Optimized antiangiogenic reprogramming of the tumor microenvironment potentiates CD40 immunotherapy.

Cancer immunotherapies are increasingly combined with targeted therapies to improve therapeutic outcomes. We show that combination of agonistic anti-CD40 with antiangiogenic antibodies targeting 2 proangiogenic factors, vascular endothelial growth factor A (VEGFA) and angiopoietin 2 (Ang2/ANGPT2), induces pleiotropic immune mechanisms that facilitate tumor rejection in several tumor models. On the one hand, VEGFA/Ang2 blockade induced regression of the tumor microvasculature while decreasing the proportion of nonperfused vessels and reducing leakiness of the remaining vessels. On the other hand, both anti-VEGFA/Ang2 and anti-CD40 independently promoted proinflammatory macrophage skewing and increased dendritic cell activation in the tumor microenvironment, which were further amplified upon combination of the 2 treatments. Finally, combined therapy provoked brisk infiltration and intratumoral redistribution of cytotoxic CD8+ T cells in the tumors, which was mainly driven by Ang2 blockade. Overall, these nonredundant synergistic mechanisms endowed T cells with improved effector functions that were conducive to more efficient tumor control, underscoring the therapeutic potential of antiangiogenic immunotherapy in cancer.

Flt3 Ligand Regulates the Development of Innate Lymphoid Cells in Fetal and Adult Mice.

Flt3 ligand (Flt3L) promotes survival of lymphoid progenitors in the bone marrow and differentiation of dendritic cells (DCs), but its role in regulating innate lymphoid cells (ILCs) during fetal and adult life is not understood. By using Flt3L knockout and transgenic mice, we demonstrate that Flt3L controls ILC numbers by regulating the pool of α4ß7(-) and α4ß7(+) lymphoid tissue inducer cell progenitors in the fetal liver and common lymphoid progenitors in the bone marrow. Deletion of flt3l severely reduced the number of fetal liver progenitors and lymphoid tissue inducer cells in the neonatal intestine, resulting in impaired development of Peyer's patches. In the adult intestine, NK cells and group 2 and 3 ILCs were severely reduced. This effect occurred independently of DCs as ILC numbers were normal in mice in which DCs were constitutively deleted. Finally, we could show that administration of Flt3L increased the number of NKp46(-) group 3 ILCs in wild-type and even in Il7(-/-) mice, which generally have reduced numbers of ILCs. Taken together, Flt3L significantly contributes to ILC and Peyer's patches development by targeting lymphoid progenitor cells during fetal and adult life.

The amount of self-antigen determines the effector function of murine T cells escaping negative selection.

Autoimmune diseases develop when self-specific T cells that escaped negative selection initiate a harmful immune response against self. However, factors, which influence the initiation and progression of an autoimmune response remain incompletely understood. By establishing a double-transgenic BALB/c mouse system in which different amounts of a cell-surface neo-self-antigen are expressed under the CD11c promoter, we demonstrate that antigen dose dramatically influences T-cell tolerance mechanisms. Moderate antigen expression in both hematopoietic and nonhematopoietic cells favors the development of antigen-specific Treg cells and the establishment of a tolerogenic environment. In marked contrast, a high dose of antigen expression results in very stringent negative selection, in poor development of antigen-specific Treg cells and in the early onset of anemia and splenomegaly and the late development of arthritis and high titers of IgG auto Abs. Disease is initiated by autoreactive T cells, which escape negative selection by expressing a second TCR with a different specificity or an altered affinity. Transfer of Ag-specific Treg cells ameliorates the early onset signs of disease but does not prevent the development of long-term chronic pathologies. Altogether, our results suggest that Ag dose directly affects Treg-cell generation and thus, the set-up of T-cell tolerance.

In vivo evidence for an instructive role of fms-like tyrosine kinase-3 (FLT3) ligand in hematopoietic development.

