Finally we highlight exciting novel pre-clinical research that has the potential to translate to the clinic successfully. We approach these methods from a pathophysiology based perspective as well and touch upon strategies targeting the interaction between tissue damage induced antigens and T-cells, regimen related endothelial toxicity, T-cell co-stimulatory pathways and other T-cell modulatory approaches, T-cell trafficking, and cytokine pathways. We end this review with a critical discussion of existing data and novel therapies that may be transformative in the field in the near future as a comprehensive picture of GVHD prophylaxis in 2020. While calcineurin inhibitors remain the standard, post-transplant eparinsphamide originally developed to facilitate haploidentical transplantation is becoming an attractive alternative to traditional calcinuerin inhibitor based prophylaxis due to its ability to reduce severe forms of acute and chronic GVHD without compromising other outcomes, even in the HLA-matched setting. In addition T-cell modulation, particularly targeting some important T-cell co-stimulatory pathways have resulted in promising outcomes and may be a part of GVHD prophylaxis in the future. Novel approaches including targeting early events in GVHD pathogenesis such as interactions bvetween tissue damage associated antigens and T-cells, endothelial toxicity, and T-cell trafficking are also promising and discussed in this review. GVHD prophylaxis in 2020 continues to evolve with novel exicitng therapies on the horizon based on a more sophisticated understanding of the immunobiology of GVHD.Graft-vs. host disease (GVHD), both acute and chronic are among the chief non-relapse complications of allogeneic transplantation which still cause substantial morbidity and mortality despite significant advances in supportive care over the last few decades. The prevention of GVHD therefore remains critical to the success of allogeneic transplantation. In this review we briefly discuss the pathophysiology and immunobiology of GVHD and the current standards in the field which remain centered around calcineurin inhibitors. We then discuss important translational advances in GVHD prophylaxis, approaching these various platforms from a mechanistic standpoint based on the pathophysiology of GVHD including  GVHD prophylaxis has come a long way since the initial days of allogeneic transplantation. The improvement in GVHD outcomes has been one of the primary reasons for the reduction in non-relapse mortality over time  that hasIn HLA-matched transplantation, while the backbone of most widely used prophylactic platforms remains calcineurin-inhibitor (CNI)-based, a variety of new drugs have been added to CNI's in an attempt to improve efficacy and reduce toxicity. In this review, we discuss current standards and their evolution over time and highlight some of these translational advances. Further we touch upon novel pre-clinical advances developed on the foundation of a deeper understanding of transplant immunology and promising for translation to the clinic. We begin with a description of the immunobiology of GVHD, to better understand potential targets which have been exploited over the last few decades and currently to develop effective prophylactic therapies for GVHD.One of the first models describing the biology of GVHD was proposed by Antin and Ferrara where they described a sequential cascade initiated by conditioning regimen mediated host tissue injury with the production of inflammatory cytokines (phase 1). This is followed by activation and proliferation of effector T-lymphocytes (phase II) which eventually lead to recruitment and activation of additional mononuclear effectors and amplification of a “cytokine storm” (Phase III) . FurtherBoth neutrophils and monocytes are involved in the initial inflammatory response in the pathogenesis of GVHD. Monocytes are activated by molecules called damage-associated molecular patterns (DAMPs) such as uric acid, ATP, Heparan sulfate, HMGB-1 or IL-33 which can initiate and perpetuate a non-infectious inflammatory response involving the innate immune system. In contrast pathogen-associated molecular patterns (PAMPs) such as bacterial lipopolysaccharides cause infection-associated inflammation. The DAMP and PAMP mediated inflammatory responses result in activation of the innate immune system (monocytes and neutrophils) which then cause local tissue damage mediated by reactive oxygen species. This eventually culminates in interaction of antigen-presenting cells (APCs) in the innate and adaptive immune and activation of cytokine cascades  leading to the “cytokine storm  which bind corresponding host tissue ligands presenting a potential target for intervention. The T-cell activation process and proliferation process is a crucial target for GVHD prevention.These events lead to a self-potentiating T-lymphocyte activation causing tissue damage via direct cellular cytotoxicity and indirectly via release of soluble mediators  .A number of additional pathways have since been implicated in GVHD pathogenesis including the canonical NOTCH pathway . It has via release of anti-inflammatory cytokines such as IL-10 and TGF-β  are important in immunologic tolerance, partly nd TGF-β . Cytokinnd TGF-β . In addiD deaths . These aChronic GVHD remains the most common late toxicity of allogeneic transplantation with significant morbidity and quality of life implications. cGVHD has its own distinctive immunobiology. Briefly we can conceptualize the pathophysiology of cGVHD in three phases: (1) Inflammation leading to tissue damage (2) chronic inflammation, thymic injury, dysregulated B- and T-cell immunity (3) tissue repair with fibrosis , 12. AltPotential targets for developing novel prophylactic platforms have been identified based on our current and more comprehensive understanding of the biology of GVHD. In this review we discuss both current standards and important translational advances as well as exciting new potential therapies which may be translated to the clinic in the future.The effective prevention of GVHD is critical to the success of allogeneic transplantation. Based on the understanding that aGVHD is primarily mediated by effector T-lymphocytes, prophylactic strategies have focused on T-cell suppression in the recipient. Calcineurin inhibitors (tacrolimus/Tac and cyclosporine/CyA) inhibit the proliferation and activation of T-cells and have been used in combination with either methotrexate (MTX) or mycophenolate mofetil (MMF) as standard prophylaxis in HLA-matched HSCT. In two randomized controlled trials (RCT) in the 1990s, the combination of Tac/MTX was found to be significantly superior to CyA/MTX is the prevention of grade II-IV aGVHD and extensive chronic GVHD in HLA-matched sibling and unrelated donors, although a benefit in overall survival (OS) was not shown , 14. Furin-vivo has been the basis for the development of a number of novel GVHD prophylaxis strategies. These have typically been incorporated into regimens where the backbone comprises CNIs. We summarize some of these approaches below.T-cell depletion or modulation Transplantation across HLA barriers historically has been difficult due to high rates of graft rejection and severe GVHD secondary to strong bidirectional alloreactive responses between donor and recipient. The introduction of post-transplant cyclophosphamide (PTCy) in the context of haploidentical transplantation has been a gamechanger and allowed us to perform such mismatched transplants safely and effectively, typically from related donors.First pioneered at Johns Hopkins, cyclophosphamide is given at doses of 50 mg/kg on days +3 and +4 following the infusion of haploidentical stem-cells. In initial studies reported by the Hopkins group, reduced intensity HSCT with PTCy along with Tac and MMF as GVHD prophylaxis resulted in engraftment in 87% of patients with acceptable rates of grade II-IV (34%) and III-IV aGVHD (6%). Further, rates of chronic severe GVHD were found to be particularly low. Relapse rates were in the 50% range . NumerouThe earlier more simplistic hypotheses postulated that PTCy results in the deletion of alloreactive T-cells with some debate regarding the effect it may have on the T-cell mediated graft-vs. leukemia (GVL) effect. However, more recently it has been shown in mice that Tregs are preserved and that T-effector cell exhaustion may play an important role . A comprThe beneficial effect of PTCy on severe aGVHD and cGVHD has led to its adoption into the HLA-matched setting more recently. In a Phase II RCT (BMT CTN 1203) which compared three different GVHD prophylaxis regimens in RIC MUD HSCT  with standard Tac/MTX prophylaxis, the PTCy arm fared the best with comparable grade II-IV aGVHD (27%) but lower rates of grade III-IV aGVHD (2%) and cGVHD requiring immunosuppression 22%) outcomes. Relapse rates in the PTCy arm were 28%. Overall GVHD and relapse free survival (GRFS) was superior in the PTCy arm meeting the primary end-point of the trial . Again i% outcomeBolstered by this data, PTCy/Tac/MMF is now being compared to standard Tac/MTX prophylaxis in RIC HLA-matched HSCT in a large phase III RCT (BMT CTN 1703). PTCy does have the potential to eliminate the effect of donor mismatch on GVHD outcomes, however, CNI based prophylaxis remains the standard until phase III data is available.in-vivo TCD with the aim to reduce both acute and chronic GVHD with varying success.The polyclonal immunoglobulin product obtained from the sera of rabbits and horses immunized with human thymocytes or T-cell lines is called anti-thymocyte globulin or ATG. ATG has been used as part of transplant conditioning to effect Of the four RCTs that have evaluated ATG in combination with standard CNI/MTX prophylaxis, the first used horse ATG and showed a reduction in aGVHD; however, there were higher rates of infection with resultant no difference in NRM or OS. Importantly, there was a reduction in chronic severe GVHD , 25. TheRabbit ATG is generally considered to deplete T-cells more effectively as well as allow greater expansion of regulatory T-cells (Tregs) . The benIn this context, there is evidence that increased doses or prolonged dosage schedules of ATG may have immunosuppressive toxicity with increased NRM and relapse . IndividIn the realm of matched related donor transplantation, a recent multi-center randomized study from China demonstrated improved acute and chronic GVHD rates without compromising relapse or treatment-related mortality  and meriin-vitro studies appeared to spare regulatory T-lymphocytes. A favorable ratio of Tregs:Teff has been shown to be associated with better GVHD outcomes and hence sirolimus has an immunologic profile that was thought to be potentially beneficial for GVHD prevention. In addition, it has a distinct toxicity profile compared to tacrolimus and is not nephrotoxic. In a large RCT in myeloablative transplants with HLA-matched donors, sirolimus in combination with tacrolimus was compared with the standard Tac/MTX platform. There was no difference in grades II-IV aGVHD and cGVHD, but better grade III-IV aGVHD outcomes with sirolimus/Tac were seen. Non-relapse mortality (NRM) and OS were similar as well  negative selection  through the use of monoclonal antibodies with or without complement  pmplement . The twoex-vivo CD34 selection to PTCy + MMF and conventional Tac/MTX prophylaxis, the results of which are eagerly awaited.An early concern with TCD was that it could affect the powerful GVL effect in HSCT which is also believed to be T-cell mediated. In