Chimeric Antigen Receptor-Engineered Human Gamma Delta T Cells: Enhanced Cytotoxicity with Retention of Cross Presentation.

Gamma delta T (γδT) lymphocytes are primed for rapid function, including cytotoxicity toward cancer cells, and are a component of the immediate stress response. Following activation, they can function as professional antigen-presenting cells. Chimeric antigen receptors (CARs) work by focusing T cell function on defined cell surface tumor antigens and provide essential costimulation for robust activation. Given the natural tropism of γδT cells for the tumor microenvironment, we hypothesized that their transduction with CARs might enhance cytotoxicity while retaining their ability to migrate to tumor and act as antigen-presenting cells to prolong the intratumoral immune response. Using a GD2-targeting CAR as a model system, we showed that γδT cells of both Vδ1 and Vδ2 subsets could be expanded and transduced to sufficient numbers for clinical studies. The CAR added to the cells' innate cytotoxicity by enhancing GD2-specific killing of GD2-expressing cancer cell lines. Migration toward tumor cells in vitro was not impaired by the presence of the CAR. Expanded CAR-transduced Vδ2 cells retained the ability to take up tumor antigens and cross presented the processed peptide to responder alpha beta T (αßT) lymphocytes. γδ CAR-T cell products show promise for evaluation in clinical studies of solid tumors.

Avoidance of On-Target Off-Tumor Activation Using a Co-stimulation-Only Chimeric Antigen Receptor.

Chimeric antigen receptors (CARs) combine T cell activation with antibody-mediated tumor antigen specificity, bypassing the need for T cell receptor (TCR) ligation. A limitation of CAR technology is on-target off-tumor toxicity caused by target antigen expression on normal cells. Using GD2 as a model cancer antigen, we hypothesized that this could be minimized by using T cells expressing Vγ9Vδ2 TCR, which recognizes transformed cells in a major histocompatibility complex (MHC)-unrestricted manner, in combination with a co-stimulatory CAR that would function independently of the TCR. An anti-GD2 CAR containing a solitary endodomain derived from the NKG2D adaptor DAP10 was expressed in Vγ9Vδ2+ T cells. Differential ligation of the CAR and/or TCR using antibody-coated beads showed that pro-inflammatory cytokine response depended on activation of both receptors. Moreover, in killing assays, GD2-expressing neuroblastoma cells that engaged the Vγ9Vδ2 TCR were efficiently lysed, whereas cells that expressed GD2 equivalently but did not engage the Vγ9Vδ2 TCR were untouched. Differentiation between X-on tumor and X-off tumor offers potential for safer immunotherapy and broader target selection.

Cell electrospinning: an in vitro and in vivo study.

Cell electrospinning and aerodynamically assisted bio-threading are novel bioplatforms for directly forming large quantities of cell-laden scaffolds for creating living sheets and vessels in three-dimensions. The functional biological architectures generated will be useful in both the laboratory and the clinic.

Human γδ T lymphocytes are licensed for professional antigen presentation by interaction with opsonized target cells.

Activated human blood γδ T cells have also been previously demonstrated to behave as professional APCs, although the processes that control APC function have not been characterized. n this study, we show that the acquisition of potent APC function by human blood γδ T cells is achieved after physical interaction with an Ab-coated target cell, a process that we refer to as licensing. In cancer models, licensing of γδ T cells by tumor-reactive mAbs promotes the uptake of tumor Ags and professional presentation to tumor-reactive αß T cells. We propose that licensing by Ab is a mechanism whereby the adaptive properties of γδ T cells are induced by their innate functions in a spatially and temporally controlled manner.

Comparison of nanocomplexes with branched and linear peptides for siRNA delivery.

Efficient delivery of small interfering RNA (siRNA) remains the greatest technological barrier to the clinical implementation of RNA interference strategies. We are investigating the relationship between the biophysical properties of siRNA nanocomplexes and their transfection efficiency as an approach to the generation of improved formulations. Peptide-based formulations are of great interest, and so in this study we have compared nanocomplex formulations for siRNA delivery containing linear and branched oligolysine or oligoarginine peptides. Peptides were combined with cationic liposomes in siRNA formulations and compared for transfection efficiency, siRNA packaging efficiency, biophysical properties, and particle stability. Nanocomplexes containing linear peptides were more condensed and stable than branched peptide formulations; however, their silencing activity was lower, suggesting that their greater stability might limit siRNA release within the cell. Thus, differences in transfection appeared to be associated with differences in packaging and stability, indicating the importance of optimizing this feature in siRNA nanocomplexes.