Cytokines are essential regulators of hematopoiesis, acting in an instructive or permissive way. Fms-like tyrosine kinase 3 ligand (FLT3L) is an important cytokine for the development of several hematopoietic populations. Its receptor (FLT3) is expressed on both myeloid and lymphoid progenitors and deletion of either the receptor or its ligand leads to defective developmental potential of hematopoietic progenitors. In vivo administration of FLT3L promotes expansion of progenitors with combined myeloid and lymphoid potential. To investigate further the role of this cytokine in hematopoietic development, we generated transgenic mice expressing high levels of human FLT3L. These transgenic mice displayed a dramatic expansion of dendritic and myeloid cells, leading to splenomegaly and blood leukocytosis. Bone marrow myeloid and lymphoid progenitors were significantly increased in numbers but retained their developmental potential. Furthermore, the transgenic mice developed anemia together with a reduction in platelet numbers. FLT3L was shown to rapidly reduce the earliest erythroid progenitors when injected into wild-type mice, indicating a direct negative role of the cytokine on erythropoiesis. We conclude that FLT3L acts on multipotent progenitors in an instructive way, inducing their development into myeloid/lymphoid lineages while suppressing their megakaryocyte/erythrocyte potential.

Soluble BAFF levels inversely correlate with peripheral B cell numbers and the expression of BAFF receptors.

The TNF family member protein BAFF/BLyS is essential for B cell survival and plays an important role in regulating class switch recombination as well as in the selection of autoreactive B cells. In humans, increased concentrations of soluble BAFF are found in different pathological conditions, which may be as diverse as autoimmune diseases, B cell malignancies, and primary Ab deficiencies (PAD). Because the mechanisms that regulate BAFF levels are not well understood, we newly developed a set of mAbs against human BAFF to study the parameters that determine the concentrations of soluble BAFF in circulation. Patients with PAD, including severe functional B cell defects such as BTK, BAFF-R, or TACI deficiency, were found to have higher BAFF levels than asplenic individuals, patients after anti-CD20 B cell depletion, chronic lymphocytic leukemia patients, or healthy donors. In a comparable manner, mice constitutively expressing human BAFF were found to have higher concentrations of BAFF in the absence than in the presence of B cells. Therefore, our data strongly suggest that BAFF steady-state concentrations mainly depend on the number of B cells as well as on the expression of BAFF-binding receptors. Because most patients with PAD have high levels of circulating BAFF, the increase in BAFF concentrations cannot compensate defects in B cell development and function.

Prophylactic and Therapeutic Effects of Interleukin-2 (IL-2)/Anti-IL-2 Complexes in Systemic Lupus Erythematosus-Like Chronic Graft-Versus-Host Disease.

Murine chronic graft-versus-host-disease (cGvHD) induced by injection of parental lymphocytes into F1 hybrids results in a disease similar to systemic lupus erythematosus. Here, we have used DBA/2 T cell injection into (C57BL/6 × DBA/2)F1 (BDF1) mice as a model system to test the prophylactic and therapeutic effects of interleukin-2 (IL-2)/anti-IL-2 immune complexes on the course of cGvHD. Our findings demonstrate that pretreatment with Treg inducing JES6/IL-2 complexes render BDF1 mice largely resistant to induction of cGvHD, whereas pretreatment with CD8+ T cell/NK cell inducing S4B6/IL-2 complexes results in a more severe cGvHD. In contrast, treatment with JES6/IL-2 complexes 4 weeks after induction had no beneficial effect on disease symptoms. However, similar treatment with S4B6/IL-2 complexes led to a significant amelioration of the disease. This therapeutic effect seems to be mediated by donor CD8+ T cells. The fact that a much stronger cGvHD is induced in BDF1 mice depleted of donor CD8+ T cells strongly supports this conclusion. The contrasting effects of the two different IL-2 complexes are likely due to different mechanisms.

Replicating viral vector platform exploits alarmin signals for potent CD8+ T cell-mediated tumour immunotherapy.