a RCT in patients transplanted with marrow grafts, TCD was compared to conventional prophylaxis with CyA/MTX; the 3-year disease free survival (DFS) was similar in both groups with lower rates of grade III-IV aGVHD with TCD. Relapse rates were however, higher with TCD, particularly in patients with chronic myelogenous leukemia (CML) . A largein-vivo TCD in the form of anti-thymocyte globulin (ATG), particularly in the haploidentical setting can potentially be helpful in achieving better engraftment rates (The other notable issue with pan-TCD has been a higher incidence of graft failure and slower immune reconstitution (IR) leading to higher rates of infectious complications, particularly viral infections  . The usent rates . Other snt rates  or the unt rates .Given these issues with pan-TCD, more selective methods of T-cell depletion aimed at preventing GVHD while preserving GVL are being explored with the availability of sophisticated clinical grade cell separation techniques.While a number of strategies have been attempted for selective TCD, they did not meet with lasting success. Depletion of CD5+ T-cells and CD8+ T-cells were tried in the 1990s; while rates of GVHD were encouraging, rates of relapse were high leading to the abandonment of these strategies. CD6 depletion is separately discussed in the section on T-cell modulation.More recently Bleakley et al. have looked at CD45RA (naïve) TCD with the understanding that it is primarily the naïve T-cells in an allograft that are alloreactive. Unfortunately, this strategy has not produced encouraging results thus far. Bleakley et al. reported a first in human trial where they performed CD45RA (naïve) TCD via a two-step immunomagnetic bead-based procedure in 35 adult patients. Although 34/35 patients engrafted, rates of aGVHD were high in the 66% range. This was not improved when naïve TCD was combined with a/b TCD (described below) either and this approach is not widely used at this time.While a majority of T-cells express α/β receptors T-cell receptors (TCR), 2–10% of T-cells express γ/δ TCR. These γ/δ T-cells are believed to have important innate immune effects characterized by rapid cytokine release and killing of viral infected and tumor cells . This maDuring T-cell activation, following initial engagement of antigen by the TCR, a number of co-stimulatory and co-inhibitory signals come into play mediated by receptors on T-cells and APCs. This is true in acute GVHD as well. Hence the modulation of these co-stimulatory and co-inhibitory interactions is one of the new frontiers in the prophylaxis of GVHD.The most promising of these has been blockade of the CD28/CTLA-4 axis. CD28 and CTLA-4 are both receptors on the T-cell which bind to B7-1/CD80 and B7-2/CD86 ligands on the APC; however, while CD28 is co-stimulatory, CTLA-4 is co-inhibitory. Abatacept (CTLA4-Ig) is the soluble extra-cellular portion of CTLA-4 complexed with immunoglobulin heavy chain which blocks CD28 and CTLA-4, with more of an effect on CD28 leading ultimately to an inhibitory signal. Murine models from Blazar et al. showed that CD28/CTLA-4 blockade could reduce aGVHD lethality . Kean etSince this approach blocks both stimulatory and inhibitory pathways, the concern for unwanted T-cell activation has been raised; hence more selective approaches to blocking CD28 are also being investigated. As an example, FR104 (CD28-specific pegylated-Fab') with and without sirolimus are being investigated in non-human primate models .ex-vivo expansion of Tregs is possible  comprise a unique subset of T-lymphocytes that can be derived from the thymus or converted from CD4+CD25- cells (inducible or iTregs). Tregs play an important role in immune homeostasis and a favorable balance between Tregs and effector T-cells may be important to prevent GVHD. While possible , there aneficial .Hence, rather than the direct infusion of Tregs, other approaches have been attempted which can upregulate Tregs or enhance their functionality in the post-transplant immune milieu. One such approach involves invariant natural killer T (iNKT) cells.ex-vivo expanded iNKT cells is also an area of investigation.iNKT cells are unique in that they co-express both T and NK cell markers and therefore straddle both the innate and adaptive immune system with a semi-invariant TCR that recognizes glycolipid antigens presented by the major histocompatibility complex (MHC) class I-like molecule CD1d. They modulate the immune system via IL-4 and IL-10. In murine models, iNKT cells reduced GVHD both by a switch to a Th2 cytokine profile and/or IL-4-dependent Treg expansion. These mice were conditioned with a regimen incorporating total lymphoid irradiation plus ATG (TLI-ATG) , 61. ThiIn this contect, REGiMMUNE is a compound in which KRN7000, a synthetic alpha-galactosylceramide derivative and a CD1d ligand, is embedded in a lipid bilayer. REGiMMUNE has been shown to reduce aGVHD mortality by expanding Tregs via iNKT cells in murine models . In a PhAlloreactive CD8+ T-cells bound for the intestines express high levels of integrin β7 (α4β7) that binds to its ligand mucosal addressin cell adhesion molecule 1 (MAdCAM 1) in Peyer's patches and gut-associated lymphoid tissue (GALT) in the intestinal mucosa. Vedolizumab is a humanized moAb which prevents T-cell trafficking to the gut by targeting α4β7integrins on the T-cells. Early proof-of-concept and restrospective analyses have shown promising efficacy with vedolizumab in steroid refractory aGVHD . Given iMaraviroc is an antagonist of CCR5, a chemokine receptor that has been implicated in T-cell trafficking during GVHD pathogenesis. Maraviroc appears to block lymphocyte chemotaxis without actually affecting T-cell function which made it an attractive candidate as a prophylactic agent. However, in a prospective non-randomized study from the BMT CTN, maraviroc in combination with standard Tac/MTX was not superior to standard of care and in this trial the PTCy/Tac/MMF arm fared the best .Interleukin-6 (IL-6), an inflammatory cytokine has been shown to be one of the chief mediators of aGVHD in murine models . TherefoThere are novel therapies which have not yet been successfully translated to clinical practice but hold great promise. These therapies are based on innovative targets based on a more intricate understanding of the pathophysiology of GVHD.As mentioned above in the section on the immunobiology of GVHD, conditioning regimen associated tissue damage exposes antigens which comprise pathogens or Pathogen-Associated Molecular Patterns (PAMPs) and components of damaged cells (Danger-Associated Molecular Patterns or DAMPs) which trigger activation of the innate immune system. Conversely Sialic-acid-binding-immunoglobulin-like lectins (Siglecs) are a particular class of pattern recognition receptors that downregulate innate immune responses . A numbeA role for Siglecs in modulating adaptive T-cell mediated immune responses has also been proposed. Reddy et al. have shown that Siglec-G interacts with CD24c in murine models and this interaction CD24 suppresses TNF-α, IL-1β, and IL-6 via NFkB and therefore is promising in the domain of GVHD prophylaxis .Defibrotide is a polydisperse mixture of predominantly single-stranded polydeoxyribonucleotides which in pre-clinical and human studies has demonstrated profibrinolytic, antithrombotic, anti-inflammatory and angio-protective effects ultimately resulting in stabilization of endothelial cells . DefibroThe canonical NOTCH pathway has been shown to play a critical role in T-cell activation, differentiation, and function in aGVHD pathogenesis . Using hn = 25) and larger studies are needed to further study the effect of MSCs on preventing GVHD. A randomized phase II trial has also shown some beneficial effect on cGVHD and need to be further studied (Attempts have been made to utilize the immunomodulatory properties of MSCs for GVHD prophylaxis based on murine models of HLA-mismatched transplantation with co-transplantation of hematopoietic stem-cells and MSCs . Ning et studied .−/−donor or recipient mice resulted in similar reduction in GVHD (OX40 (CD134) is a co-stimulatory receptor found on T-cells while its ligand OX40L is expressed on dendritic cells, B-cells, and endothelial cells. In 2003 Blazar et al. investigated the OX40 regulation of GVHD in murine models and found that antagonistic anti OX40L moAb or the use of OX40 in GVHD . Further in GVHD  and ther in GVHD  then havvia TNF-alpha and lipopolysaccharides (LPS). In murine models of GVHD, blockade of the ICOS:ICOSL interactions via moAb or ICOS−/− mice resulted in significant decrease in GVHD and GVHD related mortality both mediated by CD4+ and CD8+ cells (Inducible Costimulator (ICOS) is a member of the CD28/CTLA-4 family expressed on activated T-cells while the ICOS ligand (ICOSL), a B7 family member is constitutively expressed on B-cells, macrophages and dendritic cells and upregulated on APCs 8+ cells .ALPN101 is a novel Fc fusion protein of a human ICOSL variant immunoglobulin domain (single domain vIgD™) binding both ICOS and CD28 at higher affinity than wild-type molecules, designed to inhibit both the CD28 and ICOS pathways to dampen co-stimulatory responses during alloreactive T-cell activation. In a murine dose-ranging study, ALPN101 inhibited GVHD responses at all doses and more significantly than the comparator belatacept, an approved CTLA-4-Fc protein CD28 pathway inhibitor .A phase 1/2 dose finding study (BALANCE) is ongoing in patients with steroid sensitive and steroid refractory aGVHD (NCT04227938) investigating this potentially transformative effect and this is another promising target for GVHD prophylaxis.n = 112) with aGVHD rates in the 18% range using bone marrow grafts (CD6 is a co-stimulatory receptor on T-cells that binds to activated leukocyte cell adhesion molecule (ALCAM), a ligand on APCs and is involved in T-cell activation and trafficking. Historically CD6 T-cell depletion using a monoclonal antibody (moAb) was evaluated in a single-arm trial  which is constitutively expressed on the vascular endothelium of skin and bone marrow and inducibly expressed on other cells during inflammation. P-selectin is a receptor for PSGL-1, a glycoprotein strongly expressed on all leukocytes . PSGL-1 in-vivo induction of Treg. In preclinical aGVHD models, AAT reduced inflammatory cytokines, altered the ratio of effector and regulatory T-cells and reduced levels of DAMPs  involved in downstream transduction of various cytokine pathways . They arSo far clinical data is limited to a pilot study in myelofibrosis patients where a combination of ATG, ruxolitinib, and PTCy was used as GVHD prophylaxis with acceptable engraftment rates  and a siin vivo depletion of T cells and natural killer (NK) cells and suppresses T cell receptor activation.Monoclonal antibodies conjugated with immunotoxins is a method of selective TCD that has been attempted in the past with CD5 as a target among others as described above. T-guard is a immunotoxin combination comprised of a mixture of anti-CD3 and anti-CD7 antibodies separately conjugated to recombinant ricin A (CD3/CD7-IT), which induces This was first evaluated in a phase I/II trial in humans in SR aGVHD with a 50% response rate and manageable toxicities albeit with evidence of capillary leak and thrombotic microangiopathy . This maAlthough cGVHD is a distinct clinical and immunologic entity from aGVHD, there are limited interventions that specifically target the prevention of cGVHD. In general we know that patients who have less aGVHD will likely get less cGVHD and so the