Enhanced Cytokine Release Research Articles

Abstract 12: TCR-gated control of costimulatory switch protein (CSP) activation in rTCR-T cells expressing PD1-41BB

Abstract Chimeric antigen receptor (CAR-)/T cell receptor (TCR-)T-based immunotherapies have shown promising results in the treatment of patients suffering from hematological malignancies. However, targeting solid cancer patients with CAR-T/TCR-T cells represents a more challenging approach, due to various factors present within the hostile tumor micro-environment (TME), inhibiting T cell functionality and leading to T cell exhaustion. Programmed cell death ligand-1 (PD-L1) is expressed on tumor cells as well as various cell types within the TME and binds to programmed cell death protein 1 (PD-1) expressed on the surface of activated T cells, thereby inducing T cell exhaustion and mediating tumor immune escape. To impair this inhibitory mechanism and improve TCR-T cell efficacy for the treatment of solid tumors, we use the chimeric costimulatory switch protein (CSP) PD1-41BB that turns the inhibitory signal mediated via the PD-1-PD-L1 axis into a costimulatory one. The CSP-mediated costimulatory signal is TCR-gated, whereby costimulation can only occur if a specific peptide-HLA complex present on a target cell interacts with a recombinant TCR expressed in T cells. In vitro co-cultures of rTCR-T cells expressing the CSP (CSP rTCR-T cells) and antigen-negative or -positive cell lines carrying or lacking PD-L1 demonstrated that CSP rTCR-T cells display functionality only if the respective target antigen is expressed in target cells, but not in the absence of target antigen. PD-L1-positive, antigen-negative tumor cells are not lysed by CSP rTCR-T cells, not even in the presence of antigen-positive tumor cells, showing gated functionality and a favorable safety profile of CSP-armored TCR-T cells. CSP rTCR-T cells displayed enhanced cytokine release and killing after encounter of PD-L1-positive tumor cell lines, compared to tumor cells lacking PD-L1, and showed the capacity to recognize and lyse a large panel of tumor cell lines expressing various levels of HLA-A*02, target antigen and PD-L1 in vitro. 3D spheroids derived from PD-L1-positive, target antigen-positive tumor cell lines mimicking cell-cell interactions within a solid tumor mass efficiently triggered CSP rTCR-T cells, leading to specific IFN-γ release and lysis of tumor spheroids. Furthermore, CSP rTCR-T cells showed enhanced proliferative capacity and expansion in co-cultures with PD-L1-positive tumor cells, compared to tumor cells lacking PD-L1 expression. Importantly, various target antigen-negative healthy cell types expressing or lacking PD-L1 and representing different vital organs were not cross-recognized by CSP rTCR-T cells in vitro. Our preclinical in vitro assessments of CSP rTCR-T cell functionality and safety proved their superior and gated functionality as well as favorable safety profile, showing the great potential for the treatment of cancer patients suffering from advanced solid tumor malignancies. Citation Format: Maja Buerdek, Petra U. Prinz, Kathrin Mutze, Andrea Coluccio, Stefanie Tippmer, Miriam Bosch, Giulia Longinotti, Mario Catarinella, Kathrin Davari, Christiane Geiger, Barbara Loesch, Kirsty Crame, Dolores J. Schendel. TCR-gated control of costimulatory switch protein (CSP) activation in rTCR-T cells expressing PD1-41BB [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 12.

Tumor nano-lysate activates dendritic cells to evoke a preventative immune response

A tumor nano-lysate “TNL” vaccine comprised of sonicated 4 T1 cells was developed, characterized and implemented for the prevention of triple-negative breast cancer. This study aims to gain a better understanding of the immune response behind the success of the vaccine in vivo, through use of ex vivo and in vivo assays. Here, we analyze the activation of various immune cells isolated from healthy mouse spleens and find that antigen-presenting cells (APCs) such as dendritic cells (DCs) are being activated following 24 h incubation with 1:10 mg TNL/mg splenocytes. These cells were further explored to determine the pathway by which activation is occurring, and it was observed that TNL are phagocytosed by DCs to activate NF-kB and c-Fos pathways, resulting in enhanced cytokine release after 24 h. An in vivo temporal analysis was performed in mice to understand the immune response at 1, 3, 7 and 10 days after one 100 μL dose of TNL consisting of 105 sonicated 4 T1 cells via cardiac puncture and splenocyte and peripheral blood mononuclear cell (PBMC) analysis. Changes were observed for up to one week. A multiple dose study was performed comparing mice that were vaccinated with one dose of TNL administered every ten days for 3 doses total, as well as a PBS vehicle control. Survival for TNL-vaccinated mice was enhanced compared to the PBS control, and there was an average delay of 10 days in the onset of metastasis. The differences between the groups at the end of the study demonstrate the potential for TNL as a preventative therapeutic.

