Background Post-transplant cyclophosphamide (PTCy) and abatacept each are standard GVHD prophylaxes. PTCy uniquely prevents chronic GVHD, but otherwise the relative effects of each are undefined. Combining PTCy with abatacept clinically has shown early promising results. Objective To assess the relative impact of PTCy vs abatacept or ICOS-blockade vs combinatorial therapy in our B6C3F1→B6D2F1 MHC-haploidentical murine HCT. Results Anti-ICOS given on days 0/+3/+4 was ineffective with universal mortality. Yet, similarly high survival was seen when giving anti-ICOS on days +3/+4 as when giving it serially on days +3/+4/+7/+10/+13. Even so, anti-ICOS was less effective than PTCy 25 mg/kg/day on days +3/+4. The combination of PTCy and ICOS (both on days +3/+4) produced better weights but similar clinical GVHD scores as PTCy alone; histopathologic GVHD was slightly improved at day +7 with the combination but similar by day +21. Compared with PTCy, ICOS blockade did not achieve the relative skewing of liver-infiltrating donor T cells towards CD8+ T cells, did not effectively reduce conventional CD4+ T cell (Tcon) proliferation at day +7, and resulted in lower Treg percentages. Both PTCy and the combination reduced activation and differentiation of T cells, but the combination did not effectively reduce Tcon proliferation at day +7, led to a higher frequency of alloreactive Tcons at days +7 and +21, led to more T cells with an exhausted phenotype at day +21, and inhibited the preferential Treg expansion at day +21 seen with PTCy alone.Abatacept at the standard dosing schedule (days -1, +5, +14, +28) was ineffective with universal fatality, but abatacept given on days +3/+4 resulted in 30% survival. At day +7, PTCy vs. abatacept days +3/+4 vs. combinations had similar histopathologic GVHD; at day +21, PTCy alone had the least histopathologic GVHD. Abatacept alone at either schedule produced less skewing towards CD8+ T cells, less reductions in Tcon activation and proliferation at day +7, and lower Treg percentages. Like PTCy alone, the combinations were able to reduce Tcon activation and proliferation and restrain T-cell differentiation, but the combination also resulted in lower Treg percentages at day +21, increased exhausted Tcons at day +21, and increased frequency of alloreactive Tcons at days +7 and +21. Conclusions PTCy appears superior to co-stimulatory blockade with anti-ICOS or abatacept. Combination with PTCy improved weights but did not improve GVHD and also increased alloreactive Tcons. Co-stimulatory blockade is: 1) maximally effective on days +3/+4 with minimal benefit from prolonged administration, and 2) results in worse outcomes when started pre-HCT; of note, abatacept is first dosed on day -1 clinically and ongoing efforts are focused on extending the duration. These results need to be confirmed in human HCT, wherein each agent is combined with adjunct immunosuppressants.

