Peptide Stimulation Research Articles

Dendritic cells to prevent cancer: Immune responses against neoantigens after dendritic cell vaccination of Lynch Syndrome patients

Abstract Lynch syndrome (LS) is an autosomal dominantly inherited syndrome caused by monoallelic germline aberrations affecting a DNA mismatch repair (MMR) gene, which leads to the development of microsatellite instability. Individuals with LS are 60–80% more prone to colorectal cancer (CRC). MMR deficiency in tumor DNA causes shifts in the translational reading frame resulting in the production of altered peptides called neoantigens. In a phase I/II clinical trial we vaccinated 20 healthy LS mutation carriers and 3 LS patients with surgically resected CRC with monocyte-derived dendritic cells (DC) loaded with neoantigens of TGF-βRII and caspase-5, and CEA. The objective was to analyze the presence and functionality of antigen specific CD8+ T-cells in peripheral blood and biopsies of post-treatment skin tests. Cell lines stably expressing the (neo)antigen were generated and used in calcein based killing assays with expanded (neo)antigen specific patient CD8+ T cells. The vaccination induced antigen specific T cell responses in 20/23 patients. Multifunctional antigen specific T-cells producing multiple cytokines and high amounts of interferon-γ upon peptide stimulation were detectable in peripheral blood. Moreover patient T cells of 3/4 patients tested demonstrated cytotoxicity against tumor cells presenting TGFβRII neoantigen, the T cell receptor repertoires of which had shared motifs in the clonotypes between patients as identified by Illumina MiSeq sequencing. Conclusion-DC vaccination with (neo)antigens is well tolerated in LS patients and induces functional (neo)antigen specific T cells capable of eliciting a cytotoxic immune response thus able to enhance antitumor immunity in a prophylactic and therapeutic setting.

Robust and prototypical immune responses towards COVID-19 BNT162b2 vaccines in Indigenous people

Abstract SARS-CoV-2 has led to >270 million infections and >5 million deaths globally. Indigenous people are disproportionately affected by infectious diseases, therefore also more susceptible to the COVID-19 pandemic. There are an estimated 476 million indigenous people globally, including an estimated 798,365 Aboriginal and Torres Strait Islander in Australia. With the high vulnerability to COVID-19, this knowledge is urgently needed to better protect indigenous populations. We evaluated a breadth of immune responses in indigenous (n=57) and non-indigenous (n=49) individuals after COVID-19 vaccination. We tested RBD antibodies, spike/RBD-probe-specific B cells, peptide stimulations with activation-induced marker (AIM) assay and intracellular cytokine staining. We found 22% and 34% seroconversion rates after 1st dose of BNT162b2 vaccine for Indigenous and non-indigenous individuals, respectively, which increased to 100% at 1-mth after 2nd dose for both groups. RBD-specific IgG levels in indigenous individuals at 1-mth after 2nd dose positively correlated with their body mass index. At 1-mth after the 2nd COVID-19 vaccination, CD4+ and CD8+ T cell responses via AIM expression and IFN-γ+TNF+ production was comparable between indigenous and non-indigenous individuals. We are also going to assess the longevity of antibodies and T cells. Therefore, COVID-19 vaccination induced similar immune responses in indigenous and non-indigenous individuals.

CD8 T cell responses to conserved DNAJB1-PRKACA fusion neoantigens in fibrolamellar carcinoma

Abstract Fibrolamellar carcinoma (FLC) is an often-lethal liver malignancy of adolescents and young adults. No systemic therapies are currently approved for FLC, so novel treatments are needed to improve patient outcomes. A promising therapeutic vulnerability in FLC is a highly-conserved gene fusion between DNAJB1 and PRKACA. The resultant fusion protein is identical in more than 90% of patients, making it a potentially ideal neoantigen target for T cell-based immunotherapy. The goal of our study was to test if DNAJB1-PRKACA neoantigens could elicit T cell responses and serve as targets for immunotherapy. Spatial transcriptomic analyses of FLC tumor tissue indicated that CD8 T cells could infiltrate FLC tumors, but expressed exhaustion markers such as TOX. Nevertheless, we expanded T cells from an FLC patient tumor ex vivo and detected T cells that produced IFNγ and TNFα in response to stimulation with a fusion neoantigen. We then used functional response after peptide stimulation or peptide-MHC tetramer staining to identify fusion-specific T cell receptors (TCRs) in both FLC patients and healthy donors. We expressed these TCRs in primary human T cells and found that cells expressing fusion-specific TCRs bound to their cognate tetramer and produced IFNγ, TNFα, and IL-2 in response to stimulation with their cognate peptide. TCR-expressing cells also specifically killed target cells presenting their cognate fusion peptide in vitro. Ongoing experiments will test if cells expressing these TCRs can control growth of fusion-expressing tumors in vivo. Together, these studies have defined the first reported fusion-specific T cell response in an FLC patient, as well as fusion-specific TCRs that hold promise for development in adoptive T cell therapies. Supported by grants from NIH (R01 AI136514, F31 CA254423) and The Mark Foundation for Cancer Research (Aspire Award)