C5a enhances inflammation and chemotaxis of γδ T cells in malignant pleural effusion

BackgroundThe inhibitory effect of γδT17 cells on the formation of murine malignant pleural effusions (MPE) has been established. However, there is limited understanding regarding the phenotypic characterization of γδ T cells in MPE patients and their recruitment to the pleural cavity. MethodsWe quantified γδ T cell prevalence in pleural effusions and corresponding peripheral blood from malignant and benign patients using immunohistochemistry and flow cytometry. The expression of effector memory phenotype, stimulatory/inhibitory/chemokine receptors and cytokines on γδ T cells in MPE was analyzed using multicolor flow cytometry. The infiltration of γδ T cells in MPE was assessed through immunofluorescence, ELISA, flow cytometry and transwell migration assay. ResultsWe observed a significant infiltration of γδ T cells in MPE, surpassing the levels found in blood and benign pleural effusion. γδ T cells in MPE exhibited heightened expression of CD56 and an effector memory phenotype, while displaying lower levels of PD-1. Furthermore, γδ T cells in MPE showed higher levels of cytokines (IFN-γ, IL-17A and IL-22) and chemokine receptors (CCR2, CCR5 and CCR6). CCR2 expression was notably higher in the Vδ2 subtype compared to Vδ1 cells. Moreover, the complement C5a enhanced cytokine release by γδ T cells, upregulated CCR2 expression in Vδ2 subsets, and stimulated the production of chemokines (CCL2, CCL7 and CCL20) in MPE. In vitro utilizing CCR2 neutralising and C5aR antagonist significantly reduced the recruitment of γδ T cells. Conclusionsγδ T cells infiltrate MPE by overexpressing CCR2 and exhibit hightened inflammation, which is further augmented by C5a.

Analysis of SARS-CoV-2 isolates, namely the Wuhan strain, Delta variant, and Omicron variant, identifies differential immune profiles.

There is an urgent need to better understand the impact of different severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants on immune response and disease dynamics to facilitate better intervention strategies. Here, we show that SARS-CoV-2 variants differentially affect host immune responses. The magnitude and quantity of cytokines and chemokines were comparable in those infected with the Wuhan strain and the Delta variant. However, individuals infected with the Omicron variant had significantly lower levels of these mediators. We also found an elevation of plasma galectins (Gal-3, Gal-8, and Gal-9) in infected individuals, in particular, in those with the original strain. Soluble galectins exert a proinflammatory role in COVID-19 pathogenesis. This was illustrated by their correlation with the plasma levels of sCD14, sCD163, enhanced TNF-α/IL-6 secretion, and increased SARS-CoV-2 infectivity in vitro. Moreover, we observed enhanced CD4+ and CD8+ T cell activation in Wuhan strain-infected individuals. Surprisingly, there was a more pronounced T cell activation in those infected with the Omicron in comparison to the Delta variant. In line with T cell activation status, we observed a more pronounced expansion of T cells expressing different co-inhibitory receptors in patients infected with the Wuhan strain, followed by the Omicron and Delta variants. Individuals infected with the Wuhan strain or the Omicron variant had a similar pattern of plasma soluble immune checkpoints. Our results imply that a milder innate immune response might be beneficial and protective in those infected with the Omicron variant. Our results provide a novel insight into the differential impact of SARS-CoV-2 variants on host immunity. IMPORTANCE There is a need to better understand how different SARS-CoV-2 variants influence the immune system and disease dynamics to facilitate the development of better vaccines and therapies. We compared immune responses in 140 SARS-CoV-2-infected individuals with the Wuhan strain, the Delta variant, or the Omicron variant. All these patients were admitted to the intensive care unit and were SARS-CoV-2 vaccination naïve. We found that SARS-CoV-2 variants differentially affect the host immune response. This was done by measuring soluble biomarkers in their plasma and examining different immune cells. Overall, we found that the magnitude of cytokine storm in individuals infected with the Wuhan strain or the Delta variant was greater than in those infected with the Omicron variant. In light of enhanced cytokine release syndrome in individuals infected with the Wuhan strain or the Delta variant, we believe that a milder innate immune response might be beneficial and protective in those infected with the Omicron variant.

SYST-28 FUNCTIONAL MAPPING REVEALS WIDESPREAD REMODELLING AND A NOVEL TARGETABLE PTP4A2-ROBO1 SIGNALING AXIS AT GLIOBLASTOMA RECURRENCE

Abstract Resistance to genotoxic therapies and tumor recurrence are hallmarks of glioblastoma (GBM), an aggressive brain tumor. Here, we explore the functional drivers of post-treatment recurrent GBM. By conducting genome-wide CRISPR-Cas9 screens in patient-derived GBM models, we uncover distinct genetic dependencies in recurrent tumor cells that were absent in their patient-matched primary predecessors, accompanied by increased mutational burden and differential transcript and protein expression. These analyses map a multilayered genetic response to drive tumor recurrence, identifying protein tyrosine phosphatase 4A2 (PTP4A2) as a novel modulator of self-renewal, proliferation and tumorigenicity at GBM recurrence. Mechanistically, genetic perturbation or small molecule inhibition of PTP4A2 represses axon guidance activity through a dephosphorylation axis with roundabout guidance receptor 1 (ROBO1), exploiting a functional dependency on ROBO signaling. Roundabout guidance receptor 1 (ROBO1) protein is involved in axonal guidance during neurodevelopment and aberrant ROBO1 signaling axis is associated with higher tumorigenicity in GBM. ROBO1 was highly expressed on the surface of malignant and treatment-refractory brain tumor initiating cells (BTICs) in rGBM, brain metastasis (BM) and medulloblastoma (MB). Importantly, engineered anti-ROBO1 single-domain antibodies also mimic the effects of PTP4A2 inhibition. We therefore developed and validated second-generation CAR-T cells against ROBO1, which demonstrated upregulation of activation markers, enhanced cytokine release, markedly increased proliferation, and induction of potent and specific tumor cell death compared to untransduced cells in all the three brain cancers. These findings were further validated in vivo in rGBM, MB and BM models, where ROBO1 CAR-T cells showed significant reduction in tumor burden and increase in survival of treated mice. We conclude that functional reprogramming drives tumorigenicity and dependence on a multi-targetable PTP4A2-ROBO1 signaling axis at GBM recurrence.