prevention of aGVHD is important in the prevention of cGVHD. In terms of donor and transplant related interventions, younger same-sex donors and the use of bone marrow product rather than PBSC has been shown to reduce rates of cGVHD .T-cell directed approaches that have been quite successful include ATG and more recently PTCy as outlined above. B-cell directed approaches are an area of interest given our current understanding of the important role that B-cells play in the pathophysiology of cGVHD. Rituximab, a monoclonal antibody targeting CD20 was evaluated in a phase II trial with cGVHD rates in the 48% range cGVHD requiring immunosuppression in the 31% range . This waAnother area of interest is the augmentation of tolerance by the use of low-dose IL-2  to enhance Treg reconstitution creating a favorable immunologic milieu for the prevention of cGVHD. This strategy has had success in the therapy of steroid-refractory cGVHD  and may in-vivo and ex-vivo pan T-cell depletion strategies to more directed immunomodulatory strategies based on a more comprehensive understanding of GVHD immunobiology. Nevertheless, the basic backbone of CNI based prophylaxis has survived the test of time. We have touched upon this current standard in this review and further discussed important translational advances and exciting pre-clinical strategies which may be a part of future prophylactic regimens.GVHD prophylaxis has evolved over the last few decades from direct in vivo T-cell depletion, mechanistic studies have indicated that PTCy has a far more complex impact on the post-transplant immune system including a Treg sparing role which is of great interest in the community , the results of which are eagerly awaited as well. In the last decade the spotlight has shifted to methods of selective ex-vivo TCD with limited success in the clinical setting. a/b TCD which attempts to reduce GVHD without affecting GVL and can be performed without the use of post-transplant immunosuppression is promising and may be an important modality in the future.In the domain of in-vivo or ex-vivo), a new frontier in GVHD prophylaxis is targeting immune checkpoints which regulate T-cell activation. Given the dramatic success of checkpoint inhibitors in the world of solid tumor oncology, there has been tremendous interest and a much better understanding of these checkpoints in recent years. In the case of GVHD of course, researchers have tried to downregulate rather than upregulate T-cell activation following initial antigen engagement by the T-cell receptor complex. Although there are a number of molecules being tested at the bench and detailed in this review, the most promising of these has been blockade of the CD28/CTLA-4 axis with Abatacept (CTLA-Ig) with an eventual inhibitory signal downstream to the T-cell. Results from a RCT with pediatric and adult patients has shown a dramatic reduction in severe acute and chronic GVHD including in mismatched unrelated donors (Separate from direct TCD (d donors . With FDTargeting T-cell trafficking, an early event in GVHD prophylaxis, is being tried with integrin blockers such as vedolizumab. It is logical that inhibiting the very movement of effector T-lymphocytes to target organs should better prevent GVHD rather than trying to arrest the widespread inflammation and cytokine cascades which characterize the final common pathway in GVHD pathogenesis. As a testimony to that, when tocilizumab an IL-6 blocker was evaluated, despite promising phase II single-arm data, was not more effective than standard prophylaxis in a phase III RCT . This haWithin the limitations of this review, we have highlighted some of the exciting pre-clinical science that has the potential to translate into effective prophylactic therapies which target GVHD pathogenesis beyond direct T-cell depletion. Targeting the interaction between DAMPs/ PAMPs on APCs and Siglecs with a downstream inhibitory effect on cytokine cascades as well as investigating a role for Siglecs in modulating adaptive T-cell mediated immunity are areas of interest . EndothePathways critical in T-cell modulation during activation and proliferation such as the Notch pathway has been an area of great interest although not ready for translation at this time . BolsterIn conclusion, while GVHD prophylaxis in 2020 still incorporates the traditional paradigms of CNI based prophylaxis, PTCy is knocking on the door and a number of exciting new translational therapies and pre-clinical advances are on the horizon which promise to challenge the established paradigms.JHA supervised and edited manuscript along with MG. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The principle of constraint-induced therapy is widely practiced in rehabilitation. In hemiplegic cerebral palsy (CP) with impaired contralateral corticospinal projection due to unilateral injury, function improves after imposing a temporary constraint on limbs from the less affected hemisphere. This type of partially-reversible impairment in motor control by early brain injury bears a resemblance to the experience-dependent plastic acquisition and modification of neuronal response selectivity in the visual cortex. Previously, such mechanism was modeled within the framework of BCM (Bienenstock-Cooper-Munro) theory, a rate-based synaptic modification theory. Here, we demonstrate a minimally complex yet sufficient neural network model which provides a fundamental explanation for inter-hemispheric competition using a simplified spike-based model of information transmission and plasticity. We emulate the restoration of function in hemiplegic CP by simulating the competition between cells of the ipsilateral and contralateral corticospinal tracts. We use a high-speed hardware neural simulation to provide realistic numbers of spikes and realistic magnitudes of synaptic modification. We demonstrate that the phenomenon of constraint-induced partial reversal of hemiplegia can be modeled by simplified neural descending tracts with 2 layers of spiking neurons and synapses with spike-timing-dependent plasticity (STDP). We further demonstrate that persistent hemiplegia following unilateral cortical inactivation or deprivation is predicted by the STDP-based model but is inconsistent with BCM model. Although our model is a highly simplified and limited representation of the corticospinal system, it offers an explanation of how constraint as an intervention can help the system to escape from a suboptimal solution. This is a display of an emergent phenomenon from the synaptic competition.The online version contains supplementary material available at 10.1007/s10827-021-00782-9. Constraint-induced movement therapy (CIMT) improves limb use in patients after unilateral cerebrovascular accidents (CVA)  have been shown to contribute to significant corticospinal tract (CST) repair and motor recovery  leads to persistent deficits that are manifested by structural changes in connectivity downstream from the causative abnormality. In other words, a change in behavior or cortical stroke can lead to persistent plastic changes in the otherwise unaffected spinal cord. The effectiveness of CIMT is therefore based upon two hypotheses: (1) some component of the persistent deficit is due to persistent yet reversible effects of plasticity, and (2) the component of the deficit due to plasticity can be ameliorated by interventions that changes the ratio of CNS activity between the injured and uninjured hemispheres.This phenomenon is similar to what is seen in amblyopia caused by asymmetric and temporary visual disruption during early development  theory . With FPGA, the neuronal activity can be accelerated to 190 × real-time, therefore we are able to observe weeks of neural development within hours. The BCM-like rate-based model requires an explicit homeostatic plasticity mechanism, whereas spike-timing-dependent plasticity (STDP) in this study shows that no additional mechanism is required. It is advantageous to use spike-based emulation because the behavior of spiking algorithm could be different from the rate-base algorithm. This will be particularly true in the context of injury, which affects individual cells and is more difficult to emulate accurately in rate-based models. Our purpose is to support a mechanistic hypothesis to explain CIMT by showing that STDP is sufficient to predict: (1) persistence of plasticity-mediated deficits following temporary injury, (2) reversal of plasticity-mediated components of deficit via constraint therapy, and (3) persistence of improved function following termination of constraint therapy. Although our model is a highly simplified and limited representation of the motor system, it allows us to understand how the principle of constraint-induced therapy can be explained in terms of synaptic competition through activity-dependent plasticity. By finding a minimally complex, yet sufficient condition to demonstrate the effect of constraint-induced intervention could shed light on the effective practices of clinical therapies.https://github.com/wonjsohn/stdp_synaptic_competition) for public dissemination.We customized a two-layer neuronal network from our recently developed neuromorphic hardware ::2):1\docv: membrane potential.u: membrane recovery variable.I: synaptic currents or injected dc-current.The timing of the presynaptic and postsynaptic spikes determines the strengthening and weakening of the connection according to a spike timing dependent plasticity (STDP) model  and and4) anmentclass2pt{minimThe representation of the descending corticospinal system is modeled in two layers of neurons Fig. . The inpWe first demonstrate the three example cases in the activity-dependent synaptic competition. The effect of the initial conditions of the synaptic weights of two input neurons that are under synaptic competition are shown. In these example cases, the input neurons (R/L) receive stochastic input currents that are statistically independent of each other.Noisy current input to the neuron is used as an activity generator. The pseudorandom values drawn from the standard uniform distribution on the interval  causes the ipsilateral projection from the active hemisphere (w3) to grow because the suppression from the activity in the dominant projection is removed, resulting in the development of bilateral projection from the same hemisphere . The contralateral projection from the blocked hemisphere weakens (w2) due to inactivity and synaptic decay.Bilateral activation shows the system’s inability to spontaneously restore the diminished contralateral projectionstuck in a hemiplegic state  back to the initial healthy state. In other words, the state is  as Fig. a. This iReverse inactivation enables the diminished contralateral projection to be restoredReverse inactivation (blocking the input current to L) enables the diminished contralateral projection (w2) to be restored by transiently removing the suppression from the now-dominant ipsilateral projection (w3)  is effective in producing improvement in limb use of patients after cerebrovascular accidents (CVA)  update mechanisms dictate that correlated pre- and postsynaptic spikes cause potentiation and uncorrelated pre- and postsynaptic spikes cause depression of the synaptic weight, (2) postsynaptic firing rates are not in the range that results in net depression. Although the time constant of synaptic current and weight decay is not sensitive to the qualitative results for this study directly, the time constant for synaptic weight decay Below is the link to the electronic supplementary material.


