In the Absence of a TCR Signal IL-2/IL-12/18-Stimulated γδ T Cells Demonstrate Potent Anti-Tumoral Function Through Direct Killing and Senescence Induction in Cancer Cells.

Abundant IFN-γ secretion, potent cytotoxicity, and major histocompatibility complex-independent targeting of a large spectrum of tumors make γδ T cells attractive candidates for cancer immunotherapy. Upon tumor recognition through the T-cell receptor (TCR), NK-receptors, or NKG2D, γδ T cells generate the pro-inflammatory cytokines TNF-α and IFN-γ, or granzymes and perforin that mediate cellular apoptosis. Despite these favorable potentials, most clinical trials testing the adoptive transfer of pharmacologically TCR-targeted and expanded γδ T cells resulted in a limited response. Recently, the TCR-independent activation of γδ T cells was identified. However, the modulation of γδ T cell's effector functions solely by cytokines remains to be elucidated. In the present study, we systematically analyzed the impact of IL-2, IL-12, and IL-18 in parallel with TCR stimulation on proliferation, cytokine production, and anti-tumor activity of γδ T cells. Our results demonstrate that IL-12 and IL-18, when combined, constitute the most potent stimulus to enhance anti-tumor activity and induce proliferation and IFN-γ production by γδ T cells in the absence of TCR signaling. Intriguingly, stimulation with IL-12 and IL-18 without TCR stimulus induces a comparable degree of anti-tumor activity in γδ T cells to TCR crosslinking by killing tumor cells and driving cancer cells into senescence. These findings approve the use of IL-12/IL-18-stimulated γδ T cells for adoptive cell therapy to boost anti-tumor activity by γδ T cells.

Tumor-targeted IL-12 combined with local irradiation leads to systemic tumor control via abscopal effects in vivo.

NHS-IL12 is an immunocytokine, a fusion protein of IL12's functional domains and a necrosis-targeting antibody, which has shown significant effects against human rhabdomyosarcoma xenografts in a humanized tumor model, including terminal growth arrest and differentiation of the tumor cells. Here, we locally irradiated the tumors, increasing necrosis and consequently intratumoral immune cytokine availability, and asked whether this effect may surmount efficacy of single treatment modality. Humanized mice bearing bilateral rhabdomyosarcoma xenografts were evaluated for tumor burden and survival after irradiation, systemic NHS-IL12 therapy or a combination of both. Intratumoral immune compartments were characterized by immunohistochemistry and molecular methods. TH1-cytokine dependency of underlying effector mechanisms were investigated in vitro in several human tumor cell lines. NHS-IL12 when combined with irradiation terminally arrested tumor growth and significantly improved survival. Combination treatment induced dense intratumoral T-cell infiltrates, clonal epitope-specific T-cell expansions, expression of cytotoxins, decreased pro-tumorigenic cytokines and induced senescence and differentiation in the cancer cells. Senescence and differentiation were reproduced in vitro and confirmed to be dependent on TH1 cytokines IFNγ and TNF-α. NHS-IL12 and irradiation together induced broad intratumoral TH1 biased NK and T-cell compartments, established antitumoral cytokine profiles and irreversibly growth arrested tumor cells, leading to systemic cancer control and improved survival. For the first time, we describe immune-induced senescence as a novel mechanism resulting from a treatment regimen combining irradiation with immunotherapy.

Isolation and Ex Vivo Culture of Vδ1+CD4+γδ T Cells, an Extrathymic αßT-cell Progenitor.