Background Post-transplantation cyclophosphamide (PTCy) reduces severe graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (HCT). Additionally, the gut microbiota taxa and diversity have been associated with occurrence of GVHD and mortality in HCT patients. Methods To understand better the impact of PTCy on the gut microbiota and the importance of the gut microbiota for PTCy's mechanisms of GVHD prevention, we used our T-cell-replete MHC-haploidentical murine HCT model (B6C3F1→B6D2F1). PTCy 25mg/kg/day or vehicle (PBS) was given on days +3/+4. Results PTCy treatment was associated with increased frequencies of bacterial taxa known to be protective against GVHD, such as ruminococcacae, clostridiales, lachnospiraceae, and blautia. This change coincided with increased levels of plasma butyrate, a metabolite known to protect against GVHD; butyrate levels positively correlated with percentages of regulatory T cells (Tregs) at day +21 in mice treated with or without PTCy. Many of the bacterial taxa modulations induced by PTCy were not affected by prophylactic treatment with levofloxacin. PTCy also led to faster numerical recovery of bacteria over time. To assess the mechanistic importance of PTCy modulation of the gut microbiota, we performed fecal microbiota transplants (FMT), in which mice did not receive post-HCT treatment themselves, but rather pre-HCT received FMT via oral gavage using stool from mice treated with PTCy or PBS. Results showed that mice receiving stool from PTCy-treated mice had significantly better weights at early post-HCT time points and a trend towards better overall survival. We repeated these experiments with germ-free (GF) recipient mice reconstituted with FMT via oral and rectal gavage using stool from donors transplanted under specific pathogen-free conditions; GF mice that received PTCy-modified microbiota also had significantly higher weights in the first few weeks after HCT. Nevertheless, PTCy maintained its efficacy in preventing GVHD in GF recipients that were not FMT reconstituted. Moreover, neither levofloxacin treatment from days 0 to +14 nor aggressive depletion of the gut microbiota via continual treatment using oral broad-spectrum antibiotic cocktail (imipenem, vancomycin, neomycin) starting 2 weeks prior to HCT worsened clinical or histopathologic GVHD in PTCy-treated mice. Conclusion PTCy modulates the microbiome towards taxa protective against GVHD, which may contribute to Treg recovery via increased butyrate levels. Even so, antibiotic treatment does not hinder GVHD prevention by PTCy, and consequently concerns regarding antibiotic exposure perhaps may be of less concern for affecting post-HCT outcomes in PTCy-treated recipients. Overall, PTCy's modulation of the microbiota contributes to GVHD prevention, but is not necessary for the mechanisms by which PTCy prevents severe GVHD.

Monoclonal antibodies (mAbs) improve survival of patients with mature B-cell malignancies. Fcγ-receptor dependent effector mechanisms kill tumor cells but can promote antigen loss through trogocytosis, contributing to treatment failures. Cell-bound mAbs trigger the complement cascade to deposit C3 activation fragments and lyse cells. Within 24 hours after ofatumumab administration to patients with chronic lymphocytic leukemia (CLL), circulating tumor cells had lost CD20 and were opsonized with C3d. We hypothesized that C3d provides a target to eliminate residual CD20 negative tumor cells. To test this hypothesis, we generated C8xi, a mouse/human chimeric IgG1 that reacts with human but not mouse C3d. C8xi was effective in a patient-derived xenograft model against CD20 negative, C3d opsonized CLL cells from patients treated with ofatumumab. We also generated rabbit mAbs, two of which were chosen because they bound mouse and human C3d with low nanomolar affinity but were minimally cross-reactive with full-length C3. Anti-C3d rabbit/human chimeric IgG1 in combination with ofatumumab or rituximab prolonged survival of xenografted mice that model three different types of non-Hodgkin lymphoma (NHL). For example, in a diffuse large B-cell lymphoma model (SU-DHL-6), median survival with single-agent CD20 mAb was 114 days but was not reached for mAb combination treatment (P=.008). In another NHL model (SU-DHL-4), single-agent and combination mAb therapy eradicated lymphoma in most mice. In long-term survivors from both cohorts, there was no evidence of adverse effects. We propose that C3d mAbs combined with complement fixing CD20 mAbs can deliver a one-two punch and increase efficacy of mAb-based therapy.

Primary fibroblasts from six individuals with CLN3-related conditions were used to generate induced pluripotent stem cell (iPSC) lines CHDTRi001-B, CHDTRi002-B, CHDTRi003-A, CHDTRi004-B, CHDTRi005-A, and CHDTRi006-E through the expression of four reprogramming factors: human OCT3/4, KLF4, SOX2, and c-MYC. The iPSC lines were characterized to confirm their pluripotency via immunocytochemistry, flow cytometry, and teratoma formation. Genomic stability, cell line identity, and CLN3 genotype were confirmed. These iPSC lines may be used as participant-derived experimental models for further investigation of CLN3, a rare, fatal, pediatric, blindness and neurodegenerative lysosomal disorder with no cure.