ROBO1 CAR-T CELL THERAPY FOR TREATMENT REFRACTORY BRAIN CANCER

Abstract BTFC travel award recipient Glioblastoma (GBM) is the most common malignant adult brain tumor, with a dismal prognosis. Despite the multimodal treatment regimen, tumor recurrence is inevitable, and no standardized treatment exists for recurrent glioblastoma (rGBM). Neurodevelopment signaling pathways are often hijacked during tumor progression. Roundabout guidance receptor 1 (ROBO1) protein is involved in axonal guidance during neurodevelopment. Our preliminary findings implicated aberrant ROBO1 signaling axis to be associated with higher tumorigenecity in GBM, making it a functionally relevant therapeutic target. Moreover, ROBO1 was highly expressed on the surface of malignant and treatment-refractory brain tumor initiating cells (BTICs) in rGBM, brain metastasis (BM) and medulloblastoma (MB), prompting the development of an anti-ROBO1 CAR-T cell therapy. Here we report the development and validation of second-generation CAR-T cells using a single-domain antibody targeting ROBO1 expressing BTICs across three different brain tumors. We validated anti-ROBO1 CAR-T cells in-vitro and in-vivo using our established patient derived tumor models. In-vitro studies demonstrated upregulation of activation markers, enhanced cytokine release, markedly increased proliferation, and induction of potent and specific tumor cell death with ROBO1 CAR-T cells as compared to untransduced cells (UT) in all the three cancers. These findings were further validated in-vivo using 3 different BTIC lines one each from rGBM, MB and BM. ROBO1 CAR-T showed significant reduction in tumor burden and significant increase in survival of the mice treated with ROBO1 CAR-T cells in all the three cohorts. Thus, targeting ROBO1 could be a therapeutically tractable strategy for treating rGBM as well as other brain malignancies.

Human alveolar macrophages do not rely on glucose metabolism upon activation by lipopolysaccharide

Most macrophages generate energy to mount an inflammatory cytokine response by increased glucose metabolism through intracellular glycolysis. Previous studies have suggested that alveolar macrophages (AMs), which reside in a glucose-poor natural environment, are less capable to utilize glycolysis and instead rely on other substrates to fuel oxidative phosphorylation (OXPHOS) for energy supply. At present, it is not known whether AMs are capable to use glucose metabolism to produce cytokines when other metabolic options are blocked. Here, we studied human AMs retrieved by bronchoalveolar lavage from healthy subjects, and examined their glucose metabolism in response to activation by the gram-negative bacterial component lipopolysaccharide (LPS) ex vivo. The immunological and metabolic responses of AMs were compared to those of cultured blood monocyte-derived macrophages (MDMs) from the same subjects. LPS stimulation enhanced cytokine release by both AMs and MDMs, which was associated with increased lactate release by MDMs (reflecting glycolysis), but not by AMs. In agreement, LPS induced higher mRNA expression of multiple glycolytic regulators in MDMs, but not in AMs. Flux analyses of [13C]-glucose revealed no differences in [13C]-incorporation in glucose metabolism intermediates in AMs. Inhibition of OXPHOS by oligomycin strongly reduced LPS-induced cytokine production by AMs, but not by MDMs. Collectively, these results indicate that human AMs, in contrast to MDMs, do not use glucose metabolism during LPS-induced activation and fully rely on OXPHOS for cytokine production.

T-Cell Replete Peripheral Blood Haploidentical Transplant Using Immunogenic Dose of Cyclophosphamide with Fludarabine and Melphalan Results in Enhanced Cytokine Release Syndrome (CRS) with Robust Immune Recovery and Low Relapse Rates