The thymus, the primary organ for the generation of αß T cells and backbone of the adaptive immune system in vertebrates, has long been considered as the only source of αßT cells. Yet, thymic involution begins early in life leading to a drastically reduced output of naïve αßT cells into the periphery. Nevertheless, even centenarians can build immunity against newly acquired pathogens. Recent research suggests extrathymic αßT cell development, however our understanding of pathways that may compensate for thymic loss of function are still rudimental. γδ T cells are innate lymphocytes that constitute the main T-cell subset in the tissues. We recently ascribed a so far unappreciated outstanding function to a γδ T cell subset by showing that the scarce entity of CD4(+) Vδ1(+)γδ T cells can transdifferentiate into αßT cells in inflammatory conditions. Here, we provide the protocol for the isolation of this progenitor from peripheral blood and its subsequent cultivation. Vδ1 cells are positively enriched from PBMCs of healthy human donors using magnetic beads, followed by a second step wherein we target the scarce fraction of CD4(+) cells with a further magnetic labeling technique. The magnetic force of the second labeling exceeds the one of the first magnetic label, and thus allows the efficient, quantitative and specific positive isolation of the population of interest. We then introduce the technique and culture condition required for cloning and efficiently expanding the cells and for identification of the generated clones by FACS analysis. Thus, we provide a detailed protocol for the purification, culture and ex vivo expansion of CD4(+) Vδ1(+)γδ T cells. This knowledge is prerequisite for studies that relate to this αßT cell progenitor`s biology and for those who aim to identify the molecular triggers that are involved in its transdifferentiation.

Preemptive administration of human αß T cell receptor-targeting monoclonal antibody GZ-αßTCR potently abrogates aggressive graft-versus-host disease in vivo.

GVHD, both acute and chronic, remains the major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation. Thus, there is still a great need for therapeutic tools for the prevention and treatment of GVHD. Several biologics have shown promising results in salvage therapies but are attendant on an increased risk for opportunistic infections, lymphoproliferative disorders, and relapse. This is partly due to efficient T cell elimination that neither dissects alloreactive from non-alloreactive T cells nor considers functional and structural distinctiveness of pathogen- and malignancy-reactive γδ and iNKT T cells. A novel, humanized monoclonal antibody, GZ-αßTCR, specific for the human αß T cell receptor, was evaluated in a xenogeneic GVHD model for its potential to prevent or ameliorate GVHD and prolong survival. We could show that GZ-αßTCR significantly attenuated clinical signs of GVHD and prolonged survival by preferential depletion of CD4 cells and the naïve T cell compartment, the trigger and driver of GVHD. In a regimen that included a preemptive dose, GZ-αßTCR treatment sufficiently abrogated GVHD. Importantly, GZ-αßTCR's specificity spared host cell-mediated immune competence of cell types other than αßT cells: namely γδT cells. GZ-αßTCR's outstanding capacity to prevent GVHD and ameliorate an ongoing GVHD while sparing immune cells other than αßT cells strongly recommends GZ-αßTCR for the prevention and treatment of acute GVHD in clinical settings.

Cancer-targeted IL-12 controls human rhabdomyosarcoma by senescence induction and myogenic differentiation.

Stimulating the immune system to attack cancer is a promising approach, even for the control of advanced cancers. Several cytokines that promote interferon-γ-dominated immune responses show antitumor activity, with interleukin 12 (IL-12) being of major importance. Here, we used an antibody-IL-12 fusion protein (NHS-IL12) that binds histones of necrotic cells to treat human sarcoma in humanized mice. Following sarcoma engraftment, NHS-IL12 therapy was combined with either engineered IL-7 (FcIL-7) or IL-2 (IL-2MAB602) for continuous cytokine bioavailability. NHS-IL12 strongly induced innate and adaptive antitumor immunity when combined with IL-7 or IL-2. NHS-IL12 therapy significantly improved survival of sarcoma-bearing mice and caused long-term remissions when combined with IL-2. NHS-IL12 induced pronounced cancer cell senescence, as documented by strong expression of senescence-associated p16INK4a and nuclear translocation of p-HP1γ, and permanent arrest of cancer cell proliferation. In addition, this cancer immunotherapy initiated the induction of myogenic differentiation, further promoting the hypothesis that efficient antitumor immunity includes mechanisms different from cytotoxicity for efficient cancer control in vivo.

Chronic graft-versus-host-disease in CD34(+)-humanized NSG mice is associated with human susceptibility HLA haplotypes for autoimmune disease.