Introduction Ex vivo gene therapy for treatment of Inborn errors of Immunity (IEIs) have demonstrated significant clinical benefit in multiple Phase I/II clinical trials. Current approaches rely on engineered retroviral vectors to randomly integrate copy(s) of gene-of-interest in autologous hematopoietic stem/progenitor cells (HSPCs) genome permanently to provide gene function in transduced HSPCs and their progenies. To circumvent concerns related to potential genotoxicities due to the random vector integrations in HSPCs, targeted correction with CRISPR-Cas9-based genome editing offers improved precision for functional correction of multiple IEIs. Methods We compare the two approaches for integration of IL2RG transgene for functional correction of HSPCs from patients with X-linked Severe Combined Immunodeficiency (SCID-X1 or XSCID); delivery via current clinical lentivector (LV)- IL2RG versus targeted insertion (TI) of IL2RG via homology-directed repair (HDR) when using an adeno-associated virus (AAV)- IL2RG donor following double-strand DNA break at the endogenous IL2RG locus. Results and discussion In vitro differentiation of LV- or TI-treated XSCID HSPCs similarly overcome differentiation block into Pre-T-I and Pre-T-II lymphocytes but we observed significantly superior development of NK cells when corrected by TI (40.7% versus 4.1%, p = 0.0099). Transplants into immunodeficient mice demonstrated robust engraftment (8.1% and 23.3% in bone marrow) for LV- and TI- IL2RG HSPCs with efficient T cell development following TI- IL2RG in all four patients’ HSPCs. Extensive specificity analysis of CRISPR-Cas9 editing with rhAmpSeq covering 82 predicted off-target sites found no evidence of indels in edited cells before ( in vitro ) or following transplant, in stark contrast to LV’s non-targeted vector integration sites. Together, the improved efficiency and safety of IL2RG correction via CRISPR-Cas9-based TI approach provides a strong rationale for a clinical trial for treatment of XSCID patients.

Supplementary Table S1 from Selenium Deficiency Abrogates Inflammation-Dependent Plasma Cell Tumors in Mice

Posttransplantation cyclophosphamide (PTCy), given on days +3 and +4, reduces graft -versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (HCT), but its immunologic underpinnings are not fully understood. In a T-cell-replete, major histocompatibility complex-haploidentical murine HCT model (B6C3F1 -> B6D2F1), we previously showed that PTCy rapidly induces suppressive mechanisms sufficient to prevent GVHD induction by non-PTCy-exposed donor splenocytes infused on day +5. Here, in PTCy-treated mice, we found that depleting Foxp3+ regulatory T cells (Tregs) in the initial graft but not the day +5 splenocytes did not worsen GVHD, yet depleting Tregs in both cellular compartments led to fatal GVHD induced by the day +5 splenocytes. Hence, Tregs were necessary to control GVHD induced by new donor cells, but PTCy's impact on Tregs appeared to be indirect. Therefore, we hypothesized that myeloid-derived suppressor cells (MDSCs) play a complementary role. Functionally suppressive granulocytic and monocytic MDSCs were increased in percentages in PTCy-treated mice, and MDSC percentages were increased after administering PTCy to patients undergoing HLA-haploidentical HCT. PTCy increased colony-stimulating factors critical for MDSC development and rapidly promoted the generation of MDSCs from bone marrow precursors. MDSC reduction via anti-Gr1 treatment in murine HCT did not worsen histopathologic GVHD but resulted in decreased Tregs and inferior survival. The clinical implications of these findings, including the potential impact of expanded MDSCs after PTCy on engraftment and cytokine release syndrome, remain to be elucidated. Moreover, the indirect effect that PTCy has on Tregs, which in turn play a necessary role in GVHD prevention by initially transplanted or subsequently infused T cells, requires further investigation.