Unmanned Aerial Vehicle (UAV)-assisted cellular networks over the millimeter-wave (mmWave) frequency band can meet the requirements of a high data rate and flexible coverage in next-generation communication networks. However, higher propagation loss and the use of a large number of antennas in mmWave networks give rise to high energy consumption and UAVs are constrained by their low-capacity onboard battery. Energy harvesting (EH) is a viable solution to reduce the energy cost of UAV-enabled mmWave networks. However, the random nature of renewable energy makes it challenging to maintain robust connectivity in UAV-assisted terrestrial cellular networks. Energy cooperation allows UAVs to send their excessive energy to other UAVs with reduced energy. In this paper, we propose a power allocation algorithm based on energy harvesting and energy cooperation to maximize the throughput of a UAV-assisted mmWave cellular network. Since there is channel-state uncertainty and the amount of harvested energy can be treated as a stochastic process, we propose an optimal multi-agent deep reinforcement learning algorithm (DRL) named Multi-Agent Deep Deterministic Policy Gradient (MADDPG) to solve the renewable energy resource allocation problem for throughput maximization. The simulation results show that the proposed algorithm outperforms the Random Power (RP), Maximal Power (MP) and value-based Deep Q-Learning (DQL) algorithms in terms of network throughput. Unmanned Aerial Vehicles (UAVs) are aircrafts without a human pilot on board. UAVs are able to establish on-demand wireless connectivity faster than terrestrial communications and they can adjust their height and position to provide robust channels with short-range line-of-sight links . TherefoUAVs are identified as an important component of future-generation (5G/B5G) wireless networks due to their salient attributes  .In UAV-assisted wireless communications, UAVs are employed to provide wireless access for terrestrial users.(1)UAV-aided ubiquitous coverage: UAVs act as aerial base stations to achieve seamless coverage for a given geographical area. Some related applications are a fast communication service recovery in disaster scenarios and temporary traffic offloading in cellular hotspots.(2)UAV-aided relaying: UAVs are employed as aerial relays between far-apart terrestrial users or user groups. Some examples of applications include UAV-enabled cellular coverage extension and emergency response.(3)UAV-aided information dissemination and data collection: UAVs are used as aerial access points (APs) to disseminate (or collect) information to (from) ground nodes. Some related applications are UAV-aided wireless sensor networks and IoT communications.We distinguish between three use cases :(1)UAV-aThe use of UAVs as aerial nodes to provide wireless sensing support has several advantages compared to ground sensing . UAV-basUAV-based sensing has a wide range of potential applications, such as precision agriculture, smart logistics, 3D environment map construction, search and rescue, and military operations. There is a growing interest in the development of UAV-based sensing applications.In Ref. , a complUAVs are a popular and cost-effective technology to capture high spatial and temporal resolution remote sensing (RS) images for a wide range of precision agriculture applications . UAVs eqIn IoT communications , UAVs haIn search and rescue operations, a real-time human detection and gesture recognition system based on a UAV with a camera is proposed . The sysUAV-enabled mmWave networks offer a lot of potential advantages. On the one hand, the large available spectrum resources of mmWave communication and flexible beamforming can meet the requirements of high data rate and flexible coverage for UAVs serving as base stations (UAV-BSs) in UAV-assisted cellular networks . These nEnergy harvesting (EH) is a viable solution to reduce the energy cost of UAV-enabled mmWave networks; green energy can be harvested from renewable energy sources  to power UAVs. Energy-harvesting powered UAVs can prolong longer their operational duration as well as the wireless connectivity services they offer ,17,18. HWe consider a UAV-assisted mmWave cellular network. Some UAVs will have plenty of energy because their flight duration is shorter or because they have harvested abundant energy due to better environmental conditions . Energy cooperation allows that these UAVs can send their excessive energy to other UAVs with reduced energy.In the literature, several works have investigated energy cooperation in renewable energy harvesting-enabled cellular networks. In Ref. , an adapSeveral authors proposed using energy transfer in UAV-enabled wireless communication systems. In Ref. , the totIn this paper, we propose a power allocation algorithm based on energy harvesting and energy cooperation to maximize the throughput of a UAV-assisted mmWave cellular network. This optimal power allocation and energy transfer problem can be regarded as a discrete-time Markov Decision Process (MDP)  with conWe study optimal power allocation strategies for UAV-assisted mmWave cellular networks when there is channel-state uncertainty and the amount of harvested energy can be treated as a stochastic process.We formulate the renewable energy resource allocation problem for throughput maximization using multi-agent DRL and propose an MADDPG-based multi-UAV power allocation algorithm based on energy cooperation to solve this problem.Simulation results show that our proposed algorithm outperforms the Random Power (RP), Maximal Power (MP) and value-based Deep Q-Learning (DQL) algorithms and achieves a higher average network throughput.The contributions of this paper are summarized as follows:The paper is structured as follows. In Our network architecture is shown in sity λi  . We alsoUAVs are powered by hybrid energy sources. Onboard energy, along with a part of the harvested energy, is used to maintain the flight while the rest harvested energy is used to support the communication modules of the UAVs. The imbalance of energy harvesting between UAVs is compensated through energy cooperation.j, jL only associated with UAVj. For simplicity, the typical user set is associated with the closest UAV-BS; that is, the UAV that maximizes the average received SNR.The UAVs and user sets are denoted as N time slots. It is assumed that all UAVs communicate without the assistance of a central controller and have no global knowledge of wireless channel communication environments. This means that the channel state information (CSI) between a UAV and the mobile devices of the users is known locally. This paper focuses on the design of an optimal power allocation strategy to maximize the throughput for multi-UAV networks over A major challenge in mmWave communications is the blockage effect , namely,The path loss law in the UAV network is given by :(2)Lht,hThe amplitude of the received UAV-to-ground mmWave signal can be modelled as a Nakagami-At time slot When the transmission has the maximum antenna gain, the channel gain is Beamforming, also known as spatial filtering, concentrates the signal energy over a narrow beam by means of highly directional signal transmission or reception; this way, the spectral efficiency is improved . The narreceiver .We consider that the UAV-BS and user set antennas adopt a sectorized model that is shown in The directivity gain at one receiver located at The transmitter and receiver should adjust their antenna directions towards each other to achieve the maximum beamforming gain j and user j at time slot j that satisfies The UAV-to-ground user pair communication is affected by the interference signals from the remaining UAVs. Therefore, the received signal-to-interference-plus-noise ratio (SINR) from UAVIn this section, we investigate the optimal power allocation and energy transfer problem for throughput maximization in UAV-enabled mmWave networks, which can be regarded as MDP. Since the real channel state and energy harvesting arrival are not easily observable, traditional model-based methods are infeasible to tackle with this MDP. Therefore, we reformulate this problem using multi-agent DRL to make it solvable.We introduce the following notation:N time slots.j at time slot j.j at time slot j to serve all its users.j at time slot The theoretical downlink rate of user The total throughput at timeslot j stores mainly the renewable energy harvested during the current time slot, the energy transferred by other UAVs during the energy cooperation process, and the remaining energy from the last time slot. The current battery capacity of each UAVThe charging rate of the energy storage is usually less than the energy arrival rate, because of the limited energy conversion efficiency of the circuits. We consider that the charging rate and energy arrival rate are expressed as After j at the time j to UAVj’, j’ to UAVj and The battery energy level of UAVThe problem can be formulated as follows:P1. Throughput optimization problemFind:Max:Subject to:P1 aims at finding the best values of P1 is a non-linear optimization problem.The objective function of problem j to other UAVs should not exceed the current battery energy level. The same applies to Constraint (14) for the total transferred energy transferred from UAVj’ to other UAVs.Constraint (10) limits the harvested energy. Constraint (11) determines that the total energy consumed by each UAV should not exceed its battery level. Constraint (12) refers to the total allocated power by UAVThe optimization problem may be solved only if the complete information about energy harvesting arrival and channel state information (CSI) is known. RL algorithms can achieve near-optimal performance even without prior knowledge about the CSI, the user arrival, the energy arrival, etc. . In whatThe proposed problem can be considered an MDP. Therefore, multi-agent RL can be adopted to solve this problem efficiently.Each UAV can be regarded as an agent in the proposed system and all the network settings can be regarded as the environment. UAVs can be characterized by a tuple state space including all possible states of UAVs in the system at each time slot. The state of the j, the link’s corresponding downlink rate j, respectively.action space consisting on all the available actions of state transition function, which maps the state spaces and the action spaces of all UAVs in the current time slot to their state spaces in the next time slot.reward function of the policy of a UAV is defined as a mapping from its state space to its action space, denoted by Each UAV is motivated to maximize the throughput by making decisions on power allocation. In our system, the P2. Maximization of the total accumulated rewardsMax:Our target is to design an efficient algorithm for power allocation and energy transfer for UAVs that maximizes the throughput. Existing approaches such as dynamic programming is not suitable for such challenging tasks. Therefore, we adopt an RL algorithm to cope with the problem (P2). DRL has a better performance on tasks that have a sophisticated state space and time-varying environment than traditional reinforcement learning. There are different kinds of DRL that could deal with different situations, e.g., Deep Q-Learning (DQL) could work well with a limited action space and deep deterministic policy gradient (DDPG ) has a rMADDPG is an actor-critic algorithm  designedMADDPG adopts a strategy based on centralized training and distributed execution. Each UAV works as an agent and has an actor network The loss function of the critic network is calculated byThe gradient of the expected reward for agent Algorithm 1: MADDPG-based multi-UAV power allocation algorithm based on energy cooperationInput: The structures of the actor network, critic network, and their target networks; Number of episodes Output: Policy1: Initialize the replay memoryX. 