Generation of Non-Alloreactive Antiviral Central Memory CD8 Human Veto T Cells for Cell Therapy.

Previous studies in mice demonstrated that CD8 T cells exhibit marked veto activity enhancing engraftment in several models for T cell-depleted bone marrow (TDBM) allografting. To reduce the risk of graft-versus-host disease (GVHD) associated with allogeneic CD8 veto T cells, these studies made use of naive CD8 T cells stimulated against third-party stimulators under cytokine deprivation and subsequent expansion in the presence of IL-15. More recently, it was shown that mouse CD8 veto T cells can be generated by stimulating CD8 memory T cells from ovalbumin immunized mice under cytokine deprivation, using ovalbumin as a third-party antigen. These cells also exhibited substantial enhancement of BM allografting without GVHD. In this study, we tested the hypothesis that stimulation and expansion of human CD8 memory T cells under IL-15 and IL-7 deprivation during the early phase of activation against recall viral antigens can lead to substantial loss of alloreactive T clones while retaining marked veto activity. Memory CD8 T cells were enriched by removal of CD45RA+, CD4+, and CD56+ cells from peripheral blood of cytomegalovirus (CMV)- and Epstein-Barr virus (EBV)-positive donors. In parallel, CD14+ monocytes were isolated; differentiated into mature dendritic cells (mDCs); pulsed with a library of CMV, EBV, adenovirus, and BK virus peptides; and irradiated. The CD8 T cell-enriched fraction was then cultured with the pulsed mDCs in the presence of IL-21 for 3 days, after which IL-15 and IL-7 were added. After 12 days of culture, the cells were tested by limiting dilution analysis for the frequency of alloreactive T cell clones and their veto activity. In preclinical runs using GMP reagents, we established that within 12 days of culture, a large number of highly homogenous CD8 T cells, predominantly expressing a central memory phenotype, could be harvested. These cells exhibited marked veto activity in vitro and >3-log depletion of alloreactivity. Based on these preclinical data, a phase 1-2 clinical trial was initiated to test the safety and efficacy of these antiviral CD8 central memory veto cells in the context of nonmyeloablative (NMA) T cell-depleted haploidentical hematopoietic stem cell transplantation (HSCT). In 2 validation runs and 11 clinical runs using GMP reagents, >1 × 1010 cells were generated from a single leukapheresis in 12 out of 13 experiments. At the end of 12 days of culture, there were 97 ± 2.5% CD3+CD8+ T cells, of which 84 ± 9.0% (range, 71.5% to 95.1%) exhibited the CD45RO+CD62L+ CM phenotype. Antiviral activity tested by intracellular expression of INF-γ and TNF-α and showed an average of 38.8 ± 19.6% positive cells on 6 hours of stimulation against the viral peptide mixture. Our results demonstrate a novel approach for depleting alloreactive T cell clones from preparations of antiviral CD8 veto cells. Based on these results, a phase 1-2 clinical trial is currently in progress to test the safety and efficacy of these veto cells in the context of NMA haploidentical T cell-depleted HSCT. Studies testing the hypothesis that these non-alloreactive CD8 T cells could potentially offer a platform for off-the-shelf veto chimeric antigen receptor T cell therapy in allogenic recipients, are warranted.

Postinfectious Inflammation, Autoimmunity, and Obsessive-Compulsive Disorder: Sydenham Chorea, Pediatric Autoimmune Neuropsychiatric Disorder Associated with Streptococcal Infection, and Pediatric Acute-Onset Neuropsychiatric Disorder.

Postinfectious neuroinflammation has been implicated in multiple models of acute-onset obsessive-compulsive disorder including Sydenham chorea (SC), pediatric acute-onset neuropsychiatric syndrome (PANS), and pediatric autoimmune neuropsychiatric disorders associated with streptococcal infection (PANDAS). These conditions are associated with a range of autoantibodies which are thought to be triggered by infections, most notably group A streptococci (GAS). Based on animal models using huma sera, these autoantibodies are thought to cross-react with neural antigens in the basal ganglia and modulate neuronal activity and behavior. As is true for many childhood neuroinflammatory diseases and rheumatological diseases, SC, PANS, and PANDAS lack clinically available, rigorous diagnostic biomarkers and randomized clinical trials. In this review article, we outline the accumulating evidence supporting the role neuroinflammation plays in these disorders. We describe work with animal models including patient-derived anti-neuronal autoantibodies, and we outline imaging studies that show alterations in the basal ganglia. In addition, we present research on metabolites, which are helpful in deciphering functional phenotypes, and on the implication of sleep in these disorders. Finally, we encourage future researchers to collaborate across medical specialties (e.g., pediatrics, psychiatry, rheumatology, immunology, and infectious disease) in order to further research on clinical syndromes presenting with neuropsychiatric manifestations.