Supplementary Table S2 from Selenium Deficiency Abrogates Inflammation-Dependent Plasma Cell Tumors in Mice

Changes in metabolism of macrophages are required to sustain macrophage activation in response to different stimuli. We showed that the cytokine TGF-β (transforming growth factor–β) regulates glycolysis in macrophages independently of inflammatory cytokine production and affects survival in mouse models of sepsis. During macrophage activation, TGF-β increased the expression and activity of the glycolytic enzyme PFKL (phosphofructokinase-1 liver type) and promoted glycolysis but suppressed the production of proinflammatory cytokines. The increase in glycolysis was mediated by an mTOR–c-MYC–dependent pathway, whereas the inhibition of cytokine production was due to activation of the transcriptional coactivator SMAD3 and suppression of the activity of the proinflammatory transcription factors AP-1, NF-κB, and STAT1. In mice with LPS-induced endotoxemia and experimentally induced sepsis, the TGF-β–induced enhancement in macrophage glycolysis led to decreased survival, which was associated with increased blood coagulation. Analysis of septic patient cohorts revealed that the expression of PFKL , TGFBRI (which encodes a TGF-β receptor), and F13A1 (which encodes a coagulation factor) in myeloid cells positively correlated with COVID-19 disease. Thus, these results suggest that TGF-β is a critical regulator of macrophage metabolism and could be a therapeutic target in patients with sepsis.

The stomach-derived hormone ghrelin regulates essential physiological functions. The ghrelin receptor (GHSR) has ligand-independent actions, therefore, GHSR gene deletion may be a reasonable approach to investigate the role of this system in feeding behaviors and diet-induced obesity (DIO). Here we investigated the effects of a long-term (12 month) high-fat (HFD) versus regular diet on obesity-related measures in global GHSR-KO and wild type (WT) Wistar male and female rats. Our main findings were that the GHSR gene deletion protects against DIO and decreases food intake during HFD in male but not in female rats. GHSR gene deletion increased thermogenesis and brain glucose uptake in male rats and modified the effects of HFD on brain glucose metabolism in a sex-specific manner, as assessed with small animal positron emission tomography. RNA-sequencing was also used to show that GHSR-KO rats had upregulated expression of genes responsible for fat oxidation in brown adipose tissue. Central administration of a novel GHSR inverse agonist, PF-5190457, attenuated ghrelin-induced food intake, but only in male, not in female mice. HFD-induced binge-like eating was reduced by inverse agonism in both sexes. Our results support GHSR as a promising target for new pharmacotherapies for obesity.

Since first reported in 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is rapidly acquiring mutations, particularly in the spike protein, that can modulate pathogenicity, transmission and antibody evasion leading to successive waves of COVID19 infections despite an unprecedented mass vaccination necessitating continuous adaptation of therapeutics. Small animal models can facilitate understanding host-pathogen interactions, target selection for therapeutic drugs, and vaccine development, but availability and cost of studies in BSL3 facilities hinder progress. To generate a BSL2-compatible in vivo system that specifically recapitulates spike protein mediated disease we used replication competent, GFP tagged, recombinant Vesicular Stomatitis Virus where the VSV glycoprotein was replaced by the SARS-CoV-2 spike protein (rVSV-SARS2-S). We show that infection requires hACE2 and challenge of neonatal but not adult, K18-hACE2 transgenic mice (hACE2tg) leads to productive infection of the lungs and brains. Although disease progression was faster in SARS-CoV-2 infected mice, infection with both viruses resulted in neuronal infection and encephalitis with increased expression of Interferon-stimulated Irf7, Bst2, Ifi294, as well as CxCL10, CCL5, CLC2, and LILRB4, and both models were uniformly lethal. Further, prophylactic treatment targeting the Spike protein (Receptor Binding Domain) with antibodies resulted in similar levels of protection from lethal infection against rVSV-SARS2-S and SARS-CoV-2 viruses. Strikingly, challenge of neonatal hACE2tg mice with SARS-CoV-2 Variants of Concern (SARS-CoV-2-α, -β, ϒ, or Δ) or the corresponding rVSV-SARS2-S viruses (rVSV-SARS2-Spike-α, rVSV-SARS2-Spike-β, rVSV-SARS2-Spike-ϒ or rVSV-SARS2-Spike-Δ) resulted in increased lethality, suggesting that the Spike protein plays a key role in determining the virulence of each variant. Thus, we propose that rVSV-SARS2-S virus can be used to understand the effect of changes to SARS-CoV-2 spike protein on infection and to evaluate existing or experimental therapeutics targeting spike protein of current or future VOC of SARS-CoV-2 under BSL-2 conditions.