2: Initialize criticsInitialize target networks with random weights3: Receive initial state4: fordo5:  for6:   Each agent selects an action7:   Receive reward8:   Store9:   ifdo10:    Sample a batch of random samples11:    Set with (17). 12:    Update the actor of the estimated network13:    Update the critic of the estimated networkUpdate the target network parameters with14:  end for15: end for16: Return17: Choose optimal actionThe MADDPG algorithm is shown as Algorithm 1.The computation complexity and the space complexity for the proposed MADDPG algorithm can be estimated by the replay memory and the neural networks’ architecture. In MADDPG, the training network of each agent consists of two sets of actor networks and two sets of critic networks. The time complexity (computations) is given with regard to the floating-point operations per second (FLOPS). The neural networks are fully connected layer networks. For dot products of a We consider that Therefore, the time complexity of the training is:Space is needed to store the learning transition. The memory replay in MADDPG occupies some space to store the state sets; therefore, the space complexity is In this section, we present the simulation results of the multi-UAV power allocation and energy transfer algorithm. The simulation parameters are given in Next, we compare the proposed algorithm MADDPG with the Random Power (RP), Maximal Power (MP), Multi-Armed Bandit (MAB) and value-based Deep Q-Learning (DQL) algorithms. Upper confidence bound (UCB) is used to solve the MAB problem. RP and MP are two classical algorithms, whereas MADDPG, DQL and MAB are RL-based algorithms. Maximal Power (MP) consumes as much energy as possible in each time slot to improve its immediate throughput regardless of the future and the performance of the other UAVs. Random Power (RP) only consumes a part of the energy chosen randomly in each time slot. The average throughput for the five power allocation schemes is shown in The average throughput for the different policies is shown in The average throughput as a function of the number of users is illustrated in The energy transfer efficiency between two UAVs is shown in The average throughput for the RL-algorithms is shown in The average throughput for the RL-algorithms is shown in The average throughput for the RL-algorithms is shown in The convergence behavior of the reinforcement-based algorithms in terms of average reward is shown in In this paper, the optimal power allocation strategies for UAV-assisted mmWave cellular networks were analyzed. A power allocation algorithm based on energy harvesting and energy cooperation is proposed to maximize the throughput of a UAV-assisted mmWave cellular network. Since there is channel-state uncertainty and the amount of harvested energy can be treated as a stochastic process, we propose an optimal multi-agent deep reinforcement learning algorithm (DRL) named Multi-Agent Deep Deterministic Policy Gradient (MADDPG) to solve the renewable energy resource allocation problem for throughput maximization. The simulation results show that the proposed algorithm outperforms the Random Power (RP), Maximal Power (MP), Multi-Armed Bandit (MAB) and value-based Deep Q-Learning (DQL) algorithms in terms of network throughput. Besides, the RL-based algorithms outperform the traditional RP and MP algorithms and show improved generalization performance, since they can adjust the transmission power in a smart way. The average throughput is increased with the number of UAVs, the energy arrival, the battery capacity and the energy transfer efficiency between two UAVs. When the battery capacity is increased the throughput values for the RL policies tend to stabilize since the value of MADDPG can be applied to many tasks with discrete or continuous state/action space and joint optimization problems of multiple variables. It can successfully solve user scheduling, channel management and power allocation problems in different types of communication networks. The optimization of the locations of UAVs and their trajectories  is an im


Varicella-zoster virus (VZV) infections cause a substantial disease burden, which is underestimated due to incomplete reporting data and lack of serological surveillance. In this post-hoc analysis of a randomized, Phase IIIb clinical trial (NCT00226499) with a ten-year follow-up period, we report anti-VZV antibody levels and persistence in non-vaccinated children, as a varicella infection estimate in ten European countries with endemic varicella. The present analysis specifically focuses on clinical and serological data from the control group, which included 827 healthy participants aged 12–22 months who received two doses of measles-mumps-rubella (MMR) vaccine. The per-protocol cohort included 744 children for whom varicella occurrence was evaluated by clinical definitions, epidemiological links and PCR test outcomes. Anti-VZV antibody levels were assessed by ELISA. The primary objective of this analysis was to correlate varicella occurrence with anti-VZV antibody levels. Varicella was confirmed in 47% of MMR recipients. Among participants without reported varicella, the percentage of anti-VZV seropositive children increased to 75% and average anti-VZV antibody concentrations increased to 250 mIU/mL at year ten after vaccination, suggesting infection or exposure. An eight-fold increase in anti-VZV antibody concentrations between two consecutive visits, which is also observed after confirmed varicella, was detected in 37% of these participants during the follow-up period. About one-third of children not vaccinated against varicella and not diagnosed with varicella developed an anti-VZV immune response, suggesting subclinical varicella occurrence. Longitudinal studies combining serology and disease incidence are necessary to reliably estimate total varicella burden of infection. These vaccines have proved efficacious in preventing varicella.9 Varicella diagnosis relies primarily on clinical detection, based on the acute appearance of an itchy blistering rash with any number of skin vesicles as the main symptom of the disease.2 Previous studies also included confirmation of varicella infection via epidemiological links with valid index cases and detection of varicella virus DNA in vesicles.10–12 No other methods  are currently recommended for varicella diagnosis or routine screening.2Introduction of mandatory varicella vaccination in broader geographical regions has been hampered by the lack of reliable disease burden estimations.13 However, these studies offer only a snapshot of the seroprevalence status13 and, to our knowledge, there are no prospective serological studies assessing the natural history of VZV infection in young children and its relationship with clinical disease over extended periods of time. All European studies published to date were retrospective and estimated varicella incidence through different modeling approaches.14–16 To date, the proportion of seropositive individuals having unknowingly experienced varicella infection remains unknown, and the contribution of such cases to the total varicella burden of infection and transmission potential is therefore difficult to measure reliably.14 In addition, the question of how anti-VZV antibody concentrations evolve over time in subclinical primary varicella cases has yet to be answered.Serological testing for varicella-specific (anti-VZV) antibodies is a complementary approach to epidemiological studies enabling the reliable estimation of total varicella infection incidence. In countries of temperate climate regions with endemic varicella, VZV seropositivity in adults is above 90%.11 These children, included in the Active Control group to which varicella vaccine recipients (MMR+V and MMRV groups) were compared, received two doses of MMR vaccine in their second year of life.10–12Previous publications from a Phase IIIb randomized trial reported that 73% of children who were not vaccinated against varicella and for whom a varicella event was not detected or reported were seropositive for anti-VZV antibodies at the end of the ten-year follow-up period.In the present post-hoc analysis of this Phase IIIb trial focusing on the aforementioned Active Control group, the immunological response profiles to natural varicella exposure and disease were evaluated in children from 1 year of age onwards. The evolution of varicella seropositivity and anti-VZV antibody concentrations were assessed in this population over a ten-year period. To our knowledge, this is the first prospective serological study extending over a ten-year period and aiming to characterize seropositivity prevalence and persistence of naturally induced anti-VZV antibodies in children not vaccinated against varicella and without overt clinical varicella in European countries with endemic varicella.A summary contextualizing the outcomes of this publication is displayed in 11 and is schematically presented in 12The study design was previously described in detail10–12 Parents and guardians of the study participants provided written informed consent prior to enrollment.The study enrolled healthy children in their second year of life (between 12 and 22 months of age) who were followed until ten years post-vaccination. Eligibility criteria were previously described in detail.Randomization was described previously and is outlined in The present post-hoc analysis focused on the Active Control group which remained observer-blind until completion of the study.10–12 Results of the post-hoc analysis presented here are descriptive and include the assessment of (i) the frequency of varicella occurrence, based on varicella case reports and classification, in the Active Control group, and (ii) anti-VZV antibody levels and persistence in the Active Control group.Primary and secondary objectives of the clinical trial were previously published for the follow-up periods of three, six, and ten years.Priorix, GSK; see publication of Prymula and colleagues for details12) by subcutaneous injection in the deltoid region of the left arm.All participants from the Active Control group received the same MMR vaccine lot . Varicella-specific antibody concentrations measured by the anti-VZV ELISA were shown to be a reliable proxy for estimating protection against varicella in young children.17Blood samples were collected before vaccination, 6 weeks after each vaccine dose , one and two years after the second vaccine dose and every 2 years thereafter until study completion at 10 years post-vaccination . Presence of anti-VZV IgG antibodies in the serum was assessed using the commercial enzyme-linked immunosorbent assay (ELISA) In participants with suspected varicella skin eruptions, DNA was extracted from dermal vesicle samples. Extracted DNA was tested by polymerase chain reaction (PCR) test coupled with Restriction Fragment Length Polymorphism analysis of the PCR products to