CD8+ T cell impairment drives severe human metapneumovirus respiratory disease in aged mice.

Abstract Objective Human metapneumovirus (HMPV) is a leading cause of severe respiratory disease in children <5, adults >65, and the immunocompromised. CD8+ T lymphocytes are the primary cell responsible for clearing HMPV infection in the lung. We tested the hypothesis that CD8+ T cell impairment in the aged host drives severe respiratory infection and delayed viral clearance. Methods B6 aged 72–73wk mice and young 6–7wk mice were infected with 2×106 PFU HMPV. Seven days post infection, mice were euthanized and lung, bronchoalveolar lavage (BAL), spleen, and draining lymph nodes were collected for flow cytometry. Lung and BAL supernatant was collected for viral titer, histology, and cytokine measurement. Results Aged mice lost more weight, had delayed viral clearance, and increased clinical scores and lung histopathology. Aged mice produced fewer HMPV-tetramer+ cells in lung and BAL. Aged CD8+ T lymphocytes in BAL co-expressed inhibitory receptors PD-1, TIM-3, LAG-3, and 2B4. Aged CD8+ T lymphocytes also upregulated senescence markers, T-bet and EOMES. Upon ex vivo peptide stimulation, aged CD8+ T cells produced significantly less granzyme B compared to young CD8+ T cells. Adoptive transfer of young T cells into aged mice rescued this severe disease phenotype. Young CD8+ cells in the aged host produced more tetramer+ cells and granzyme B and had less senescent marker expression. Conclusion Aged mice exhibit more severe respiratory disease when infected with HMPV. Aged CD8+ T cells appear to be impaired and upregulate senescence markers that contribute to severe HMPV infection in the aged host. Supported by: NIH/NIAID 5R01 AI085062-09, John V. Williams, MD NIH T32GM008208 Medical Scientist Training Program, University of Pittsburgh School of Medicine

Examining effector functions of lung CD8+ tissue resident memory T cells in humans.

Abstract Due to their position in the lung tissue, CD8+ tissue resident memory T cells (TRM) act as sentinels of the respiratory tract that rapidly respond to, and mediate protection against, respiratory viruses. In mice, TRM have been shown to mediate protection at barrier sites by producing cytokines, chemokines, and performing cell lysis. In the lungs specifically, our lab has shown that influenza-specific CD8+ TRM rapidly produce IFNγ, but airway TRM are poorly cytolytic in mice. In humans, less is known about the effector functions of virus-specific CD8+ TRM in the lungs, and thus this study seeks to fill that gap in knowledge. Using cells from healthy human lungs, we first identified and quantified the frequency of antigen-specific cells in our lung donors by performing intracellular cytokine staining (IFNγ+) and activation induced marker assays (CD137+ CD25+). Then, by performing a series of in vitro peptide stimulation and cytokine neutralization experiments, we investigated which cytokines are produced by lung CD8+ TRM, and how those cytokines impact local innate and epithelial cells. Initial results show that, when stimulated with their cognate antigen, lung CD8+ TRM produce cytokines that directly activate innate immune cells. Results of this study suggests that human CD8+ TRM in the lungs act to rapidly reprogram local immune cells, and these data will ultimately help us understand how CD8+ TRM fit into the overall immune response to respiratory viruses. Supported by grants from the NIH/NHLBI (R35 HL150803) and by NIH/NIAD through the Centers of Excellence for Influenza Research and Response (75N93019R00028).