Viral infections lead to alarmin release and elicit potent cytotoxic effector T lymphocyte (CTLeff) responses. Conversely, the induction of protective tumour-specific CTLeff and their recruitment into the tumour remain challenging tasks. Here we show that lymphocytic choriomeningitis virus (LCMV) can be engineered to serve as a replication competent, stably-attenuated immunotherapy vector (artLCMV). artLCMV delivers tumour-associated antigens to dendritic cells for efficient CTL priming. Unlike replication-deficient vectors, artLCMV targets also lymphoid tissue stroma cells expressing the alarmin interleukin-33. By triggering interleukin-33 signals, artLCMV elicits CTLeff responses of higher magnitude and functionality than those induced by replication-deficient vectors. Superior anti-tumour efficacy of artLCMV immunotherapy depends on interleukin-33 signalling, and a massive CTLeff influx triggers an inflammatory conversion of the tumour microenvironment. Our observations suggest that replicating viral delivery systems can release alarmins for improved anti-tumour efficacy. These mechanistic insights may outweigh safety concerns around replicating viral vectors in cancer immunotherapy.

Trastuzumab emtansine (T-DM1) renders HER2+ breast cancer highly susceptible to CTLA-4/PD-1 blockade.

Targeted drug delivery with antibody-drug conjugates such as the HER2-directed ado-trastuzumab emtansine (T-DM1) has emerged as a powerful strategy for cancer therapy. We show that T-DM1 is particularly effective in eliciting antitumor immunity in patients with early breast cancer (WSG-ADAPT trial) and in a HER2-expressing orthotopic tumor model. In the latter, despite primary resistance to immunotherapy, combined treatment with T-DM1 and anti-CTLA-4/PD-1 (cytotoxic T lymphocyte-associated protein-4/programmed cell death protein-1) was curative because it triggered innate and adaptive immunity. Tumor rejection was accompanied by massive T cell infiltration, TH1 (T helper 1) cell polarization, and, notably, a substantial increase in regulatory T cells. Depletion of regulatory T cells resulted in inflammation and tissue damage, implying their essential role in protecting the host during therapy. This study provides insights into the mechanisms of T-DM1's therapeutic activity and a rationale for potential therapeutic combination strategies with immunotherapy.

Reconstitution of the peripheral B lymphocyte compartment in patients with ANCA-associated vasculitides treated with rituximab for relapsing or refractory disease.

While in patients with rheumatoid arthritis B-cell repopulation starts within 9 months after rituximab (RTX) therapy, a delayed B-cell repopulation was reported in some RTX-treated patients with ANCA-associated vasculitides (AAV). To date, the frequency of AAV patients with impaired peripheral B-cell regeneration and the mechanisms leading to the constricted regenerative capacity are unknown. We analyzed the B-cell repopulation kinetic in 37 AAV patients treated with RTX followed by maintenance immunosuppressants. We report on serum concentrations of the B-cell-activating factor BAFF, immunoglobulins and B-cell subpopulations in patients that relapsed after RTX. B-cells were re-detectable in only one patient within 9 months after RTX. In 14 patients (41%), B-cell repopulation started later, after a mean observation time of 21 months. Only seven of these patients had detectable B-cells within the first year after RTX. Twenty patients (59%) had no B-cell reconstitution within the observation period. BAFF was increased in RTX-treated AAV patients compared to healthy controls and correlated inversely with peripheral B-cell numbers, IgG- and IgA concentrations. Immunoglobulin concentrations declined significantly after RTX and the IgG concentration correlated with B-cell numbers. Thirteen patients relapsed after RTX. Relapses occurred exclusively either after B-cell reconstitution had started or were accompanied by rising ANCA titres. In relapsed patients, the B-lymphocyte compartment consisted mainly of switched memory B-cells. Our data indicate that RTX treatment can induce secondary immunodeficiency in AAV, with hypogammaglobulinemia and long-lasting B-lymphopenia. Further studies are needed to define the pathophysiology of the impaired B-cell development in RTX-treated AAV patients.