Chronic graft-versus-host disease (cGVHD) is a significant hurdle to long-term hematopoietic stem-cell transplantation success. Insights into the pathogenesis and mechanistical investigations of novel therapeutic strategies are limited as appropriate animal models are missing. The immunodeficient NSG mouse - when humanized with human bone marrow, fetal liver and thymus (BLT NSG) - is prone for cGVHD, yet mainly affects the skin. In contrast, the NSG mouse humanized exclusively with CD34(+)-selected, CD3(+)-depleted stem cells (CD34(+)NSG) has neither been described for acute nor chronic GVHD so far. This is the first report about the development of systemic autoimmune cGVHD ≥24 weeks post stem cell receipt involving lung, liver, skin, gingiva and intestine in two NSG cohorts humanized with CD34(+) grafts from different donors. Affected mice presented with sclerodermatous skin, fibrotic lung, severe hepatitis, and massive dental malformation/loss. CD4(+)-dominated, TH2-biased, bulky T-cell infiltrates featured highly skewed T cell receptor (TCR) repertoires, clonal expansions, and autoreactive TCRs. In affected tissues profibrotic IL-13 and -4 dominated over TH1 cytokines IFN-γ and TNF-α. Thus, the time point of manifestation and the phenotype match human systemic pleiotropic sclerodermatous GVHD. The CD34(+)NSG-model's intrinsic deficiency of thymus, thymus-derived regulatory T cells (nTreg) and B cells emphasizes the role of the genetic polymorphism and the cytokines in the pathogenesis of cGVHD. Importantly, the only factor discriminating diseased versus non-diseased CD34(+)NSG cohorts were two risk HLA haplotypes that in human mediate susceptibility for autoimmune disease (psoriasis). Thus, the CD34(+)NSG model may serve as a platform for addressing issues related to the pathophysiology and treatment of human autoimmunity and chronic GVHD.

Human Peripheral CD4(+) Vδ1(+) γδT Cells Can Develop into αßT Cells.

The lifelong generation of αßT cells enables us to continuously build immunity against pathogens and malignancies despite the loss of thymic function with age. Homeostatic proliferation of post-thymic naïve and memory T cells and their transition into effector and long-lived memory cells balance the decreasing output of naïve T cells, and recent research suggests that also αßT-cell development independent from the thymus may occur. However, the sites and mechanisms of extrathymic T-cell development are not yet understood in detail. γδT cells represent a small fraction of the overall T-cell pool, and are endowed with tremendous phenotypic and functional plasticity. γδT cells that express the Vδ1 gene segment are a minor population in human peripheral blood but predominate in epithelial (and inflamed) tissues. Here, we characterize a CD4(+) peripheral Vδ1(+) γδT-cell subpopulation that expresses stem-cell and progenitor markers and is able to develop into functional αßT cells ex vivo in a simple culture system and in vivo. The route taken by this process resembles thymic T-cell development. However, it involves the re-organization of the Vδ1(+) γδTCR into the αßTCR as a consequence of TCR-γ chain downregulation and the expression of surface Vδ1(+)Vß(+) TCR components, which we believe function as surrogate pre-TCR. This transdifferentiation process is readily detectable in vivo in inflamed tissue. Our study provides a conceptual framework for extrathymic T-cell development and opens up a new vista in immunology that requires adaptive immune responses in infection, autoimmunity, and cancer to be reconsidered.

Twinning in tufted capuchins (Cebus apella): rate, survivorship, and weight gain.

We calculated the rate of twinning across four captive collections of tufted capuchins (Cebus apella) to be 2.4%. This rate contrast with previous reports that twinning in tufted capuchins is rare. Additionally, we present data on the survival and weight gain of twins in this species as compared to singletons. Twins face their greatest risk of mortality on or before the first day of life, when 45% will die compared to 16% of singletons. After the first day of life, twins and singletons demonstrate comparable survival rates. This, in conjunction with the finding that at no time during the first year of life do twins and singletons differ significantly in their weights, suggests that twinning is a viable reproductive form for these animals, especially in the captive setting where nutritional demands are met.