Switchable targeting of solid tumors by BsCAR T cells.

The development of chimeric antigen receptor (CAR) T cell therapy has become a critical milestone in modern oncotherapy. Despite the remarkable in vitro effectiveness, the problem of safety and efficacy of CAR T cell therapy against solid tumors is challenged by the lack of tumor-specific antigens required to avoid on-target off-tumor effects. Spatially separating the cytotoxic function of CAR T cells from tumor antigen recognition provided by protein mediators allows for the precise control of CAR T cell cytotoxicity. Here, the high affinity and capability of the bacterial toxin-antitoxin barnase-barstar system were adopted to guide CAR T cells to solid tumors. The complementary modules based on (1) ankyrin repeat (DARPin)-barnase proteins and (2) barstar-based CAR (BsCAR) were designed to provide switchable targeting to tumor cells. The alteration of the DARPin-barnase switches enabled the targeting of different tumor antigens with a single BsCAR. A gradual increase in cytokine release and tunable BsCAR T cell cytotoxicity was achieved by varying DARPin-barnase loads. Switchable BsCAR T cell therapy was able to eradicate the HER2+ ductal carcinoma in vivo. Guiding BsCAR T cells by DARPin-barnase switches provides a universal approach for a controlled multitargeted adoptive immunotherapy.

Antigen-Specific Stimulation and Expansion of CAR-T Cells Using Membrane Vesicles as Target Cell Surrogates.

Development of CAR-T therapy led to immediate success in the treatment of B cell leukemia. Manufacturing of therapy-competent functional CAR-T cells needs robust protocols for ex vivo/in vitro expansion of modified T-cells. This step is challenging, especially if non-viral low-efficiency delivery protocols are used to generate CAR-T cells. Modern protocols for CAR-T cell expansion are imperfect since non-specific stimulation results in rapid outgrowth of CAR-negative T cells, and removal of feeder cells from mixed cultures necessitates additional purification steps. To develop a specific and improved protocol for CAR-T cell expansion, cell-derived membrane vesicles are taken advantage of, and the simple structural demands of the CAR-antigen interaction. This novel approach is to make antigenic microcytospheres from common cell lines stably expressing surface-bound CAR antigens, and then use them for stimulation and expansion of CAR-T cells. The data presented in this article clearly demonstrate that this protocol produced antigen-specific vesicles with the capacity to induce stronger stimulation, proliferation, and functional activity of CAR-T cells than is possible with existing protocols. It is predicted that this new methodology will significantly advance the ability to obtain improved populations of functional CAR-T cells for therapy.

TGFbeta signalling in control of T-cell-mediated self-reactivity.

In the immune system, transforming growth factor-beta (TGFbeta) affects multiple cell lineages by either promoting or opposing their differentiation, survival and proliferation. Understanding the cellular mechanisms of TGFbeta-mediated regulation is complicated due to a broad distribution of TGFbeta receptors on the surface of different immune-cell types. Recent studies using in vivo genetic approaches revealed a critical role for TGFbeta signalling in T cells in restraining fatal autoimmune lesions. Here, we review recent advances in our understanding of a role for TGFbeta signalling in the regulation of T-cell differentiation in the thymus and in the periphery, with a particular emphasis on TGFbeta-mediated control of self-reactive T cells.

Regulatory T cell-derived interleukin-10 limits inflammation at environmental interfaces.

The regulatory T (Treg) cells restrain immune responses through suppressor-function elaboration that is dependent upon expression of the transcription factor Foxp3. Despite a critical role for Treg cells in maintaining lympho-myeloid homeostasis, it remains unclear whether a single mechanism or multiple mechanisms of Treg cell-mediated suppression are operating in vivo and how redundant such mechanisms might be. Here we addressed these questions by examining the role of the immunomodulatory cytokine IL-10 in Treg cell-mediated suppression. Analyses of mice in which the Treg cell-specific ablation of a conditional IL-10 allele was induced by Cre recombinase knocked into the Foxp3 gene locus showed that although IL-10 production by Treg cells was not required for the control of systemic autoimmunity, it was essential for keeping immune responses in check at environmental interfaces such as the colon and lungs. Our study suggests that Treg cells utilize multiple means to limit immune responses. Furthermore, these mechanisms are likely to be nonredundant, in that a distinct suppressor mechanism most likely plays a prominent and identifiable role at a particular tissue and inflammatory setting.