The immune suppressive properties of high-dose Cyclophosphamide are well known; however, a growing literature has described its immune stimulatory properties when applied in low dose, mediated through proliferation of effector T cells and abrogation of regulatory T cells. In order to exploit the immunogenic property of low dose Cyclophosphamide we hypothesized that its addition to Melphalan and Fludarabine in the absence of the immune suppressive effect of radiation would result in robust effector T cells proliferation through enhanced cytokine release and antigen alloreactivity. (1)We examined the clinical outcomes and immune recovery of 14 consecutive haploidentical transplant patients with high risk hematological malignancies who received their first allogeneic transplant between 7/8/2015 and 1/10/2017. The immune recovery markers were assessed at day 60, 120 and 180 post-transplant and included absolute lymphocyte count (ALC), CD3+ CD4+, CD8+, activated T cells (CD3+HLA Dr+), CD19+ and NK cells (CD56/16+). Patients and graft characteristics are as shown in Table-1. The median duration of follow up for survivors is 15 months (range 6-25); and all but 3 patients have completed 1 year of follow up. The conditioning regimen was low dose Cyclophosphamide 14.5 mg/m2 on day -6 & day -5, Fludarabine 30 mg/m2 on days -6 to Days -2 and Melphalan 70 mg/m2 on day -3 & -2. Graft versus host disease (GVHD) prophylaxis was post-transplant Cyclophosphamide 50 mg/Kg on day +3 and day +4 along with Tacrolimus and Mycophenolate starting at day +5. Tacrolimus continued until day +180 (Target level 5-8) and Mycophenolate continued until Day+30. Chimerism studies were performed at day +30 and +100 post-transplant by variable number tandem repeat PCR analysis of peripheral blood & bone marrow. All patients engrafted except one patient who died at day 25 prior to engraftment. Median time to neutrophil engraftment was 21 days (range 12-57). All of the evaluable 13 patients for engraftment achieved 100% donor chimerism in bone marrow (unsorted) and peripheral blood (CD3, CD33 & CD 56) by day 100. The day100 and one year overall survival were 92.8% and 85.7% respectively. Only 1/14 patients has relapsed, and the cumulative incidence of relapse is 7.74% at 1 year post- transplant. Similarly, the cumulative incidence of treatment related mortality (TRM) is 7% at 1 year post transplant. Overall 3 patients have died (2 Patients TRM and 1 Patient Relapse related mortality). TRM was related to severe CRS grade 4 in one patient at day +25 and pneumonia at day +540 in another patient. Acute GVHD grade 2-4 developed in 4 patients of whom 2 had grade 3-4. Chronic GVHD developed in 8 patients (57%) but was extensive in only 3 patients. All patients developed CRS grade as previously proposed by Lee et al.Grade 4 CRS developed in 4/14 patients requiring ventilator support and dialysis, which led to one death during the first year post-transplant at day 25. Grade 1 CRS developed in 7 patients while 3 patients developed grade 2 CRS. Two of 4 patients with grade 4 CRS had delayed neutrophilic engraftment at day +34 and +57, and both achieved 100% donor chimerism by day 100 in bone marrow and peripheral blood. Post-Transplant immune recovery is shown in Fig-1. There was significant early recovery at day +60 in all T cell subsets. This recovery was most pronounced in activated T cells, giving support to our hypothesis. While we have observed a progressive reduction in the median number of early activated T cells at day +120 & +180, the number of helper T cells (CD4) and NK cells (CD56/16) maintained its early immune recovery. Additionally, we found more robust NK cell recovery in patients with grade 2-4 CRS (#6) compared to patients with less severe CRS grade 1 (#7). In contrast, CD4 recovery was more robust in patients with less severe CRS (Table 2)Conclusion:Fludarabine/Melphalan/Cyclophosphamide as a conditioning regimen prior to T-cell replete peripheral blood haploidentical transplant results in early robust immune recovery and may have the potential to decrease the risk of relapse in high risk hematological malignancies. The observed robust NK recovery in particular in patients with more severe CRS may be attributed to the lower risk of relapse. Further confirmation in a larger cohort of patients is needed to confirm these results.(1) Madondo MT, Quinn M, Plebanski M. Low dose cyclophosphamide: mechanisms of T cell modulation. Cancer Treat Rev 2016; 42: 3-9. [Display omitted] DisclosuresChandler:Vector Oncology: Consultancy; BMS: Consultancy; Janssen: Consultancy, Speakers Bureau.

Dysregulation of Connexin Expression Plays a Pivotal Role in Psoriasis.

Background: Psoriasis, a chronic inflammatory disease affecting 2–3% of the population, is characterised by epidermal hyperplasia, a sustained pro-inflammatory immune response and is primarily a T-cell driven disease. Previous work determined that Connexin26 is upregulated in psoriatic tissue. This study extends these findings. Methods: Biopsies spanning psoriatic plaque (PP) and non-involved tissue (PN) were compared to normal controls (NN). RNA was isolated and subject to real-time PCR to determine gene expression profiles, including GJB2/CX26, GJB6/CX30 and GJA1/CX43. Protein expression was assessed by immunohistochemistry. Keratinocytes and fibroblasts were isolated and used in 3D organotypic models. The pro-inflammatory status of fibroblasts and 3D cultures was assessed via ELISA and RnD cytokine arrays in the presence or absence of the connexin channel blocker Gap27. Results: Connexin26 expression is dramatically enhanced at both transcriptional and translational level in PP and PN tissue compared to NN (>100x). In contrast, CX43 gene expression is not affected, but the protein is post-translationally modified and accumulates in psoriatic tissue. Fibroblasts isolated from psoriatic patients had a higher inflammatory index than normal fibroblasts and drove normal keratinocytes to adopt a “psoriatic phenotype” in a 3D-organotypic model. Exposure of normal fibroblasts to the pro-inflammatory mediator peptidoglycan, isolated from Staphylococcus aureus enhanced cytokine release, an event protected by Gap27. Conclusion: dysregulation of the connexin26:43 expression profile in psoriatic tissue contributes to an imbalance of cellular events. Inhibition of connexin signalling reduces pro-inflammatory events and may hold therapeutic benefit.

Rhinovirus impairs the immune response of alveolar macrophages to facilitate Streptococcus pneumonia infection.