Metabolomic Characterization of Pediatric Acute-Onset Neuropsychiatric Syndrome (PANS).

INTRODUCTION: PANS is a controversial clinical entity, consisting of a complex constellation of psychiatric symptoms, adventitious changes, and expression of various serological alterations, likely sustained by an autoimmune/inflammatory disease. Detection of novel biomarkers of PANS is highly desirable for both diagnostic and therapeutic management of affected patients. Analysis of metabolites has proven useful in detecting biomarkers for other neuroimmune-psychiatric diseases. Here, we utilize the metabolomics approach to determine whether it is possible to define a specific metabolic pattern in patients affected by PANS compared to healthy subjects. DESIGN: This observational case-control study tested consecutive patients referred for PANS between June 2019 to May 2020. A PANS diagnosis was confirmed according to the PANS working criteria (National Institute of Mental Health [NIMH], 2010). Healthy age and sex-matched subjects were recruited as controls. METHODS: Thirty-four outpatients referred for PANS (mean age 9.5 years; SD 2.9, 71% male) and 25 neurotypical subjects matched for age and gender, were subjected to metabolite analysis. Serum samples were obtained from each participant and were analyzed using Nuclear Magnetic Resonance (NMR) spectroscopy. Subsequently, multivariate and univariate statistical analyses and Receiver Operator Curves (ROC) were performed. RESULTS: Separation of the samples, in line with the presence of PANS diagnosis, was observed by applying a supervised model (R2X = 0.44, R2Y = 0.54, Q2 = 0.44, p-value < 0.0001). The significantly altered variables were 2-Hydroxybutyrate, glycine, glutamine, histidine, tryptophan. Pathway analysis indicated that phenylalanine, tyrosine, and tryptophan metabolism, as well as glutamine and glutamate metabolism, exhibited the largest deviations from neurotypical controls. CONCLUSION: We found a unique plasma metabolic profile in PANS patients, significantly differing from that of healthy children, that suggests the involvement of specific patterns of neurotransmission (tryptophan, glycine, histamine/histidine) as well as a more general state of neuroinflammation and oxidative stress (glutamine, 2-Hydroxybutyrate, and tryptophan-kynurenine pathway) in the disorder. This metabolomics study offers new insights into biological mechanisms underpinning the disorder and supports research of other potential biomarkers implicated in PANS.

Veto cells for safer nonmyeloablative haploidentical HSCT and CAR T cell therapy.

Haploidentical donors are a readily available source for mismatched hematopoietic bone marrow transplantation. The application of this regimen is constantly increasing with the advent of methods that overcome T-cell alloreactions that occur due to human-leukocyte-antigen disparity between host and donor. One successful method to overcome both graft rejection and graft-vs-host disease is transplantation of large numbers T-cell-depleted (TCD) haploidentical stem cell grafts (haploSCT), after myeloablative conditioning. The success of stem cell dose escalation is attributed to a unique immunoregulatory cell-property, termed "veto-activity." However, engraftment of mismatched hematopoietic stem cells following reduced-intensity conditioning still represents a major challenge. Here, we describe how the addition of post-transplant high-dose cyclophosphamide can promote immune tolerance induction after megadose TCD haploSCT, following nonmyeloablative conditioning. We also discuss ways of harnessing the immune regulatory properties of adoptively transferred "veto" cells to support mixed chimerism further and confer tolerance to cell-therapies, such as CAR-T cells. These approaches will soon be tested in phase 1-2 clinical studies and may prove to be a safe and efficacious treatment for many disorders such as hemoglobinopathies, autoimmune diseases, and as a prelude for organ tolerance. Moreover, this approach could pave the way for "off-the-shelf" cell-therapy agents, making them cheaper and easily obtainable.

Molecular signature of Epstein-Barr virus infection in MS brain lesions.