TGF-beta-induced Foxp3 inhibits T(H)17 cell differentiation by antagonizing RORgammat function.

T helper cells that produce IL-17 (T(H)17 cells) promote autoimmunity in mice and have been implicated in the pathogenesis of human inflammatory diseases. At mucosal surfaces, T(H)17 cells are thought to protect the host from infection, whereas regulatory T (T(reg)) cells control immune responses and inflammation triggered by the resident microflora. Differentiation of both cell types requires transforming growth factor-beta (TGF-beta), but depends on distinct transcription factors: RORgammat (encoded by Rorc(gammat)) for T(H)17 cells and Foxp3 for T(reg) cells. How TGF-beta regulates the differentiation of T cells with opposing activities has been perplexing. Here we demonstrate that, together with pro-inflammatory cytokines, TGF-beta orchestrates T(H)17 cell differentiation in a concentration-dependent manner. At low concentrations, TGF-beta synergizes with interleukin (IL)-6 and IL-21 (refs 9-11) to promote IL-23 receptor (Il23r) expression, favouring T(H)17 cell differentiation. High concentrations of TGF-beta repress IL23r expression and favour Foxp3+ T(reg) cells. RORgammat and Foxp3 are co-expressed in naive CD4+ T cells exposed to TGF-beta and in a subset of T cells in the small intestinal lamina propria of the mouse. In vitro, TGF-beta-induced Foxp3 inhibits RORgammat function, at least in part through their interaction. Accordingly, lamina propria T cells that co-express both transcription factors produce less IL-17 (also known as IL-17a) than those that express RORgammat alone. IL-6, IL-21 and IL-23 relieve Foxp3-mediated inhibition of RORgammat, thereby promoting T(H)17 cell differentiation. Therefore, the decision of antigen-stimulated cells to differentiate into either T(H)17 or T(reg) cells depends on the cytokine-regulated balance of RORgammat and Foxp3.

Two-dimensional high-throughput on-cell screening of immunoglobulins against broad antigen repertoires.

Identifying high-affinity antibodies in human serum is challenging due to extremely low number of circulating B cells specific to the desired antigens. Delays caused by a lack of information on the immunogenic proteins of viral origin hamper the development of therapeutic antibodies. We propose an efficient approach allowing for enrichment of high-affinity antibodies against pathogen proteins with simultaneous epitope mapping, even in the absence of structural information about the pathogenic immunogens. To screen therapeutic antibodies from blood of recovered donors, only pathogen transcriptome is required to design an antigen polypeptide library, representing pathogen proteins, exposed on the bacteriophage surface. We developed a two-dimensional screening approach enriching lentiviral immunoglobulin libraries from the convalescent or vaccinated donors against bacteriophage library expressing the overlapping set of polypeptides covering the spike protein of SARS-CoV-2. This platform is suitable for pathogen-specific immunoglobulin enrichment and allows high-throughput selection of therapeutic human antibodies.

Importance of group X-secreted phospholipase A2 in allergen-induced airway inflammation and remodeling in a mouse asthma model.

Arachidonic acid metabolites, the eicosanoids, are key mediators of allergen-induced airway inflammation and remodeling in asthma. The availability of free arachidonate in cells for subsequent eicosanoid biosynthesis is controlled by phospholipase A(2)s (PLA(2)s), most notably cytosolic PLA(2)-alpha. 10 secreted PLA(2)s (sPLA(2)s) have also been identified, but their function in eicosanoid generation is poorly understood. We investigated the role of group X sPLA(2) (sPLA(2)-X), the sPLA(2) with the highest in vitro cellular phospholipolysis activity, in acute and chronic mouse asthma models in vivo. The lungs of sPLA(2)-X(-/-) mice, compared with those of sPLA(2)-X(+/+) littermates, had significant reduction in ovalbumin-induced infiltration by CD4(+) and CD8(+) T cells and eosinophils, goblet cell metaplasia, smooth muscle cell layer thickening, subepithelial fibrosis, and levels of T helper type 2 cell cytokines and eicosanoids. These data direct attention to sPLA(2)-X as a novel therapeutic target for asthma.