Pneumonia is one important cause of mortality in neonates. However, the mechanism remains still unclear. Viral infection greatly enhances the morbidity of Streptococcus pneumonia. In this study, we tried to understand how human rhinovirus (HRV) would accelerate Streptococcus pneumonia infection. Alveolar macrophages (AMs) were isolated from neonatal mice. Cytokine concentrations were detected using ELISA. The phagocytosis of Streptococcus pneumonia by AMs was indicated by immunofluorescence. Toll-like receptor 3 (TLR3) and CD68 expression in isolated AMs or infected mice were determined by western blot or immunochemistry. The mortality was explored using Kaplan-Meier analysis. HRV infection enhanced cytokine release by AMs, and decreased Streptococcus pneumonia-induced TNF-α, IL-1β and IL-6 release by AMs, while has no influence on IL-10 release. HRV infection impaired phagocytosis of Streptococcus pneumonia in AMs. Mechanically, HRV infection up-regulated TLR3 expression in AMs. Mortality and pneumococcal burden decreased in TLR3-/- neonatal mice and inflammation and phagocytosis were restored in TLR3-/- AMs. Neonatal rhinovirus impairs the immune response of alveolar macrophages to facilitate Streptococcus pneumonia infection via TLR3 signaling.

Interdomain spacing and spatial configuration drive the potency of IgG-[L]-scFv T cell bispecific antibodies.

T cell-bispecific antibodies (BsAbs) couple cytotoxic T lymphocytes to tumor cells, inducing their destruction. Although there are more than 60 classes of BsAbs in development, the relative importance of parameters such as interdomain spacing or spatial configuration is largely unknown. Here, we dissected a symmetric dual bivalent BsAb platform (IgG-[L]-scFv: antitumor IgG with anti-CD3 scFv fused to the light chains) to explore the importance of valency and spatial configuration for BsAb-induced T cell cytotoxicity. Our results revealed that placing tumor and T cell binding domains on the same side of a BsAb (cis-configuration) elicited substantially stronger antitumor activity, in vitro and in vivo, compared to positioning them on opposite sides (trans-configuration). Moreover, using two cis-modules in the same BsAb further improved cytotoxicity (up to 2000-fold). In addition, separating antigen-binding components with a single Ig domain (CL) markedly enhanced cytokine release and in vivo tumor responses compared to smaller (G4S1) or larger (CH1-CH2-CH3) spacers. These findings provide guidelines for improving BsAb function and highlight the importance of spatial configuration and dual bivalency as development parameters.

Neuropeptide regulation of secretion and inflammation in human airway gland serous cells.

Airway submucosal gland serous cells are sites of expression of the cystic fibrosis transmembrane conductance regulator (CFTR) and are important for fluid secretion in conducting airways. To elucidate how neuropeptides regulate serous cells, we tested if human nasal turbinate serous cells secrete bicarbonate (HCO3 -), important for mucus polymerisation and antimicrobial peptide function, during stimulation with cAMP-elevating vasoactive intestinal peptide (VIP) and if this requires CFTR. Serous cells stimulated with VIP exhibited a ∼15-20% cAMP-dependent decrease in cell volume and a ∼0.15 unit decrease in intracellular pH (pHi), reflecting activation of Cl- and HCO3 - secretion, respectively. HCO3 - secretion was directly dependent on CFTR and was absent in cells from CF patients. In contrast, neuropeptide Y (NPY) reduced VIP-evoked cAMP increases, CFTR activation, and Cl-/HCO3 - secretion. Culture of primary serous cells in a model that maintained a serous phenotype confirmed the activating and inhibiting effects of VIP and NPY, respectively, on fluid and HCO3 - secretion. Moreover, VIP enhanced antimicrobial peptide secretion and antimicrobial efficacy of secretions while NPY reduced antimicrobial efficacy. In contrast, NPY enhanced cytokine release while VIP reduced cytokine release through a mechanism requiring CFTR. As levels of VIP and NPY are up-regulated in diseases like allergy, asthma, and chronic rhinosinusitis, the balance of these two peptides in the airway may control mucus rheology and inflammatory responses in serous cells. Furthermore, the loss of CFTR conductance in serous cells may contribute to CF pathophysiology by increasing serous cells inflammatory responses in addition to directly impairing Cl- and HCO3 - secretion.

T-Cell Replete Peripheral Blood Haploidentical Transplant Using Immunogenic Dose of Cyclophosphamide with Fludarabine/Melphalan Results in Enhanced Cytokine Release Syndrome (CRS) with Robust Immune Recovery and Low Relapse Rate