identify and characterize VZV serotypes. This analysis enabled the distinction between wild type and vaccine strain infections.10–12 and is presented in 18 The IDMC classified cases according to the modified scale of Vazquez and colleagues.19 A varicella case was considered as confirmed if it met the clinical case definition with a positive varicella PCR result, or if it met the clinical definition, was IDMC confirmed, and epidemiologically linked to a valid index case.10–12Reporting and evaluation of possible varicella cases was conducted as previously described10–12 Varicella antibody persistence was evaluated in the same cohort, in participants for whom valid serology results from at least two consecutive study visits were collected. The anti-VZV ELISA cutoff value was set at 25 mIU/mL, to enable distinction between seronegative children (<25 mIU/mL), seropositive children (≥25 mIU/mL) and children with seroconversion . Seroresponse was defined as an increase in anti-VZV antibody levels from <25 mIU/mL before to ≥50 mIU/mL after a defined blood sampling timepoint. The proportion of seropositive participants was calculated as the proportion of participants seronegative prior to vaccination displaying antibody concentrations above the cutoff value after vaccination. For geometric mean concentration (GMC) calculations, values below the cutoff were arbitrarily set to half the cutoff value. GMCs were calculated as the anti-log of the mean logarithmic concentration measured by ELISA.Varicella cases were followed up starting from the recorded rash onset date and were assessed in the per-protocol cohort for efficacy which included children with completed vaccinations and fulfilled protocol requirements see .10–12 VaParticipants who were lost to follow-up or were withdrawn from the study were considered for the analyses up to the point of the last contact.20All computations were conducted in SAS software , including the calculations of 95% confidence intervals (CIs) by the method of Clopper.Enrollment occurred between September 2005 and May 2006. All 5,803 enrolled children were vaccinated, and 827 were included in the Active Control group . The per31 The participant population was overall homogeneous and comparable between countries in terms of mean age, gender distribution, and ethnicity  were characterized as likely varicella rashes and, thereafter, assessed by the IDMC and laboratory tested. Following PCR testing and assessment of a link with a clinical index case, 39 cases (5%) were excluded as non-varicella and non-HZ cases, leaving 352 (47%) of the per-protocol cohort for efficacy participants diagnosed with a confirmed varicella case . The perAnti-VZV antibody concentrations in participants from the Active Control group with and without a diagnosed varicella case were analyzed over time .Table 2.One year after the second MMR dose, on average 8% of Active Control group participants without a confirmed varicella case were seropositive for anti-VZV antibodies. The percentage of seropositive participants without a diagnosed or reported varicella increased during the study. At Y10, 75% of children from the Active Control group without a confirmed varicella case were seropositive . In partAnti-VZV antibody concentration increases for Active Control group participants with and without a confirmed varicella case were further categorized in two-fold incremental steps . When looking at increases in anti-VZV antibody concentrations between two consecutive visits, at least an eight-fold increase was detected in 97% of participants with a confirmed varicella case. A similar increase was detected in 116 out of 318 (37%) children without a confirmed varicella infection (per-protocol cohort for efficacy).In children without a confirmed varicella case, there was an increase in anti-VZV seropositivity, with anti-VZV antibody concentrations spanning wide ranges . This inThis publication, reporting a post-hoc analysis of data from a Phase IIIb clinical trial with a ten-year follow-up period, describes the unexpected anti-VZV serological evolution over a 10-year period in children not vaccinated against varicella and without overt clinical varicella in countries with endemic varicella.10–12 As previously described, the GMCs at Y10 timepoint were comparable in the Active Control group without censoring of the post-infection data and the MMRV group.11Previous publications from this clinical trial reported that seropositivity for anti-VZV antibodies in MMRV and MMR+V groups was characterized by a persistent and progressive increase of anti-VZV antibody levels from Y1 to Y10.10–12 These undetected infection events are either truly asymptomatic, or with minimal, atypical or partial symptoms (e.g. fever only), thus not leading to a suspicion and diagnosis of varicella. While both subclinical primary VZV infections and subclinical reactivation cases have been described,21–25 to our knowledge, this is the first study reporting the long-term serological detection of primary VZV infection.Overall, the participant population was nearly exclusively Caucasian (European ethnicity), and varicella person-year rate did not differ markedly between countries suggesting homogeneous exposure to circulating wild-type virus, thus making the grouping of countries for the analysis in this paper acceptable and relevant. In the present post-hoc analysis, the anti-VZV antibody levels increased in the Active Control group over several years. The percentage of anti-VZV seropositive children without a confirmed varicella case increased from 8% at Y1 to 75% at Y10. The increases in seropositivity rates and anti-VZV concentrations over time are unexpected for the Active Control group, as participants with a history of confirmed varicella were censored. Moreover, among the participants without a confirmed varicella case, throughout the ten-year follow-up, about one-third (37%) of the children experienced an eight-fold increase in anti-VZV antibody concentration between two consecutive visits, such an increase being similar to what usually happens to children having confirmed varicella. This could have been a consequence of misclassification of “no cases”  or of the presence of subclinical varicella cases. In addition, the numbers of participants without a varicella case progressively decreased during the follow-up and anti-VZV seropositivity at Y10 was observed in 96 of the 744 efficacy cohort participants. Nevertheless, these results strongly suggest that varicella infection, detected serologically by anti-VZV antibody ELISA, may occur without overt clinical skin symptoms which served as the primary method of suspicion in this and related studies.26 Ideally, systematic screening for presence of anti-VZV antibodies in any given population would provide valuable insight into true VZV prevalence. However, such testing should only be conducted if it is necessary to measure exposure to circulating virus and if it is deemed feasible, given the logistical and financial burden it poses to study design and its participants.Overall varicella infection incidence is likely underestimated in most epidemiological surveys, as serologic screening is not systematically done. Consequently, varicella vaccine efficacy, herd protection, and benefit/risk profile may be higher than originally estimated. Furthermore, it is unknown if minimally symptomatic varicella cases may contribute to further viral transmission. It was already reported that unknown varicella or HZ disease history may hinder precise estimations of the risk of HZ occurrence.9 A recent review summarizing seroprevalence data from 16 European countries found that between 73% and 97% of children below the age of ten were seropositive for anti-VZV antibodies before the introduction of national varicella vaccination programs.8 These data are consistent with an earlier European study.27 A further comprehensive review of varicella seroprevalence in Europe revealed high variability in burden of disease in different countries, associated to social mixing patterns and healthcare seeking behaviors.9 In this review, authors estimate that, in the absence of universal varicella vaccination, the highest incidence of disease is expected in children below the age of five, with more than five million infections per year at the European level.While the present post-hoc analysis was based on clinical trial data, to our knowledge, most studies on varicella prevalence are retrospective and rely on modeling to estimate the evolution of varicella seropositivity in different age groups.14–16 The findings of one study from Italy indicated that varicella seroprevalence might be up to eight-fold higher than expected based solely on case reporting.14 The analysis was based on the discrepancy between the serological data and the reported cases. Since 2010, there are still no European standards for varicella reporting and surveillance.28 Therefore, future prospective and longitudinal follow-up studies would be informative to obtain accurate data on the total varicella burden of disease in Europe.Other studies compared seroprevalence data with varicella case report frequencies across different age groups.11 This could suggest that these participants are protected to the same level as children who received one dose of varicella vaccine. Similarly, the anti-VZV antibody concentrations were comparable between Active Control group participants with a varicella case since previous visit at Y10, obtained in this study, and the MMRV recipients at Day 84 timepoint published previously.11 These results are strongly supportive of a two-dose immunization schedule against varicella.The profile of the anti-VZV antibody concentration kinetics in seropositive Active Control group participants having no confirmed varicella cases was similar to the previously published profile in MMR+V recipients.The strengths of the study include the plurality of the involved countries and centers, a long follow-up period of the same participants, and stringent controls for assessment of vaccine safety and varicella case definition.30The limitations of this study were the ethnically homogeneous population of participants and the two-year periods between serological assessments during the long-term follow-up. Despite regular contacts with the children’s families, it is possible that the time of rash onset was not always precisely determined. Another limitation was the availability of antibody concentrations only as immunological read-out, as it has been demonstrated that cellular immunity is implicated in long-term protection against varicella.To our knowledge, this is the first prospective long-term serological cohort allowing to characterize seropositivity prevalence and persistence of naturally induced anti-VZV antibodies in children not vaccinated against varicella in European countries with endemic varicella, over a ten-year period. About one-third of children not vaccinated against varicella developed an anti-VZV immune response although no varicella was detected or reported. While syndromic and seroprevalence surveys are important tools to assess the large-scale impact of varicella, used independently, such analyses are likely to reveal only part of the picture, with seroprevalence data failing to provide robust information about burden of disease and syndromic surveys potentially underestimating incidence.Click here for additional data file.