Protein phosphorylation, controlled by the coordinated actions of phosphatases and kinases, is an important regulatory mechanism in synaptic transmission and other neurophysiological processes. Ionotropic glutamate receptors are known targets of phosphorylation on serine, threonine and tyrosine residues, with functional consequences for cell excitability, plasticity and toxicity. While phosphorylation of metabotropic glutamate receptors (mGluRs) also impacts critical cellular processes, there has been no evidence for direct tyrosine phosphorylation of mGluRs. In the present study, anti-phosphotyrosine and specific mGluR antibodies were used to detect tyrosine-phosphorylated mGluRs in rat brain. In particular, we found that mGluR5 is an abundant phosphotyrosine protein in vivo as well as in primary striatal neurons and tissue slices in vitro. The protein phosphatase inhibitor pervanadate robustly increased the amount of tyrosine-phosphorylated mGluR5, suggesting the receptor is subject to an endogenous, active cycle of phosphorylation and dephosphorylation. Furthermore, NMDA treatment also increased the amount of tyrosine-phosphorylated mGluR5, suggesting these endogenous phosphorylation regulatory mechanisms can be used to mediate crosstalk between synaptic glutamate receptors. While mGluR5-stimulated phosphoinositide hydrolysis appears to be unaltered by pervanadate treatment, tyrosine phosphorylation of mGluR5 may be important in trafficking, anchoring, or signaling of the receptor through G protein-independent pathways.

Post-transplantation cyclophosphamide (PTCy) reduces the incidence and severity of graft-versus-host disease (GVHD), thereby improving the safety and accessibility of allogeneic hematopoietic cell transplantation (HCT). We have shown that PTCy works by inducing functional impairment and suppression of alloreactive T cells. We also have identified that reduced proliferation of alloreactive CD4 + T cells at day +7 and preferential recovery of CD4 + CD25 + Foxp3 + regulatory T cells (T regs ) at day +21 are potential biomarkers associated with optimal PTCy dosing and timing in our B6C3F1→B6D2F1 MHC-haploidentical murine HCT model. To understand whether the effects of PTCy are unique and also to understand better the biology of GVHD prevention by PTCy, here we tested the relative impact of cyclophosphamide compared with five other optimally dosed chemotherapeutics (methotrexate, bendamustine, paclitaxel, vincristine, and cytarabine) that vary in mechanisms of action and drug resistance. Only cyclophosphamide, methotrexate, and cytarabine were effective in preventing fatal GVHD, but cyclophosphamide was superior in ameliorating both clinical and histopathological GVHD. Flow cytometric analyses of blood and spleens revealed that these three chemotherapeutics were distinct in constraining conventional T-cell numerical recovery and facilitating preferential T reg recovery at day +21. However, cyclophosphamide was unique in consistently reducing proliferation and expression of the activation marker CD25 by alloreactive CD4 + Foxp3 - conventional T cells at day +7. Furthermore, cyclophosphamide restrained the differentiation of alloreactive CD4 + Foxp3 - conventional T cells at both days +7 and +21, whereas methotrexate and cytarabine only restrained differentiation at day +7. No chemotherapeutic selectively eliminated alloreactive T cells. These data suggest that constrained alloreactive CD4 + Foxp3 - conventional T-cell numerical recovery and associated preferential CD4 + CD25 + Foxp3 + T reg reconstitution at day +21 may be potential biomarkers of effective GVHD prevention. Additionally, these results reveal that PTCy uniquely restrains alloreactive CD4 + Foxp3 - conventional T-cell proliferation and differentiation, which may explain the superior effects of PTCy in preventing GVHD. Further study is needed to determine whether these findings also hold true in clinical HCT.