Nuclear oncoprotein prothymosin alpha is a partner of Keap1: implications for expression of oxidative stress-protecting genes.

Animal cells counteract oxidative stress and electrophilic attack through coordinated expression of a set of detoxifying and antioxidant enzyme genes mediated by transcription factor Nrf2. In unstressed cells, Nrf2 appears to be sequestered in the cytoplasm via association with an inhibitor protein, Keap1. Here, by using the yeast two-hybrid screen, human Keap1 has been identified as a partner of the nuclear protein prothymosin alpha. The in vivo and in vitro data indicated that the prothymosin alpha-Keap1 interaction is direct, highly specific, and functionally relevant. Furthermore, we showed that Keap1 is a nuclear-cytoplasmic shuttling protein equipped with a nuclear export signal that is important for its inhibitory action. Prothymosin alpha was able to liberate Nrf2 from the Nrf2-Keap1 inhibitory complex in vitro through competition with Nrf2 for binding to the same domain of Keap1. In vivo, the level of Nrf2-dependent transcription was correlated with the intracellular level of prothymosin alpha by using prothymosin alpha overproduction and mRNA interference approaches. Our data attribute to prothymosin alpha the role of intranuclear dissociator of the Nrf2-Keap1 complex, thus revealing a novel function for prothymosin alpha and adding a new dimension to the molecular mechanisms underlying expression of oxidative stress-protecting genes.

The introduction of mesenchymal stromal cells induces different immunological responses in the lungs of healthy and M. tuberculosis infected mice.

Mesenchymal stromal cells (MSC) have strong immunomodulatory properties and therefore can be used to control inflammation and tissue damage. It was suggested recently that MSC injections can be used to treat multi-drug resistant tuberculosis (TB). However, MSC trafficking and immunomodulatory effects of MSC injections during Mycobacterium tuberculosis (Mtb) infection have not been studied. To address this issue we have analyzed MSC distribution in tissues and local immunological effects of MSC injections in Mtb infected and uninfected mice. After intravenous injection, MSC accumulated preferentially in the lungs where they were located as cell aggregates in the alveolar walls. Immunological analysis of MSC effects included detection of activated, IFN-γ and IL-4 producing CD4+ lymphocytes, the frequency analysis of dendritic cells (CD11c+F4/80) and macrophages (CD11c-F4/80+) located in the lungs, the expression of IA/IE and CD11b molecules by these cells, and evaluation of 23 cytokines/chemokines in lung lysates. In the lungs of uninfected mice, MSC transfer markedly increased the percentage of IFN-γ+ CD4+ lymphocytes and dendritic cells, elevated levels of IA/IE expression by dendritic cells and macrophages, augmented local production of type 2 cytokines (IL-4, IL-5, IL-10) and chemokines (CCL2, CCL3, CCL4, CCL5, CXCL1), and downregulated type 1 and hematopoietic cytokines (IL-12p70, IFN-γ, IL-3, IL-6, GM-CSF). Compared to uninfected mice, Mtb infected mice had statistically higher "background" frequency of activated CD69+ and IFN-γ+ CD4+ lymphocytes and dendritic cells, and higher levels of cytokines in the lungs. The injections of MSC to Mtb infected mice did not show statistically significant effects on CD4+ lymphocytes, dendritic cells and macrophages, only slightly shifted cytokine profile, and did not change pathogen load or slow down TB progression. Lung section analysis showed that in Mtb infected mice, MSC could not be found in the proximity of the inflammatory foci. Thus, in healthy recipients, MSC administration dramatically changed T-cell function and cytokine/chemokine milieu in the lungs, most likely, due to capillary blockade. But, during Mtb infection, i.e., in the highly-inflammatory conditions, MSC did not affect T-cell function and the level of inflammation. The findings emphasize the importance of the evaluation of MSC effects locally at the site of their predominant post-injection localization and question MSC usefulness as anti-TB treatment.

Sensing prothymosin alpha origin, mutations and conformation with monoclonal antibodies.

