 mice/group.</p

IL-7R signaling in non-hematopoietic cells regulates CD8⁺ T cell differentiation in response to peptide vaccination and IL-7 therapy.

<p>(A-G) The indicated BM chimeras received CD8⁺ OT-I T cells and were treated with SIINFEKL and rIL-7 as described above. Three weeks after T cell transfer, splenic CD8⁺ OT-I T cells were analyzed by flow cytometry. Bar diagrams show pooled results (±SEM) of 2–3 independent experiments with a total of (A, B, C, E, G) 10–17 or (D) 4–10 mice/group. Histogram overlays show representative results for individual mice. Grey-lined histograms represent FMO staining controls.</p