Immune cells express an array of inhibitory checkpoint receptors that are upregulated upon activation and limit tissue damage associated with excessive response to pathogens or allergens. Mouse leukocyte immunoglobulin like receptor B4 (LILRB4), also known as glycoprotein 49B (gp49B), is an inhibitory checkpoint receptor constitutively expressed in myeloid cells and upregulated in B cells, T cells, and NK cells upon activation. Here, we report that expression of LILRB4, which binds Zika virus (ZIKV), was increased in microglia and myeloid cells infiltrating the brains of neonatal mice with ZIKV-associated meningoencephalitis. Importantly, while C57BL/6 mice developed transient neurological symptoms but survived infection, mice lacking LILRB4/gp49B (LILRB4 KO) exhibited more severe signs of neurological disease and succumbed to disease. Their brains showed increased cellular infiltration but reduced control of viral burden. The reduced viral clearance was associated with altered NK cell function in the absence of LILRB4/gp49B. In naive animals, this manifested as reduced granzyme B responses to stimulation, but in ZIKV-infected animals, NK cells showed phenotypic changes that suggested altered maturation, diminished glucose consumption, reduced IFN-γ and granzyme B production, and impaired cytotoxicity. Together, our data reveal LILRB4/gp49B as an important regulator of NK cell function during viral infections.

Relapse limits the therapeutic efficacy both of chimeric antigen receptor (CAR) T cells and allogeneic hematopoietic cell transplantation (allo-HCT). Patients may undergo these therapies sequentially to prevent or treat relapsed malignancy. However, direct integration of the 2 therapies has been avoided over concerns for potential induction of graft-versus-host disease (GVHD) by allogeneic CAR T cells. We have shown in murine T-cell-replete MHC-haploidentical allo-HCT that suppressive mechanisms induced immediately after posttransplant cyclophosphamide (PTCy), given on days +3/+4, prevent GVHD induction by alloreactive T cells infused as early as day +5. Therefore, we hypothesized that allogeneic CAR T cells given in a similarly integrated manner in our murine MHC-haploidentical allo-HCT model may safely exert antitumor effects. Indeed, allogeneic anti-CD19 CAR T cells given early after (day +5) PTCy or even prior to (day 0) PTCy cleared leukemia without exacerbating the cytokine release syndrome occurring from the MHC-haploidentical allo-HCT or interfering with PTCy-mediated GVHD prevention. Meanwhile, CAR T-cell treatment on day +9 or day +14 was safe but less effective, suggesting a limited therapeutic window. CAR T cells infused before PTCy were not eliminated, but surviving CAR T cells continued to proliferate highly and expand despite PTCy. In comparison with infusion on day +5, CAR T-cell infusion on day 0 demonstrated superior clinical efficacy associated with earlier CAR T-cell expansion, higher phenotypic CAR T-cell activation, less CD4(+)CD25+Foxp3+ CAR T-cell recovery, and transcriptional changes suggesting increased activation of CD4+ CAR T cells and more cytotoxic CD8(+) CAR T cells. This study provides mechanistic insight into PTCy's impact on graft-versus-tumor immunity and describes novel approaches to integrate CAR T cells and allo-HCT that may compensate for deficiencies of each individual approach.