In contrast to Chimeric-Antigen Receptor (CAR) T-Cell therapy, there is limited literature if any that has addressed the impact of intensity of lymphodepletion rather than myeloablation on relapse after HID HCT. We hypothesize d that enhanced lympho-depletion would create a host immunogenic microenvironment that would enhance donor T cell expansion and alloreactivity after HID HCT. We utilized an intensified lympho-depleting conditioning regimen with Fludarabine (Flu) 150 mg/m2, Melphalan (Mel) 140 mg/m2 and Cyclophosphamide (CY) 29 mg/m2 prior to HID HCT to treat 15 consecutive patients with HM between July 2015 and June 2017. The intensity of lymphodepletion was confirmed in 12 evaluable patients at day (0) by complete absence of lymphocytes in peripheral smear. In contrast, myeloid elements were detected in 7/12 patients suggesting more intense lympho-depletion than myeloablation. All Patients received GVHD prophylaxis with Tacrolimus/Mycophenolate with post-transplant Cyclophosphamide according to Hopkins regimen. Patient characteristics are shown in Table 1. Eight patients had relapsed/refractory disease at time of transplant. The median duration of follow up is 29 months (range 13-37). All patients engrafted and achieved 100% Chimerism in BM and PB by day 100 except for 1 patient who died at day +25 prior to engraftment. Median time to neutrophil engraftment was 21 days (range 12-57). All evaluable patients with relapsed/refractory disease (7/8) achieved complete remission post-transplant. The day 100, 1-year & 2 year overall survival was 93%, 73% & 66% respectively. We observed very low cumulative incidence of relapse of 7% at 2 years post-HCT (only 1/15 patients). Treatment related mortality (TRM) was 6%, 20% and 27% at day 100, 1 and 2 years respectively (4/15 patients). Severe Cytokine Release Syndrome (CRS), grade 4 by Lee Criteria occurred in 4 patients, all of them have died at day 25, 258, 288 and 540. Grade II-IV acute GVHD developed in 4/15 patients (27%), 2 of whom had grade III-IV by day +180. Chronic extensive GVHD developed in 7 patients (46%). At 2 years, post HCT, only 1/7patients remain with extensive chronic GVHD requiring active treatment. Post -transplant immune reconstitution panels were obtained at day 60, 120, 180 and 360 as shown in Fig 1. We observed early recovery in all T-cell subsets at day 60 with activated T cells (CD3+, HLA-Dr+) being most pronounced. While there was a steady decline of activated T cell, the NK cells and CD4 maintained their early recovery through the first year. There was initial decline in the number of B cells at day 120 which gradually recovered by 1 year. Conclusion: Enhanced lymphodepletion prior to peripheral blood HID HCT may enhance early T cell proliferation, alloreactivity and immune reconstitution. Future strategies directed at improving management of severe CRS are needed to harness the benefits of the observed low relapse rate.

Contribution of p62/SQSTM1 to PDGF-BB-induced myofibroblast-like phenotypic transition in vascular smooth muscle cells lacking Smpd1 gene

Accumulating evidence indicates a critical role of autophagy in regulating vascular smooth muscle cell (SMC) homeostasis in atherogenesis. However, little is known about the modulatory role of autophagy in PDGF-BB-induced SMC transition towards the synthetic phenotype and extracellular matrix remodeling. We recently demonstrated that acid sphingomyelinase (ASM, encoded by Smpd1 gene) controls autophagy maturation in coronary arterial SMCs. Here, we demonstrate that PDGF-BB stimulation causes a myofibroblast-like non-canonical synthetic phenotype transition in Smpd1−/− SMCs. These non-canonical phenotypic changes induced by PDGF-BB in Smpd1−/− SMCs were characterized by increased expression of fibroblast-specific protein (FSP-1), massive deposition of collagen type I, decreased cell size, elevated inflammatory status with enhanced cytokine release and adhesion molecule expression. Mechanistically, PDGF-BB induces prolonged Akt activation that causes decreased autophagosome biogenesis and thereby exaggerates p62/SQSTM1 accumulation in Smpd1−/− SMCs. More importantly, Akt inhibition or p62/SQSTM1 gene silencing attenuates PDGF-BB-induced phenotypic changes in Smpd1−/− SMCs. This first demonstration of a p62/SQSTM1-dependent myofibroblast-like phenotypic transition in Smpd1−/− SMCs suggests that ASM-mediated autophagy pathway contributes to maintaining the arterial smooth muscle homeostasis in situation of vascular remodeling during atherosclerosis.

Regulation of fetal hemoglobin expression during hematopoietic stem cell development and its importance in bone metabolism and osteoporosis

We have shown that an altered tissue redox environment in mice lacking either murine beta Hemoglobin major (HgbβmaKO) or minor (HgbβmiKO) regulates inflammation. The REDOX environment in marrow stem cell niches also control differentiation pathways. We investigated osteoclastogenesis (OC)/osteoblastogenesis (OB), in bone cultures derived from untreated or FSLE-treated WT, HgbβmaKO or HgbβmiKO mice.Marrow mesenchymal cells from 10d pre-cultures were incubated on an osteogenic matrix for 21d prior to analysis of inflammatory cytokine release into culture supernatants, and relative OC:OB using (TRAP:BSP, RANKL:OPG) mRNA expression ratios and TRAP or Von Kossa staining. Cells from WT and HgbβmaKO mice show decreased IL-1β,TNFα and IL-6 production and enhanced osteoblastogenesis with altered mRNA expression ratios and increased bone nodules (Von Kossa staining) in vitro after in vivo stimulation of mRNA expression of fetal Hgb genes (Hgbε and Hgbβmi) by a fetal liver extract (FSLE). Marrow from HgbβmiKO showed enhanced cytokine release and preferential enhanced osteoclastogenesis relative to similar cells from WT or HgbβmaKO mice, with no increased osteoblastogenesis after mouse treatment with FSLE. Pre-treatment of WT or HgbβmaKO, but not HgbβmiKO mice, with other molecules (rapamycin; hydroxyurea) which increase expression of fetal Hgb genes also augmented osteoblastogenesis and decreased cytokine production in cells differentiating in vitro. Infusion of rabbit anti- Hgbε or anti- Hgbβmi, but not anti-Hgbα or anti- Hgbβma into WT mice from day 13 gestation for 3 weeks led to attenuated osteoblastogenesis in cultured cells. We conclude that increased fetal hemoglobin expression, or use of agents which improve fetal hemoglobin expression, increases osteoblast bone differentiation in association with decreased inflammatory cytokine release.