Genetically engineered T cells have been successfully used in the treatment of hematological malignancies, greatly increasing both progression-free and overall survival in patients. However, the outcomes of patients treated with Chimeric Antigen Receptor (CAR) T cells targeting solid tumors have been disappointing. There is an unmet clinical need for therapies which are specifically designed to overcome the challenges associated with solid tumors such as tumor heterogeneity and antigen escape. Genetic engineering employing the use of biological logic gating in T cells is an emerging and cutting-edge field that may address these issues. The advantages of logic gating include localized secretion of anti-tumor proteins into the tumor microenvironment, multi antigen targeting of tumors and a potential increase in safety when targeting tumor antigens which may not be exclusively tumor specific. In this review, we introduce the concept of biological logic gating and how this technology addresses some of the challenges of current CAR T treatment. We outline the types of logic gating circuits and finally discuss the application of this new technology to engineered T cells, in the treatment of cancer. Chimeric antigen receptor (CAR) T cell immunotherapy has initiated a new class of therapies, redirecting a patient’s T cells to recognize and destroy malignant cells. CARs are modular, synthetic receptors consisting of an antigen binding domain ) joined via a hinge to a transmembrane domain, costimulatory domain , and we untangle the relationships between avidity and antigen level expression on tumor vs healthy tissue, more sophisticated and specific methods of regulating T cell responses using logic gating are being developed.One advantage of applying logic gating to biological therapies is to trigger localized secretion of ant-tumor molecules, within the tumor microenvironment. Logic gated molecule release results in the highest concentration being delivered directly at the tumor site, rather than the molecule being sequestered in other tissues. Second, the specific delivery of molecules may allow for the application of a wider range of antitumor molecules, which may be unsuitable for systemic delivery. Additionally, well-established non-logic gated systems such as nuclear factor of activated T cells (NFAT)-dependent There are several non-specific biological agents which have demonstrated efficacy in promoting anti-tumor responses. Interleukin-12 has potent anti-tumor efficacy and has shown to synergise with CAR T cell therapy in glioblastoma,However, there must also be stringent pre-clinical evaluations of such therapies. The final doses delivered at the tumor site may remain unknown or be under limited control and therefore the half-life of these molecules must be determined to ascertain potential toxicity risks. Despite these considerations, there are numerous possibilities in the types of treatments which could be delivered via logic gating.NCT02209376) allowed patients to participate in the trial with EGFRvIII positivity ranging from 6%–96% .Antigen escape is the process of specific downregulation of a tumor expressed protein resulting from selective pressure exerted by targeted therapy or the outgrowth of pre-existing antigen negative clones. While antigen escape has been observed in hematological malignancies,One of the most difficult challenges associated with CAR T cell therapy is the selection of an appropriate tumor antigen to target. Target antigens can be broadly classified as TSA, tumor associated (TAA) or Embryonic Cancer Germline Antigens.Published results of glioblastom-specific CAR T cell clinical trials have reported only transient antitumor responses in trials targeting several different antigens.3 40While targeting TSAs in the context of solid tumors provides the greatest level of safety to avoid off tumor toxicity, the heterogeneous and potential low expression of TSAs has contributed to the lower success rate for CAR T cell therapy. However, using these antigens as induction signals of a logic gated system, rather than the target of therapy may provide greater anti-tumor function and more complete tumor cell elimination than what is induced by conventional CAR T treatment. Importantly, logic gating is still a form of targeted therapy and therefore, these systems are not immune from inducing target antigen downregulation or loss via antigen escape.The application of various logic gating systems has the potential to modulate immune responses. Depending on the type of system designed and the form of logic gating which is most suitable, immune responses such as CRS and off-tumor targeting of healthy tissue can be reduced, by the direct and local influence on tumor microenvironment.CRS is another important consideration for CAR T cell therapy, particularly for hematological malignancies. The large quantity of proinflammatory cytokines such as IFN-γ systemically produced by highly activated CAR T cells activates bystander myeloid cells to release additional proinflammatory cytokines; including the potent IL-6.Cases of CRS vary in severity, but the most severe cases can lead to multi-organ failure and death.Some degree of inflammatory cytokine release can enhance antitumor responses by activating bystander cells, however, a fine balance must be achieved to avoid excessive cytokine release and damage to tissues. Using logic gating systems, CRS could be controlled with the gated release of anti-inflammatory cytokines such as IL-10, monoclonal antibodies tocilizumab or mediators within the tumor microenvironment. This would mean that the tumor cells could still be eliminated, but the adverse patient responses of CRS may be avoided.Logic gating approaches using inhibitory signaling pathways can further aid discrimination between healthy and malignant tissue. Genetic circuits built to only be induced in the absence of the ligation of a healthy antigen targeting CAR may prevent the off-tumor toxicity which can be observed when the primary target is minimally expressed on healthy cells. These types of circuits could prevent off-tumor toxicity before they occur. The application of inhibitory logic gating circuits may also work to increase the possible pool of target antigens by providing a checkpoint of ‘no healthy antigen’ on the cells before the destruction of the tumor cell, increasing safety.Novel approaches are still needed to provide a greater level of regulation and control over engineered and infused T cells. Logic gating provides an additional level of control and safety and depending on the gating mechanism, even multi-antigen, or combination approaches. There are three main logic gating approaches described in this review and we will provide examples from the literature where they have been applied to engineered T cells and discuss some reflections on future approaches.Logic gating techniques have begun to be incorporated into the design of engineered T cells. These systems allow either multi-antigen targeting, combinatorial treatment with biological agents (such as checkpoint inhibitors or cytokines) and potentially a level of safety that could not be achieved with the current generation of CAR T cells. There are multiple types of logic circuits which can be applied to genetically engineered T cells . Biologiet alOne of the early forms of exerting control over engineered T cells was the development of ON switches. In this system, the administration of small molecules enables the completion of a circuit to allow T signalling.In this system, the antigen-binding domain and the signaling domain (containing the CD3ζ domain and ITAMs) are two separate molecules that only associate in the presence of a small molecule. This small molecule is a modified version of rapamycin with reduced immunosuppressive activity,46One year after the publication of pharmacological control of ON switched CARs, the technology was enhanced by the ‘IF-THEN’ or AND logic gating system. The concept of AND-gating requires two distinct antigens to be detected to trigger the outcome or downstream event; one to induce the logic gated system, the other to kill the target cell. The benefit of genetic approaches such as AND-gating in biological systems results in the presence of potentially potent anti-tumor molecules being modulated within the tumor environment and thus limiting systemic expression which may be intolerable to patients.24et al.In 2016, the design of the ‘AND-gated’ Synthetic Notch receptor (SynNotch) was developed by Morsut et al noted an interval of approximately 6 hours from SynNotch activation to CAR expression (as determined by GFP expression of the tagged CAR).On ligation of the scFv with its target antigen, the transcription factor Gal4-VP64 is cleaved from the SynNotch receptor by the natural Notch cleavage enzyme γ-secretase and the transcription factor is translocated into the nucleus of the cell where it becomes bound to the Upstream Activating Sequence. After binding, Gal4-VP64 drives the transcription of the desired cellular program, designed by the user . In RoybThe SynNotch system has been applied to multiple cancer models, including leukaemia,et al, who designed both a CAR and a SynNotch receptor specific for the receptor tyrosine kinase AxlThe SynNotch system has also been applied to mesothelioma and ovarian cancer using the same ‘pan’ tumor antigen; ALPPL2, as the activation signal.et alet al+ CD45RA+) and reduced expression of checkpoint molecules. Hyrenius-Wittsten et al, propose that this less differentiated state is due to the removal of tonic signaling by changing the receptor design .Logic gating has also been shown to have a positive effect on T cell phenotype. There is evidence to suggest that the fine balance of signals, received by the T cell induces changes the differentiated state and indeed, tonic signaling induced by a CAR has been shown to influence T cell fate.et al have been the dominant investigators in the SynNotch research field, thus, the research field is small but growing. Other research groups have also applied SynNotch logic gating in their studies. Stanley Riddell’s group have developed an ‘AND’ gated ROR-1 specific CAR T cell which improved the safety of targeting this TAA by requiring the coexpression of another protein; EpCAM, or, for more tumor specificity B7-H3, preventing direction of cytotoxicity towards ROR-1 expressing stromal cells.56To date, Roybal The benefit of systems such as AND-gating broadens the applicability of many anti-tumor or immune activating molecules which may have been prevented from clinical use because of their systemic adverse effects. Stringent safety testing will be required to fully characterize the induction and half-life of the induced protein production. AND-gating is yet to be tested in clinical trials and so the ‘real world’ success is to be determined.The concept of OR-gating is an example of a multi-antigen approach requiring the recognition of either one or more of the targeted CAR T cell antigens . This stMultiantigen targeting CAR T cell capabilities have demonstrated enhanced anti-tumor elimination compared with single targeting strategies.et al demonstrated that tanCAR T cells controlled tumor growth significantly longer than pooled and dual CAR T cells.59 64Dual targeting antigens can be particularly advantageous to mitigate antigen escape or antigen loss. Clinically, CD19 antigen reduction or loss following treatment with CD19-targeted immunotherapies is often observed in relapsed and/or refractory B-ALL.The benefit of the OR-gated designs of multiantigen targeted CAR T cells increases recognition capacity by CAR T cells and enables the genetic targeting of heterogeneous tumors, minimizing antigen escape. However, while these approaches offer hope to eliminate heterogeneous tumors, OR-gated designs have been associated with significant on-target/off-tumor adverse events. In studies of B-cell malignancies, the administration of dual CAR T cells resulted in significant CRS in multiple clinical trials, including CD19/CD22 dual CAR T cells,Many proteins which are highly expressed on tumor cells, may also be expressed at low levels on healthy tissue. Whist the inclusion of suicide switches in genetically