OBJECTIVE: We sought to confirm the presence and frequency of B cells and Epstein-Barr virus (EBV) (latent and lytic phase) antigens in archived MS and non-MS brain tissue by immunohistochemistry. METHODS: We quantified the type and location of B-cell subsets within active and chronic MS brain lesions in relation to viral antigen expression. The presence of EBV-infected cells was further confirmed by in situ hybridization to detect the EBV RNA transcript, EBV-encoded RNA-1 (EBER-1). RESULTS: We report the presence of EBV latent membrane protein 1 (LMP-1) in 93% of MS and 78% of control brains, with a greater percentage of MS brains containing CD138+ plasma cells and LMP-1-rich populations. Notably, 78% of chronic MS lesions and 33.3% of non-MS brains contained parenchymal CD138+ plasma cells. EBV early lytic protein, EBV immediate-early lytic gene (BZLF1), was also observed in 46% of MS, primarily in association with chronic lesions and 44% of non-MS brain tissue. Furthermore, 85% of MS brains revealed frequent EBER-positive cells, whereas non-MS brains seldom contained EBER-positive cells. EBV infection was detectable, by immunohistochemistry and by in situ hybridization, in both MS and non-MS brains, although latent virus was more prevalent in MS brains, while lytic virus was restricted to chronic MS lesions. CONCLUSIONS: Together, our observations suggest an uncharacterized link between the EBV virus life cycle and MS pathogenesis.

Immune tolerance induction by nonmyeloablative haploidentical HSCT combining T-cell depletion and posttransplant cyclophosphamide.

The establishment of safe approaches to attain durable donor-type chimerism and immune tolerance toward donor antigens represents a major challenge in transplantation biology. Haploidentical hematopoietic stem cell transplantation (HSCT) is currently used for cancer therapy either as a T-cell-depleted megadose HSCT following myeloablative conditioning or with T-cell-replete HSCT following nonmyeloablative conditioning (NMAC) and high-dose posttransplant cyclophosphamide (PTCY). The latter approach suffers from a significant rate of chronic graft-versus-host disease (GVHD), despite prolonged immunosuppression. The use of T-depleted grafts, although free of GVHD risk, is not effective after NMAC because of graft rejection. We now demonstrate in mice conditioned with NMAC that combining the power of high-dose PTCY with T-cell-depleted megadose HSCT can overcome this barrier. This approach was evaluated in 2 patients with multiple myeloma and 1 patient with Hodgkin lymphoma. The first myeloma patient now followed for 25 months, exhibited full donor-type chimerism in the myeloid and B-cell lineages and mixed chimerism in the T-cell compartment. The second myeloma patient failed to attain chimerism. Notably, the low toxicity of this protocol enabled a subsequent successful fully myeloablative haploidentical HSCT in this patient. The third patients was conditioned with slightly higher total body irradiation and engrafted promptly. All patients remain in remission without GVHD. Both engrafted patients were able to control cytomegalovirus reactivation. Enzyme-linked immunospot analysis revealed immune tolerance toward donor cells. Our results demonstrate a novel and safer nonmyeloablative haplo-HSCT offering a platform for immune tolerance induction as a prelude to cell therapy and organ transplantation.

The evolution of T-cell depletion in haploidentical stem-cell transplantation.

T-cell depletion (TCD) can prevent the onset of graft-versus-host disease (GvHD) in animal models of bone marrow transplantation; this manipulation enabled the successful application in the 1980s of T-cell depleted bone marrow (BM) for the treatment of babies with severe combined immune deficiency (SCID). However, in leukaemia patients, implementation of T-cell depletion has been more difficult, especially due to high rate of graft-rejection, leukaemia relapse and delayed immune reconstitution. These hurdles were gradually overcome by modifying the cell composition of the graft, and by reducing the toxicities associated with conditioning protocols. Although no 'gold standard' TCD method exists, T-cell depletion in its modern forms could offer clinical benefit, even for patients with a matched sibling donor.

Megadose stem cell administration as a route to mixed chimerism.

PURPOSE OF REVIEW: To discuss recent innovations for the application of mismatched haploidentical hematopoietic stem cell transplantation as a means to achieve immune tolerance prior to allograft transplantation. We highlight the advantages of utilizing novel 'veto' cells, to facilitate induction of mixed chimerism, under nonmyeloablative conditioning. RECENT FINDINGS: Haploidentical hematopoietic stem cell transplantation has been used for leukemia patients, where reduction of graft-versus-host disease risk has been routinely attained by depleting grafts of T-cells, and escalated doses of CD34 progenitors (i.e. megadose) to overcome graft rejection. These 'megadose' transplants overcome rejection through a unique immunoregulatory 'veto' capacity. The importance of attaining mixed chimerism for allograft tolerance induction in humans has been highlighted by several studies of combined haploidentical hematopoietic stem cell transplantation and kidney transplants. However, stable chimerism could only by attained by making use of T-cell replete haploidentical hematopoietic stem cell transplantation, posing a significant risk for graft-versus-host or other immune abnormalities. The use of nonalloreactive 'veto' cells, such as anti-third-party central memory CD8 T cells, could potentially overcome this barrier. SUMMARY: Achieving stable mixed chimerism after haploidentical hematopoietic stem cell transplantation, key to utilizing transplantation as a prelude for organ tolerance, may be realized by coadministration of 'veto' cells under current nonmyeloablative protocols.