510 - T-Cell Replete Peripheral Blood Haploidentical Donor Transplant (HID) Using Immunogenic Dose of Cyclophosphamide (Cy) with Fludarabine (Flu)/Melphalan (Mel) Results in Enhanced Cytokine Release Syndrome (CRS) with Robust Immune Recovery & Low Relapse Rate

510 - T-Cell Replete Peripheral Blood Haploidentical Donor Transplant (HID) Using Immunogenic Dose of Cyclophosphamide (Cy) with Fludarabine (Flu)/Melphalan (Mel) Results in Enhanced Cytokine Release Syndrome (CRS) with Robust Immune Recovery & Low Relapse Rate

CD169+ Macrophages Restrain Systemic Inflammation Induced by Staphylococcus aureus Enterotoxin A Lung Response

Abstract Alveolar macrophages (AMs) are considered the first line of defense in the airways. Exposure to harmful substances and certain infections can lead to dysfunction or depletion of AMs. Importantly, these conditions have been associated with increased risk of sepsis and acute lung injury. Staphylococcus aureus enterotoxins are superantigens that induce oligoclonal activation of T cells and a robust cytokine release, leading to systemic inflammatory response and tissue injury. In this study we investigated the relationship between S. aureus enterotoxins and AMs. Following inhalation, S. aureus enterotoxin was preferentially bound to AMs and MHC class II was not required. Furthermore, the enterotoxin was internalized and its presence in the cells decreased by 24 h after exposure. Ablation of AMs in CD169–diphtheria toxin receptor mice was associated with increased activation of enterotoxin-specific T cells and enhanced cytokine release into circulation. Thus, conditions causing depletion of AMs may increase the risk of S. aureus enterotoxin–induced diseases.

205 - Immune Reconstitution and Outcomes after Peripheral Blood Haploidentical Transplantation with Novel Conditioning and Post-Transplant Cyclophosphomide (PTCY)

The outcomes after Haploidentical HCT with Flu-CY-TBI and PTCY using BM as a graft source have been associated with high relapse and delayed immune recovery. Additionally, there is limited knowledge regarding immune reconstitution post haploidentical HCT with the use of mobilized peripheral blood. We hypothesized that the immunogenic properties of pre-transplant low dose Cyclophosphamide along with immunomodulatory effects of Melphalan would enhance early post-transplant engraftment and early peripheral T cell recovery through enhanced cytokine release and antigen alloreactivity. We examined the clinical outcomes and immune reconstitution recovery of 12 consecutive haploidentical HCT patients with high-risk hematological malignancies. Patients and Graft characteristics is as shown in Table 1. Conditioning regimen was low dose Cy 14.5 mg/m2 on day -6 & day −5, Flu 30 mg/m2 day −6 to Day −2 and Mel 70 mg/m2 on day −3 & −2. Graft versus host disease prophylaxis (GVHD) was PTCY 50 mg/Kg on day +3 and day +4 along with Tacrolimus and Mycophenolate starting at day +5. Immune reconstitution performed on days +60, +120 and +180 post HCT. All the patients treated according to an institutional protocol and records reviewed retrospectively after IRB approval.Table 1 All patients engrafted with median time to engraftment of 17 days (range 12-23). Chimerism studies revealed enhanced engraftment with 100% donor in bone marrow and peripheral blood at day 30 in all 12 patients. Post HCT immune recovery shown in Table 2. There was significant early recovery at day 60 of all T cell subsets, most pronounced in activated T cells. While we have observed a progressive reduction in the median number of early-activated T cells at day 120 & 180, the number CD4 and NK cells has improved (Figure 1).Table 2Table 2 With median duration of follow up of 323 days, range (123-451), the overall survival at day 100 and projected one year is 92% and 81% respectively. Only 2/12 had relapsed at 95 and 321 days post- transplant. Treatment related mortality (TRM) was limited to 1/12 patients. Acute GVHD grade 2-4 developed in six of 12 patients, two of whom were grade 3-4. Chronic GVHD has developed in four patients (1 serositis, 1 pericardial effusion and 2 nephrotic range proteinuria). CMV reactivation occurred in 9/12 patient with no CMV disease. Aspergillus antigen positivity in serum occurred in 4 /12 but only two of them developed clinical fungal infection. Conclusion: In this limited series of patients with high- risk hematological malignancies who underwent haploidentical HCT with Cy/Flu and Melphalan with PTCY, the regimen was well tolerated and resulted in effective disease control. The regimen has also demonstrated enhanced early engraftment and robust immune recovery in comparison to other studies, Table 3. The study provide unavailable knowledge about immune reconstitution post Haplo-identical HCT with PTCY with the use of mobilized peripheral blood.Table 3

A Genotyping and Phenotyping Study Concerning the Possible Effects of Some Inflammatory Cytokine Gene Polymorphisms on the Development of Coal Workers' Pneumoconiosis