MIRTH (Myocardial Intramural Remodeling by Transvenous Tether) is a transcatheter ventricular remodeling procedure. A transvenous tension element is placed within the walls of the beating left ventricle and shortened to narrow chamber dimensions. MIRTH uses 2 new techniques: controlled intramyocardial guidewire navigation and EDEN (Electrocardiographic Radial Depth Navigation). MIRTH caused a sustained reduction in chamber dimensions in healthy swine. Midventricular implants approximated papillary muscles. MIRTH shortening improved myocardial contractility in cardiomyopathy in a dose-dependent manner up to a threshold beyond which additional shortening reduced performance. MIRTH may help treat dilated cardiomyopathy. Clinical investigation is warranted.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare accelerated aging disorder most notably characterized by cardiovascular disease and premature death from myocardial infarction or stroke. The majority of cases are caused by a de novo single nucleotide mutation in the LMNA gene that activates a cryptic splice donor site, resulting in production of a toxic form of lamin A with a 50 amino acid internal deletion, termed progerin. We previously reported the generation of a transgenic murine model of progeria carrying a human BAC harboring the common mutation, G608G, which in the single-copy state develops features of HGPS that are limited to the vascular system. Here, we report the phenotype of mice bred to carry two copies of the BAC, which more completely recapitulate the phenotypic features of HGPS in skin, adipose, skeletal, and vascular tissues. We further show that genetic reduction of the mechanistic target of rapamycin (mTOR) significantly extends lifespan in these mice, providing a rationale for pharmacologic inhibition of the mTOR pathway in the treatment of HGPS.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare accelerated aging disorder characterized by premature death from myocardial infarction or stroke. It is caused by de novo single-nucleotide mutations in the LMNA gene that activate a cryptic splice donor site, resulting in the production of a toxic form of lamin A, which is termed progerin. Here we present a potential genetic therapeutic strategy that utilizes antisense peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) to block pathogenic splicing of mutant transcripts. Of several candidates, PPMO SRP-2001 provided the most significant decrease in progerin transcripts in patient fibroblasts. Intravenous delivery of SRP-2001 to a transgenic mouse model of HGPS produced significant reduction of progerin transcripts in the aorta, a particularly critical target tissue in HGPS. Long-term continuous treatment with SRP-2001 yielded a 61.6% increase in lifespan and rescue of vascular smooth muscle cell loss in large arteries. These results provide a rationale for proceeding to human trials.