engineered cells provides a level of safety, this activation is irreversible and binary; either on or off. Once the suicide gene has been activated, the transduced cells are destroyed, no longer exerting anti-tumor functions, or providing benefit to the patient.Unlike AND-circuits or OR-circuits which result in activation, the inhibitory NOT- logic gate prevents T cell activation on target recognition. NOT-gating can be applied to tumor antigens which may not be exquisitely tumor specific and display a low level of expression on healthy cells.Inhibitory CARs (iCARs) are designed with inhibitory signaling domains such as those found in PD-1 and CTLA-4et al demonstrated in vitro that iCARs had greatly reduced cytokine production and cytotoxic capabilities when cocultured with induced pluripotent stem cell-derived differentiated into fibroblasts which displayed both healthy (PSMA) and tumor (CD19) antigens compared with levels measured with coculture with target antigen (CD19) expressing cells.The function of iCARs has been evaluated in vitro and in vivo. Fedorov et al have shown preliminary evidence to suggest that T cells expressing both the CD93 NOT-gated CAR T cells and CD19 iCAR T cells exhibited decreases in PD-1 and TIM-3 expression compared with mock transduced and CD93-28z CAR T cells at baseline, however, further phenotypic analysis on T cell fitness post iCAR activation was not investigated.70Despite these promising findings, it is important to consider the potential implications of genetic circuits on T cell health and phenotype. The premise of the iCAR is that the inhibitory signal generated by ligation of a ‘healthy/normal protein’ by the iCAR will override any effector ‘kill’ signal induced by ligation of a ‘tumor targeting’ CAR. Therefore, the resulting T cell function is dependent on the balance of activation and inhibitory signals. Mackall Opposing most of the biological logic gating systems which promote T cell toxicity, there is another class of logic gating inhibiting T cell function. This family of receptors is referred to as OFF-switch. OFF-switches are generally pharmacologically mediated.73The authors also showed titratable function of the switch using a small molecule; asunaprevir (an anti-viral drug approved for use in combination with Daclatasvir in the treatment of Hepatitis C.Inhibitory circuits, or NOT-gates, provide an opportunity for the field to begin to investigate the anti-tumor function of therapies targeting highly expressed tumor antigens, which may have some level of expression on healthy tissue. The inbuilt safety step of either triggering an inhibitory signaling event or an inhibitory signaling event and degradation of the CAR could provide some level of reassurance that any targeting of healthy tissue can be halted before tissue destruction occurs.Genetically engineered T cells have revolutionized the treatment for many types of hematological malignancies, improving both progression-free and overall survival. However, the same success is yet to be translated to solid tumors.Approaches such as biological logic gating enable a greater level of safety to be achieved with engineered T cells. The anti-tumor payload in the form of cytokines or additional CARs can be delivered directly to the tumor site and further finetuning of molecules such as with OFF switches, pharmacological modification or NOT-gating may reduce the likelihood of leakiness out of the tumor microenvironment. Restricting expression of these molecules locally within the tumor microenvironment broadens the applicability of many anti-tumor therapeutics.There may be limitless possibilities when it comes to designing a logic gating system. Wendell Lim’s group has published on multiple effective logic gates; AND, NOT, Multiantigen AND gates, OR and even OFF-Notch; logic gating used to induce the expression of apoptotic factors within the cell.However, despite these advantages, logic gated systems have not yet made a clinical appearance. Publications by field leaders Roybal and Lim have demonstrated efficacy both in vitro and in vivo for numerous logic gated systems but currently, no clinical trials have been published. Depending on the nature of the induced molecule, an in-depth understanding of the expression and degradation kinetics will be required before progression to in-person trials.CAR T cells and T cell-based therapeutics form a critical component of immunotherapy approaches. Enhanced engineering of these systems is likely to enhance safety and potential efficacy against solid tumors by providing a multi-targeted approach directly at the tumor site. Early mechanisms of control overengineered cells include suicide switches which result in the irreversible destruction of the engineered cell. Advances in genetic engineering have resulted in the development of a range of logic gated systems. These approaches provide multiple benefits to engineered cells, including enhanced control and safety, with potential protective mechanisms against on-target/off-tumor toxicity and CRS. While a greater level of understanding of induction and degradation kinetics is still required and in patient trials, this technology has the potential to enhance patient responses to CAR T cell therapy in treating solid tumors.


Our findings highlight the importance of considering all concomitant medications in patients on ICIs who develop immune-mediated adverse reactions. In the event of some immune-related adverse reactions, it may be critical to identify the culprit antigen-forming entity that the ICIs have altered the perception of rather than simply attribute causality to the ICI itself in order to optimize both patient safety and treatment of malignancies.Many adverse reactions associated with immune checkpoint inhibitor (ICI) treatments are immunologically driven and may necessitate discontinuation of the ICI. Herein, we present a patient who had been administered the radio contrast media amidotrizoate multiple times without issue but who then developed a Stevens-Johnson syndrome reaction after coadministration of atezolizumab. Causality was confirmed by a positive re-challenge with amidotrizoate and laboratory investigations that implicated T cells. Importantly, the introduction of atezolizumab appears to have altered the immunologic response to amidotrizoate in terms of the tolerance–elicitation continuum. Proof of concept studies demonstrated enhancement of recall responses to a surrogate antigen panel following  The emergence of immune checkpoint inhibitors (ICIs) as a therapeutic option has dramatically altered the oncologic treatment landscape. Impressive efficacy data have been accruing over the last few years with the use of antibodies targeted at the PD1 and CTLA-4 coinhibitory pathways. Unfortunately, the use of checkpoint inhibitors has also been associated with a neoteric spectrum of adverse reactions, coined immune-related adverse events (IrAEs). The mechanism thought to underlie the IrAEs is ICI-mediated perturbation of self and xenobiotic related antigens. ICI mediated aberration of T cell responses to a number of compounds was first reported in various experimental models incorporating blockade/ablation of the CTLA-4 and PD pathways.in-vitro evidence that provides novel mechanistic insight.We report the case of a patient who developed Stevens-Johnson syndrome (SJS) to a previously tolerated iodinated contrast medium, amidotrizoate, after the use of atezolizumab, a PD-L1 blocker. We also present supporting The patient was recruited from the Clatterbridge Cancer Centre, Liverpool, UK, at the onset of the initial severe skin reaction considered to be due to the ICI agent. Subsequent sampling occurred at the onset of the second severe episode. Clinical data and photographic images were recorded and anonymized. Biologic samples  and skin biopsies) were obtained in a longitudinal manner periodically during both the initial and recurrent episodes. We also obtained blood samples from seven other patients prior to and 1 week following initiation of treatment with ICIs.et al.Bulk PBMC enrichment, limiting dilution, mitogen-driven expansion and initial specificity testing (for amidotrizoate) of monoclonal T cell populations was conducted as described by Gibson 5 cells/well) from healthy donors (n=9) treated with checkpoint blockade antibodies in-vitro  and PBMC samples taken from clinical visits of oncology patients ; pembrolizumab (n=3); nivolumab (n=1)) prior to and 1 week following initiation of treatment with ICIs to investigate recall responses to an antigen panel . Media control wells served as baseline controls, with all antigen specific responses normalized to a stimulation index of media control±blocking antibody in order to account for any blockade-induced baseline shift.Lymphocyte transformation tests (LTT) were conducted as outlined by Pichler and TilchThe index patient had been receiving first line ICI treatment with atezolizumab (anti-PD-L1 IgG1 mAb) for the management of metastatic renal cell carcinoma. The patient had previously undergone in excess of 20 contrast-enhanced CT scans. On each occasion, the iodinated contrast agent amidotrizoate was administered intravenously  and was well tolerated prior to the recommencement of ICI therapy. He had received 15 cycles of treatment followed by a treatment break to allow for palliative surgery. Following recovery from surgery, the patient recommenced ICI therapy and underwent a CT scan 2 weeks after. Twenty-four hours after exposure to intravenous amidotrizoate, the patient experienced a mild pruritic reaction. Following subsequent exposures, the patient experienced increasingly severe skin manifestations progressing from mild erythema multiforme (EM) to a blistering, desquamating rash consistent with Stevens-Johnson syndrome (SJS). Blistering involved both oral  and geniThe serious cutaneous adverse reaction in this patient was initially attributed to the ICI itself, but the possible role of amidotrizoate could not be excluded. Given the limitation of non-contrast enhanced imaging in malignant disease surveillance, a clinical, risk-informed decision was made to expose the patient to the contrast agent. Thus, 6 months after his previous CT scan, he was rechallenged with oral amidotrizoate . This culminated in the recrudescence of SJS with the same clinical phenotype within 2 hours of administration . This waIn-vitro blockade of PDL-1 and CTLA-4 resulted in marked enhancement of memory responses to the antigen panel relative to the results obtained in the absence of the ICIs in 6/9 healthy donors  the known on-target pharmacologic effects of ICIsex-vivo and in-vivo administration of ICIs. The interaction we have described between atezolizumab and amidotrizoate may therefore represent a novel pharmacodynamic drug–drug interaction.Our mechanistic data indicate the involvement of amidotrizoate-responsive T cells capable of secreting the cytolytic molecules granzyme B and perforin; both thought to be key mediators of SJS/TEN reactions.A clinically relevant ramification of our findings is that administration of ICI agents may inadvertently shift individuals towards an adverse immunological state in which hypersensitivity reactions are favored. While it is often reasonable to blame the ICI on its own for causing an irAE, and withdraw it, this may have negative consequences on survival from the malignancy. It is therefore also important to consider the role of concomitant therapies, particularly agents known to cause hypersensitivity reactions, in the manifestation of such reactions. The long half-life of the ICI antibodiesex-vivo functional studies in investigation of deregulation imposed by ICIs within a given individual. An idealistic future paradigm as outlined in (Ex-vivo blockade in a functional T cell assay may therefore have potential as a novel avenue of exploration for prospective biomarkers of response. Future studies to this end should evaluate the corroboration of ex-vivo ICI blockade to in-vivo for various antigens, assess the transposition of this to tumor response and toxicity outcomes and attempt to categorize responses according to outcomes.Our proof of concept study indicates utility of lined in  stipulatlined in . While s