Antigen-specific gene therapy after immunisation reduces the severity of collagen-induced arthritis.

Reestablishment of tolerance induction in rheumatoid arthritis (RA) would be an optimal treatment with few, if any, side effects. However, to develop such a treatment further insights in the immunological mechanisms governing tolerance are needed. We have developed a model of antigen-specific tolerance in collagen type II (CII) induced arthritis (CIA) using lentivirus-based gene therapy. The immunodominant epitope of CII was inserted into a lentivirus vector to achieve expression on the MHC class II molecule and the lentiviral particles were subsequently intravenously injected at different time points during CIA. Injection of lentiviral particles in early phases of CIA, that is, at day 7 or day 26 after CII immunisation, partially prevented development of arthritis, decreased the serum levels of CII-specific IgG antibodies, and enhanced the suppressive function of CII-specific T regulatory cells. When lentiviral particles were injected during manifest arthritis, that is, at day 31 after CII immunisation, the severity of arthritis progression was ameliorated, the levels of CII-specific IgG antibodies decreased and the proportion of T regulatory cells increased. Thus, antigen-specific gene therapy is effective when administered throughout the inflammatory course of arthritis and offers a good model for investigation of the basic mechanisms during tolerance in CIA.

Bacterial outer-membrane vesicles promote Vγ9Vδ2 T cell oncolytic activity.

Background: Increasing evidence suggests the immune activation elicited by bacterial outer-membrane vesicles (OMVs) can initiate a potent anti-tumor immunity, facilitating the recognition and destruction of malignant cells. At present the pathways underlying this response remain poorly understood, though a role for innate-like cells such as γδ T cells has been suggested. Methods: Peripheral blood mononuclear cells (PBMCs) from healthy donors were co-cultured with E. coli MG1655 Δpal ΔlpxM OMVs and corresponding immune activation studied by cell marker expression and cytokine production. OMV-activated γδ T cells were co-cultured with cancer cell lines to determine cytotoxicity. Results: The vesicles induced a broad inflammatory response with γδ T cells observed as the predominant cell type to proliferate post-OMV challenge. Notably, the majority of γδ T cells were of the Vγ9Vδ2 type, known to respond to both bacterial metabolites and stress markers present on tumor cells. We observed robust cytolytic activity of Vγ9Vδ2 T cells against both breast and leukaemia cell lines (SkBr3 and Nalm6 respectively) after OMV-mediated expansion. Conclusions: Our findings identify for the first time, that OMV-challenge stimulates the expansion of Vγ9Vδ2 T cells which subsequently present anti-tumor capabilities. We propose that OMV-mediated immune activation leverages the anti-microbial/anti-tumor capacity of Vγ9Vδ2 T cells, an axis amenable for improved future therapeutics.

CD68-expressing cells can prime T cells and initiate autoimmune arthritis in the absence of reactive oxygen species.

It is widely believed that DC, but not macrophages, prime naïve T cells in vivo. Here, we investigated the ability of CD68-expressing cells (commonly defined as macrophages) in priming autoreactive T cells and initiating collagen-induced arthritis (CIA) in the mouse. For this purpose, a transgenic mouse was developed (MBQ mouse) where macrophages exclusively expressed the MHC class II H2-A(q) (A(q)) on an H2-A(p) (A(p)) background. A(q), but not A(p) expression mediates susceptibility to CIA through presentation of type II collagen (CII) to T cells. CIA severity is enhanced by a mutation in the Ncf1 gene, impairing reactive oxygen species (ROS) production by the phagocyte NADPH oxidase (NOX2) complex. Expression of functional Ncf1 on macrophages was previously shown to protect from severe CIA. To study the effect of ROS on macrophage-mediated priming of T cells, the Ncf1 mutation was introduced in the MBQ mouse. Upon CII immunization, Ncf1-mutated MBQ mice, but not Ncf1 wild-type MBQ mice nor Ncf1-mutated A(p) mice, activated autoreactive T cells and developed CIA. These findings demonstrate for the first time that macrophages can initiate arthritis and that the process is negatively regulated by ROS produced via the NOX2 complex.

In vitro and in vivo interrogation of bio-sprayed cells.

Bio-sprays can directly form pre-organized cell-bearing structures for applications ranging from engineering functional tissues to the forming of cultures, most useful for modeling disease, to the discovery and development of drugs. Bio-electrosprays and aerodynamically assisted bio-jets, are leading approaches that have been demonstrated as having far-reaching ramifications for regenerative biology and medicine.

Human gamma delta T cells: a lymphoid lineage cell capable of professional phagocytosis.