Anti-CD19 CAR T cells can induce remissions in many patients. Yet, relapses can occur and so some patients may proceed to HCT either prior to or after relapse to try to achieve long-term cure. Even so, relapse limits the success of HCT. We recently showed that the suppressive mechanisms induced immediately after PTCy prevent new donor T cells from causing GVHD. Thus, we hypothesized that donor anti-CD19 CAR T cells given early after PTCy would not cause GVHD but may retain an anti-tumor effect; the premise is that the CAR T cells would induce an initial deep remission, while the polyclonal graft-versus-tumor (GVT) T-cell response would ensure long-term cure.To test this approach, we modified our T-cell-replete murine MHC-haploidentical HCT model (B6C3F1→B6D2F1) by giving 1 × 106 E2a-PBx leukemia cells 7 days prior to lethal irradiation and HCT [Figure 1A]. Importantly, E2a-PBx, as a pre-B-cell ALL cell line on a B6 background, is syngeneic with host and donor; thus, in this model only anti-CD19 CAR T cells can treat the leukemia (there is no allo response against it). T-cell-enriched B6C3F1 donor splenocytes were stimulated with anti-CD3/CD28 beads, transduced with an anti-CD19 CAR vector with a CD28 costimulatory domain, and infused at 1 × 106 transduced T cells per mouse on day +5 (24 hours after PTCy). Mice receiving leukemia, HCT, and PTCy all died of leukemia when receiving non-transduced in vitro expanded T cells, but cleared leukemia when receiving CAR T cells [Figure 2A]; this effect occurred without clinical or histopathologic GVHD or toxicity. Leukemia relapse did occur in some CAR-treated mice and was more frequent in mice treated with PTCy (∼25% of mice). Relapse events after PTCy were associated with CD19+ extramedullary tumors but partial loss of CD19 in hematopoietic organs. Measurable CAR T cells were found but had increased expression of co-inhibitory molecules, suggesting that CAR T-cell exhaustion and leukemia antigen loss may be two mechanisms of escape. Attempts to assess the impact of having both CAR and allogeneic T cells able to treat the leukemia (to mirror the intended clinical use) in a C3H→B6D2F1 HCT model were limited by permanent clearance of leukemia by the allogeneic response alone.We also explored the impact of PTCy on GVT immunity by giving CAR T cells on day 0 prior to PTCy [Figure 1B]; leukemia was cleared by mice despite PTCy and did not relapse [Figure 2B]. CAR T cells persisted after PTCy and actually increased in numbers in the bone marrow from days +3 to +7. There was no increase of GVHD in CAR-treated mice clinically or histopathologically.Our data suggest that CAR T cells can be safely and effectively administered either prior to or after PTCy, permitting an anti-tumor response without GVHD. Furthermore, these studies show that tumor-specific T cells survive PTCy, consistent with our recent findings that alloreactive T cells persist after PTCy. Anti-CD19 CAR T cells can induce remissions in many patients. Yet, relapses can occur and so some patients may proceed to HCT either prior to or after relapse to try to achieve long-term cure. Even so, relapse limits the success of HCT. We recently showed that the suppressive mechanisms induced immediately after PTCy prevent new donor T cells from causing GVHD. Thus, we hypothesized that donor anti-CD19 CAR T cells given early after PTCy would not cause GVHD but may retain an anti-tumor effect; the premise is that the CAR T cells would induce an initial deep remission, while the polyclonal graft-versus-tumor (GVT) T-cell response would ensure long-term cure. To test this approach, we modified our T-cell-replete murine MHC-haploidentical HCT model (B6C3F1→B6D2F1) by giving 1 × 106 E2a-PBx leukemia cells 7 days prior to lethal irradiation and HCT [Figure 1A]. Importantly, E2a-PBx, as a pre-B-cell ALL cell line on a B6 background, is syngeneic with host and donor; thus, in this model only anti-CD19 CAR T cells can treat the leukemia (there is no allo response against it). T-cell-enriched B6C3F1 donor splenocytes were stimulated with anti-CD3/CD28 beads, transduced with an anti-CD19 CAR vector with a CD28 costimulatory domain, and infused at 1 × 106 transduced T cells per mouse on day +5 (24 hours after PTCy). Mice receiving leukemia, HCT, and PTCy all died of leukemia when receiving non-transduced in vitro expanded T cells, but cleared leukemia when receiving CAR T cells [Figure 2A]; this effect occurred without clinical or histopathologic GVHD or toxicity. Leukemia relapse did occur in some CAR-treated mice and was more frequent in mice treated with PTCy (∼25% of mice). Relapse events after PTCy were associated with CD19+ extramedullary tumors but partial loss of CD19 in hematopoietic organs. Measurable CAR T cells were found but had increased expression of co-inhibitory molecules, suggesting that CAR T-cell exhaustion and leukemia antigen loss may be two mechanisms of escape. Attempts to assess the impact of having both CAR and allogeneic T cells able to treat the leukemia (to mirror the intended clinical use) in a C3H→B6D2F1 HCT model were limited by permanent clearance of leukemia by the allogeneic response alone. We also explored the impact of PTCy on GVT immunity by giving CAR T cells on day 0 prior to PTCy [Figure 1B]; leukemia was cleared by mice despite PTCy and did not relapse [Figure 2B]. CAR T cells persisted after PTCy and actually increased in numbers in the bone marrow from days +3 to +7. There was no increase of GVHD in CAR-treated mice clinically or histopathologically. Our data suggest that CAR T cells can be safely and effectively administered either prior to or after PTCy, permitting an anti-tumor response without GVHD. Furthermore, these studies show that tumor-specific T cells survive PTCy, consistent with our recent findings that alloreactive T cells persist after PTCy. Figure 1, Figure 2.Figure 2CAR T cells given before or after PTCy maintain anti-tumor efficacy. Using CAR T cells integrated with the B6C3F1–>B6D2F1 HCT model as in Figure 1, CAR T cells were given on (A) day +5 or (B) day 0. TCD BM= T-cell-depleted bone marrow, Splen=splenocytes.View Large Image Figure ViewerDownload Hi-res image Download (PPT)