Cytokines are important for playing a major role in several inflammatory reactions resulting in development of several diseases as well as Coal Worker’s Pneumoconiosis (CWP). Coal dust exposure stimulates inflammatory response leading to enhanced cytokine release from monocytes such as TNF-alpha and IL1. These released cytokines are the key points in the pathogenesis of CWP. The present study aimed to seek the cytokine gene profiles of Turkish coal workers by genotyping and phenotyping analysis of important CWP-related proinflammatory cytokines; TNF-alpha, IL1-alpha and IL1-beta. According to the genotyping results, TNFA –238 gene polymorphism was appeared to be a risk factor in development of CWP (OR=3.79) and regarding to the phenotyping analysis, both TNF-alpha and IL1 cytokine releases from the monocytes in CWP patients were enhanced significantly compared to the healthy workers. Therewithal, LPS and coal dust stimulated TNF-alpha release were higher significantly in allele 2 carriers than allele 1 carriers in both of the groups. These data propose that coal dust-induced TNF-alpha release from monocytes may be a valuable biomarker of CWP.

Pre-Transplant Low Dose Cyclophosphamide Enhances Engraftment and Immune Reconstitution in Haploidentical Hematopoietic Stem Cell Transplant (HCT) Recipients Receiving Total Body Radiation (TBI) Free Reduced Intensity Conditioning with Post- Transplant Cyclophosphamide (PTCY)

The outcomes after Haploidentical HCT with Fludarabine (Flu)/Cyclophosphamide (Cy)/TBI and PTCY as pioneered by the Hopkins group have been associated with higher risk of relapse and delayed immune reconstitution. Multiple groups have explored the potential benefits of more intensive conditioning regimens with or without TBI. However, none of these regimens to our knowledge has exploited the immunogenic properties of pre-transplant low dose Cy while avoiding the immune-suppressive effect of TBI. Additionally, there is limited knowledge regarding immune reconstitution post haploidentical HCT with the use of mobilized peripheral blood stem cells. Accordingly, we designed a protocol modification of the Hopkins regimen by replacing TBI with Melphalan (Mel) without adding Thiotepa in contrast to the MD Anderson regimen (Ciurea et al). We hypothesized that the immunogenic properties of pre-transplant low dose Cy along with the immunomodulatory effects of Mel would enhance early post-transplant engraftment and early peripheral T cell recovery through enhanced cytokine release and antigen alloreactivityWe examined the clinical outcomes and immune reconstitution recovery of 12 consecutive haploidentical HCT patients with high-risk hematological malignancies. Patient and graft characteristics are as shown in table-1. The conditioning regimen was low dose Cy 14.5 mg/m2 on day -6 & day -5, Flu 30 mg/m2 on days -6 to Days -2 and Mel 70 mg/m2 on day -3 & -2. Graft versus host disease prophylaxis (GVHD) was PTCY 50 mg/Kg on day +3 and day +4 along with Tacrolimus and Mycophenolate starting at day +5 as previously described by the Hopkins group. An immune reconstitution panel of absolute lymphocyte count (ALC), CD3, CD4, CD8, Activated T cell (CD3-HLA Dr + T cells) & NK cell (CD56/16) was performed by 4 color flow-cytometry on days +60, +120 and +180 post HCT. Chimerism studies were performed at day +30 and +100 post HCT by variable number tandem repeat PCR analysis of peripheral blood & bone marrow. All of the patients were treated according to an institutional protocol and records were reviewed retrospectively after IRB approval.All patients engrafted with a median time to engraftment of 17 days (range 12-23). Chimerism studies revealed enhanced engraftment with 100% donor in bone marrow (unsorted) and peripheral blood (CD3, CD33 & CD 56) at day +30 in all 12 patients. All patients with active disease at time of transplant (5 Pts) achieved complete remission at day +30 evaluation. Post HCT immune recovery is shown in table 2. There was significant early recovery at day + 60 of the median ALC and all T cell subsets. This recovery was most pronounced in activated T cells. While we have observed a progressive reduction in the median number of early activated T cells at day 120 & 180, the number of helper T cells (CD4) and NK cells (CD56/16) did not decline (Figure 1).With a median duration of follow up of 261 days (range 62-390), the overall survival at day 100 and projected one year survival is 96% and 81% respectively. Only one of 12 patients had relapsed. Acute GVHD grade 2-4 developed in six of 12 patients, two of whom were grade 3-4. Chronic GVHD developed in four patients (1 serositis, 1 pericardial effusion and 2 nephrotic range proteinuria). Cytokine storm developed in 5/12 patients after stem cell infusion and resolved after PTCY. BK cystitis developed in four patients but continuous bladder irrigation was only required in two patients. All cases of BK cystitis were transient and resolved with supportive measures. CMV reactivation occurred in 9/12 patients; no CMV disease or CMV mortality was observed. Aspergillus antigen positivity in serum occurred in 4 /12 but only two developed clinical fungal infection.Conclusion: In this limited series of patients with high- risk hematological malignancies who underwent haploidentical HCT with low dose Cy/Flu/Mel and PTCY, the regimen was well tolerated and resulted in effective disease control. The regimen has also demonstrated enhanced early engraftment and robust immune recovery in comparison to other studies, table 3. However, it is not clear if this enhanced immune recovery is related to the conditioning regimen modification or the use of mobilized stem cells. The results are intriguing and need further confirmation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.