Professional phagocytosis in mammals is considered to be performed exclusively by myeloid cell types. In this study, we demonstrate, for the first time, that a mammalian lymphocyte subset can operate as a professional phagocyte. By using confocal microscopy, transmission electron microscopy, and functional Ag presentation assays, we find that freshly isolated human peripheral blood gammadelta T cells can phagocytose Escherichia coli and 1 microm synthetic beads via Ab opsonization and CD16 (FcgammaRIII), leading to Ag processing and presentation on MHC class II. In contrast, other CD16(+) lymphocytes, i.e., CD16(+)/CD56(+) NK cells, were not capable of such functions. These findings of distinct myeloid characteristics in gammadelta T cells strongly support the suggestion that gammadelta T cells are evolutionarily ancient lymphocytes and have implications for our understanding of their role in transitional immunity and the control of infectious diseases and cancer.

Gamma Delta T Cells and Their Involvement in COVID-19 Virus Infections.

The global outbreak of the SARS-Cov-2 virus in 2020 has killed millions of people worldwide and forced large parts of the world into lockdowns. While multiple vaccine programs are starting to immunize the global population, there is no direct cure for COVID-19, the disease caused by the SARS-Cov-2 infection. A common symptom in patients is a decrease in T cells, called lymphopenia. It is as of yet unclear what the exact role of T cells are in the immune response to COVID-19. The research so far has mainly focused on the involvement of classical αß T cells. However, another subset of T cells called γδ T cells could have an important role to play. As part of the innate immune system, γδ T cells respond to inflammation and stressed or infected cells. The γδ T cell subset appears to be particularly affected by lymphopenia in COVID-19 patients and commonly express activation and exhaustion markers. Particularly in children, this subset of T cells seems to be most affected. This is interesting and relevant because γδ T cells are more prominent and active in early life. Their specific involvement in this group of patients could indicate a significant role for γδ T cells in this disease. Furthermore, they seem to be involved in other viral infections and were able to kill SARS infected cells in vitro. γδ T cells can take up, process and present antigens from microbes and human cells. As e.g. tumour-associated antigens are presented by MHC on γδ T cells to classical T-cells, we argue here that it stands to reason that also viral antigens, such as SARS-Cov-2-derived peptides, can be presented in the same way. γδ T cells are already used for medical purposes in oncology and have potential in cancer therapy. As γδ T cells are not necessarily able to distinguish between a transformed and a virally infected cell it could therefore be of great interest to investigate further the relationship between COVID-19 and γδ T cells.

Bioengineering bacterial encapsulin nanocompartments as targeted drug delivery system.

The development of Drug Delivery Systems (DDS) has led to increasingly efficient therapies for the treatment and detection of various diseases. DDS use a range of nanoscale delivery platforms produced from polymeric of inorganic materials, such as micelles, and metal and polymeric nanoparticles, but their variant chemical composition make alterations to their size, shape, or structures inherently complex. Genetically encoded protein nanocages are highly promising DDS candidates because of their modular composition, ease of recombinant production in a range of hosts, control over assembly and loading of cargo molecules and biodegradability. One example of naturally occurring nanocompartments are encapsulins, recently discovered bacterial organelles that have been shown to be reprogrammable as nanobioreactors and vaccine candidates. Here we report the design and application of a targeted DDS platform based on the Thermotoga maritima encapsulin reprogrammed to display an antibody mimic protein called Designed Ankyrin repeat protein (DARPin) on the outer surface and to encapsulate a cytotoxic payload. The DARPin9.29 chosen in this study specifically binds to human epidermal growth factor receptor 2 (HER2) on breast cancer cells, as demonstrated in an in vitro cell culture model. The encapsulin-based DDS is assembled in one step in vivo by co-expressing the encapsulin-DARPin9.29 fusion protein with an engineered flavin-binding protein mini-singlet oxygen generator (MiniSOG), from a single plasmid in Escherichia coli. Purified encapsulin-DARPin_miniSOG nanocompartments bind specifically to HER2 positive breast cancer cells and trigger apoptosis, indicating that the system is functional and specific. The DDS is modular and has the potential to form the basis of a multi-receptor targeted system by utilising the DARPin screening libraries, allowing use of new DARPins of known specificities, and through the proven flexibility of the encapsulin cargo loading mechanism, allowing selection of cargo proteins of choice.

Combining bio-electrospraying with gene therapy: a novel biotechnique for the delivery of genetic material via living cells.