A new approach for eradication of residual lymphoma cells by host nonreactive anti-third-party central memory CD8 T cells.

Generation of T cells endowed with graft-versus-leukemia (GVL) and depleted of graft-versus-host (GVH) activity represents a highly desirable goal in bone marrow transplantation (BMT). Here, we demonstrate that donor anti-third-party CD8 T cells with central memory phenotype (Tcm) exhibit marked GVL reactivity through a unique T-cell receptor-independent mechanism. Thus, in a residual disease mouse model, Tcm therapy following autologous BMT led to significant survival prolongation, with 30% to 40% of the treated mice displaying long-term tumor-free survival. A more impressive finding was that infusion of donor Tcm in an allogeneic model rapidly eliminated residual lymphoma cells and led to long-term survival of 100% in the absence of GVH disease. Collectively, the strong GVL reactivity of anti-third-party Tcm, coupled with their demonstrated enhancement of bone marrow allografting, suggests that the use of Tcm therapy in conjunction with allogeneic T-cell-depleted BMT could be of particular benefit in patients with B-cell malignancies who cannot tolerate intensive myeloablative conditioning.

Murine anti-third-party central-memory CD8(+) T cells promote hematopoietic chimerism under mild conditioning: lymph-node sequestration and deletion of anti-donor T cells.

Transplantation of T cell-depleted BM (TDBM) under mild conditioning, associated with minimal toxicity and reduced risk of GVHD, offers an attractive therapeutic option for patients with nonmalignant hematologic disorders and can mediate immune tolerance to subsequent organ transplantation. However, overcoming TDBM rejection after reduced conditioning remains a challenge. Here, we address this barrier using donorderived central memory CD8(+) T cells (Tcms), directed against third-party antigens. Our results show that fully allogeneic or (hostXdonor)F1-Tcm, support donor chimerism (> 6 months) in sublethally irradiated (5.5Gy) mice, without GVHD symptoms. Chimerism under yet lower irradiation (4.5Gy) was achieved by combining Tcm with short-term administration of low-dose Rapamycin. Importantly, this chimerism resulted in successful donor skin acceptance, whereas third-party skin was rejected. Tracking of host anti-donor T cells (HADTCs), that mediate TDBMT rejection, in a novel bioluminescence-imaging model revealed that Tcms both induce accumulation and eradicate HADTCs in the LNs,concomitant with their elimination from other organs, including the BM. Further analysis with 2-photon microcopy revealed that Tcms form conjugates with HADTCs, resulting in decelerated and confined movement of HADTCs within the LNs in an antigen-specific manner. Thus, anti-third-party Tcms support TDBMT engraftment under reduced-conditioning through lymph-node sequestration and deletion of HADTCs, offering a novel and potentially safe approach for attaining stable hematopoietic chimerism.

Induction of transplantation tolerance in haploidenical transplantation under reduced intensity conditioning: the role of ex-vivo generated donor CD8+ T cells with central memory phenotype.

Haploidentical hematopoietic stem cell transplantation (HSCT) offers the advantage of readily available family member donors for nearly all patients. A 'megadose' of purified CD34+ hematopoietic stem cells is used to overcome the host's residual immunity surviving the myeloablative conditioning, while avoiding severe GVHD. However, the number of CD34+ cells that can be harvested is insufficient for overcoming the large numbers of host T cells remaining after reduced intensity conditioning (RIC). Therefore, combining a 'megadose' of CD34+ HSCT with other tolerizing cells could potentially support and promote successful engraftment of haploidentical purified stem cell transplantation under a safer RIC. One approach to address this challenge could be afforded by using Donor CD8 T cells directed against 3rd-party stimulators, bearing an ex-vivo induced central memory phenotype (Tcm). These Tcm cells,depleted of GVH reactivity, were shown to be highly efficient in overcoming host T cells mediated rejection and in promoting fully mismatched bone-marrow (BM) engraftment, in HSCT murine models. This is likely due to the marked lymph node homing of the Tcm, their strong proliferative capacity and prolonged persistence in BM transplant recipients. Thus, combining anti 3rd-party Tcm cell therapy with a 'megadose' of purified CD34+ stem cells, could offer a safer RIC protocol for attaining hematopoietic chimerism in patients with hematological diseases and as a platform for organ transplantation or cell therapy in cancer patients.