To overcome poor immunogenicity of prothymosin alpha, a small and highly acidic nuclear protein involved in cell proliferation, production of anti-prothymosin alpha antibodies in mice immunized with free human prothymosin alpha, with prothymosin alpha coupled to different carriers and with prothymosin alpha fused to green fluorescent protein was assessed. Fusing prothymosin alpha to green fluorescent protein turned out to be the superior approach resulting in production of high titer anti-prothymosin alpha antibodies. From these studies, two highly specific anti-prothymosin alpha monoclonal antibodies recognizing epitopes within the amino terminal (2F11) and middle (4F4) portions of the human prothymosin alpha molecule were obtained and characterized. As expected, the 2F11 antibody displayed broad species specificity, whereas the 4F4 antibody appeared to be species-specific permitting discrimination of human versus rat protein. Furthermore, a combination of point mutations in prothymosin alpha that alter the properties of the protein precluded recognition by the 4F4 antibody. Intramolecular masking of the 4F4 epitope in prothymosin alpha fused to the Tat transduction peptide of human immunodeficiency virus type 1 was observed. The anti-prothymosin alpha antibodies obtained were suitable for precipitation of human prothymosin alpha from HeLa cell lysates and for immunolocalization of the endogenous prothymosin alpha within the cells. Fusion with green fluorescent protein may thus be helpful in raising antibodies against 'problematic' proteins.

Stability of the regulatory T cell lineage in vivo.

Tissue maintenance and homeostasis can be achieved through the replacement of dying cells by differentiating precursors or self-renewal of terminally differentiated cells or relies heavily on cellular longevity in poorly regenerating tissues. Regulatory T cells (T(reg) cells) represent an actively dividing cell population with critical function in suppression of lethal immune-mediated inflammation. The plasticity of T(reg) cells has been actively debated because it could factor importantly in protective immunity or autoimmunity. By using inducible labeling and tracking of T(reg) cell fate in vivo, or transfers of highly purified T(reg) cells, we have demonstrated notable stability of this cell population under physiologic and inflammatory conditions. Our results suggest that self-renewal of mature T(reg) cells serves as a major mechanism of maintenance of the T(reg) cell lineage in adult mice.

A plant caspase-like protease activated during the hypersensitive response.

To test the hypothesis that caspase-like proteases exist and are critically involved in the implementation of programmed cell death (PCD) in plants, a search was undertaken for plant caspases activated during the N gene-mediated hypersensitive response (HR; a form of pathogen-induced PCD in plants) in tobacco plants infected with Tobacco mosaic virus (TMV). For detection, characterization, and partial purification of a tobacco caspase, the Agrobacterium tumefaciens VirD2 protein, shown here to be cleaved specifically at two sites (TATD and GEQD) by human caspase-3, was used as a target. In tobacco leaves, specific proteolytic processing of the ectopically produced VirD2 derivatives at these sites was found to occur early in the course of the HR triggered by TMV. A proteolytic activity capable of specifically cleaving the model substrate at TATD was partially purified from these leaves. A tetrapeptide aldehyde designed and synthesized on the basis of the elucidated plant caspase cleavage site prevented fragmentation of the substrate protein by plant and human caspases in vitro and counteracted TMV-triggered HR in vivo. Therefore, our data provide a characterization of caspase-specific protein fragmentation in apoptotic plant cells, with implications for the importance of such activity in the implementation of plant PCD.

Apoptosis-related fragmentation, translocation, and properties of human prothymosin alpha.

Human prothymosin alpha is a proliferation-related nuclear protein undergoing caspase-mediated fragmentation in apoptotic cells. We show here that caspase-3 is the principal executor of prothymosin alpha fragmentation in vivo. In apoptotic HeLa cells as well as in vitro, caspase-3 cleaves prothymosin alpha at one major carboxy terminal (DDVD(99)) and several suboptimal sites. Prothymosin alpha cleavage at two amino-terminal sites (AAVD(6) and NGRD(31)) contributes significantly to the final pattern of prothymosin alpha fragmentation in vitro and could be detected to occur in apoptotic cells. The major caspase cleavage at D(99) disrupts the nuclear localization signal of prothymosin alpha, which leads to a profound alteration in subcellular localization of the truncated protein. By using a set of anti-prothymosin alpha monoclonal antibodies, we were able to observe nuclear escape and cell surface exposure of endogenous prothymosin alpha in apoptotic, but not in normal, cells. We demonstrate also that ectopic production of human prothymosin alpha and its mutants with nuclear or nuclear-cytoplasmic localization confers increased resistance of HeLa cells toward the tumor necrosis factor-induced apoptosis.