The investigations reported in this article demonstrate the ability of bio-electrosprays and cell electrospinning to deliver a genetic construct in association with living cells. Previous studies on both bio-electrosprays and cell electrospinning demonstrated great promise for tissue engineering and regenerative biology/medicine. The investigations described herein widen the applicability of these biotechniques by combining gene therapy protocols, resulting in a novel drug delivery methodology previously unexplored. In these studies a human cell line was transduced with recombinant self-inactivating lentiviral particles. These particles incorporated a green fluorescent protein fused to an endosomal targeting construct. This construct encodes a peptide, which can subsequently be detected on the surface of cells by specific T-cells. The transduced cell line was subsequently manipulated in association with either bio-electrospraying or cell electrospinning. Hence this demonstrates (i) the ability to safely handle genetically modified living cells and (ii) the ability to directly form pre-determined architectures bearing living therapeutic cells. This merged technology demonstrates a unique approach for directly forming living therapeutic architectures for controlled and targeted release of experimental cells/genes, as well as medical cell/gene therapeutics for a plethora of biological and medical applications. Hence, such developments could be applied to personalised medicine.

Tolerance induction using lentiviral gene delivery delays onset and severity of collagen II arthritis.

The treatment of rheumatoid arthritis remains suboptimal; thus there is considerable interest in the development of strategies that mediate tolerance to autoantigens. Using lentiviral gene transfer in vivo, we expressed the immunodominant epitope of collagen type II (CII) on major histocompatibility complex class II molecules (MHC II) in a mouse model of destructive arthritis. A sequence corresponding to amino acids 259-270 of CII was fused into the class II-associated invariant chain peptide (CLIP) position of the invariant chain to achieve efficient binding to MHC II. Transduction of cloned cells and primary antigen-presenting cells (APCs) in vitro demonstrated successful presentation of the peptide on MHC II, and a physiological glycosylation pattern. Compared with controls, mice intravenously injected with lentiviral vectors encoding this epitope displayed significantly less frequent, less severe, and less destructive arthritis, decreased lymphocyte proliferation in response to restimulation with CII, and lower CII-specific antibody levels. This was associated with an increased production of transforming growth factor-beta (TGF-beta) in vitro. We suggest that overexpression of the immunodominant CII epitope on MHC II induces T cell production of TGF-beta and leads to inhibition of arthritis by means of both antigen-specific and bystander mechanisms. Thus, antigen-specific tolerance induction using lentiviral gene delivery can ameliorate arthritis.

Broad and Effective Protection against Staphylococcus aureus Is Elicited by a Multivalent Vaccine Formulated with Novel Antigens.

The demand for a prophylactic vaccine against methicillin-resistant Staphylococcus aureus (MRSA) has motivated numerous dedicated research groups to design and develop such a vaccine. In this study, we have developed a multivalent vaccine, Sta-V5, composed of five conserved antigens involved in three important virulence mechanisms. This prototype vaccine conferred up to 100% protection against multiple epidemiologically relevant S. aureus isolates in five different murine disease models. The vaccine not only elicits functional antibodies that mediate opsonophagocytic killing of S. aureus but also mounts robust antigen-specific T-cell responses. In addition, our data implied that γδ T cells contribute to the protection induced by Sta-V5 in a murine skin infection model.IMPORTANCEStaphylococcus aureus infections, especially MRSA infections, are becoming a major global health issue and are resulting in mortality rates that are increasing every year. However, an effective vaccine is lacking due to the complexity of the infection process of S. aureus In this study, we found that the addition of two novel protein components to three well-studied vaccine candidates significantly improved the efficacy of the combined vaccine. Furthermore, the five-component vaccine not only elicits a robust antibody response but also induces cytokine secretion by T cells, making it a promising vaccine candidate to fill the void.

A novel method for ABO-incompatible heart transplantation.

BACKGROUND: Since 1996, ABO-incompatible heart transplantation has been undertaken by performing whole-body plasma exchange to remove isohemagglutinins using the cardiopulmonary bypass (CPB) circuit at the time of transplantation. This requires large volumes of donated blood and blood products, causes hemodynamic instability during the exchange transfusion, and limits practical use to small children. We sought to determine the efficacy of anti-A/B immunoadsorption within the CPB circuit on removal of isohemagglutinins in an ex vivo setting before its use clinically. METHODS: An anti-A/B immunoadsorption column was placed into a CPB circuit mimicking a typical ABO-incompatible transplant patient, which had been primed with type O whole human blood. Samples were taken for determination of isohemagglutinin titers following each plasma volume pass through the anti-A/B immunoadsorption column. RESULTS: There was a linear decrease of at least 1 dilution seen in both anti-A and anti-B IgG and IgM antibodies with each plasma volume pass through the column. This predictable removal allowed the formulation of selection criteria for ABO-incompatible heart transplantation given the reciprocal of titer and patient weight. This degree of predictability allowed us to use it successfully in the clinical setting, reducing antibodies to an undetectable level during ABO-incompatible heart transplantation. CONCLUSIONS: The incorporation of an anti-A/B immunoadsorption column into the extracorporeal circuit reduces allogeneic blood product requirement for ABO-incompatible heart transplantation, while providing efficacious removal of anti-A and anti-B isohemagglutinins. This can be undertaken within the time period of CPB before graft reperfusion and expands the potential recipient pool to larger patients with higher isohemagglutinin titers.

Effective combination treatment of GD2-expressing neuroblastoma and Ewing's sarcoma using anti-GD2 ch14.18/CHO antibody with Vγ9Vδ2+ γδT cells.

Gamma delta T lymphocytes (γδT cells) have pleiotropic properties including innate cytotoxicity, which make them attractive effectors for cancer immunotherapy. Combination treatment with zoledronic acid and IL-2 can activate and expand the most common subset of blood γδT, which express the Vγ9Vδ2 T cell receptor (TCR) (Vδ2 T cells). Vγ9Vδ2 T cells are equipped for antibody-dependent cell-mediated cytotoxicity (ADCC) through expression of the low-affinity FcγR CD16. GD2 is a highly ranked tumor associated antigen for immunotherapy due to bright expression on the cell surface, absent expression on normal tissues and availability of therapeutic antibodies with known efficacy in neuroblastoma. To explore the hypothesis that zoledronic acid, IL-2 and anti-GD2 antibodies will synergize in a therapeutic combination, we evaluated in vitro cytotoxicity and tumor growth inhibition in the GD2 expressing cancers neuroblastoma and Ewing's sarcoma. Vδ2 T cells exert ADCC against GD2-expressing Ewing's sarcoma and neuroblastoma cell lines, an effect which correlates with the brightness of GD2 expression. In an immunodeficient mouse model of small established GD2-expressing Ewing's sarcoma or neuroblastoma tumors, the combination of adoptively transferred Vδ2+ T cells, expanded in vitro with zoledronic acid and IL-2, with anti-GD2 antibody ch14.18/CHO, and with systemic zoledronic acid, significantly suppressed tumor growth compared to antibody or γδT cell-free controls. Combination treatment using ch14.18/CHO, zoledronic acid and IL-2 is more effective than their use in isolation. The already-established safety profiles of these agents make testing of the combination in GD2 positive cancers such as neuroblastoma or Ewing's sarcoma both rational and feasible.

Gene Therapy Induces Antigen-Specific Tolerance in Experimental Collagen-Induced Arthritis.

Here, we investigate induction of immunological tolerance by lentiviral based gene therapy in a mouse model of rheumatoid arthritis, collagen II-induced arthritis (CIA). Targeting the expression of the collagen type II (CII) to antigen presenting cells (APCs) induced antigen-specific tolerance, where only 5% of the mice developed arthritis as compared with 95% of the control mice. In the CII-tolerized mice, the proportion of Tregs as well as mRNA expression of SOCS1 (suppressors of cytokine signaling 1) increased at day 3 after CII immunization. Transfer of B cells or non-B cell APC, as well as T cells, from tolerized to naïve mice all mediated a certain degree of tolerance. Thus, sustainable tolerance is established very early during the course of arthritis and is mediated by both B and non-B cells as APCs. This novel approach for inducing tolerance to disease specific antigens can be used for studying tolerance mechanisms, not only in CIA but also in other autoimmune diseases.

Collagen epitope expression on B cells is sufficient to confer tolerance to collagen-induced arthritis.

BACKGROUND: The mechanisms underlying tolerance induction and maintenance in autoimmune arthritis remain elusive. In a mouse model of rheumatoid arthritis, collagen type II (CII)-induced arthritis, we explore the contribution of B cells to antigen-specific tolerance. METHODS: To generate expression of the CII-peptide specifically on B-cell major histocompatibility complex type II, lentiviral-based gene therapy including a B-cell-specific Igk promoter was used. RESULTS: Presentation of the CII-peptide on B cells significantly reduced the frequency and severity of arthritis as well as the serum levels of CII -specific IgG antibodies. Further, both frequency and suppressive function of regulatory T cells were increased in tolerized mice. Adoptive transfer of regulatory T cells from tolerized mice to naïve mice ameliorated the development of CII-induced arthritis. CONCLUSION: Our data suggest that endogenous presentation of the CII-peptide on B cells is one of the key contributors to arthritis tolerance induction and maintenance.