Characterization of SARS-CoV-2-specific CD8+ T cells in COVID-19 convalescent, vaccinated, and individuals with respect to age

Abstract COVID-19, an infectious disease caused by SARS-CoV-2, has given rise to the current global pandemic. Despite the number of infections nearing 300 million people worldwide, CD8+ T cells against SARS-CoV-2 in COVID-19 convalescent, vaccinated, and healthy adults and their changes with age have not been fully characterized. Here, we report the frequency, phenotype, and in vitro expansion capacity of circulating CD8+ T cells recognizing twenty-two epitopes from five SARS-CoV-2 proteins (S, N, M, ORF1ab, and ORF3a). Through multi-color flow cytometry using antigen-specific tetramers, we found that the frequencies of circulating CD8+ T cells ranged from undetectable to 1% in healthy, convalescent, and vaccinated individuals. Analysis of healthy adults ranging from 17 to 98 years of age revealed that the frequency of CD8+ T cells recognizing the epitope N-LLL (N222–230, LLLDRLNQL) is negatively correlated with age, particularly in the frequency of naive N-LLL-specific CD8+ T cells. Furthermore, the percentage of donors which displayed expansion of N-LLL-specific CD8+ T cells in response to in vitro peptide stimulation was significantly higher in convalescent patients than in healthy donors. These preliminary results suggest that there is an age-associated reduction of N-LLL-specific CD8+ T cells and exposure to SARS-CoV-2 primes these cells for robust expansion in response to stimulation with the N-LLL epitope. This work is still ongoing, and we will present the final results during the meeting.

Multi-functional HCV-specific CD4+ T cells associated with viral control during the postpartum period express Th1- and Tfh-biasing transcription factors

Abstract Chronic hepatitis C virus (HCV) infection is characterized by dysfunctional HCV-specific CD8+ T cells, rare CD4+ T responses, and stable high-level viremia. Recovered CD4+ T cell function and unusual declines in viremia have been observed in some women after childbirth. Whether recovered CD4+ T cells are of lineages to help B cells or CD8 T cells is unknown. Preliminary studies suggest both may be true. In 7 women with postpartum viral suppression (>= 1 log10 by 3 months postpartum, “controllers”), and 7 women with more stable viral levels, “non-controllers”, circulating HCV-tetramer+ (tet+) CD4+ T cells expressed chemokine receptors associated with two predominate populations, Th1-biased Tfh (cTfh1) cells and Th1 cells – with a higher cTfh1/Th1 ratio linked to viral control. Here we assessed HCV-specific cTfh1 and Th1 functional responses by combined surface chemokine receptor and intracellular cytokine staining (ICS) after HCV peptide stimulation, and we also assessed expression of lineage-defining transcription factors on HCV-tet+ cells. HCV-specific cytokine+ cells were predominantly cTfh1 or Th1 populations, and multi-functionality correlated with viral control. HCV-tet+ CD4+ T cell Tbet and BCL6 expression levels did not differ by degree of viral control, but both were enhanced in tet+cTfh1 cells relative to bulk cTfh1 cells, and tet+ Th1 cells expressed significantly more BCL6 than bulk Th1 cells. Together, these data point to a postpartum CD4+ T cell recovery driven by transcription factors favoring cTfh1/Th1-biased phenotypes. Understanding how these cells interact with CD8+ T cells and B cell will be vital for understanding this unique instance of natural control of a chronic viral infection. Supported by a grant from the NIH (R01 AI096882).

DNA-PKcs is required for the activation and cytotoxicity of CD8+ effector T cells

Abstract Antigen-mediated activation of naïve CD8+ T cells and their differentiation into cytotoxic cells is regulated by a series of signaling mechanisms designed to maintain T cell homeostasis and proper immune responses. Given that cytotoxic CD8+ T cells are critical to the elimination of cancerous or virally infected cells as well as mediators of autoimmune diseases, GVHD and allogenic organ rejection, it is critical that we continue to dissect these mechanisms to identify promising targets for T cell modulation in the clinic. Previously, we have shown that inhibition of the kinase DNA-dependent protein kinase catalytic subunit (DNA-PKcs) reduces activity of the transcription factors NFAT, NFκB and EGR1 and production of cytokines including IL2, IL6 and IFNγ in TCR-activated T cells. Using pharmacological inhibitor and genetic approaches, we provide evidence for a novel role of DNA-PKcs in CD8+ T cell activation. Inhibition of DNA-PKcs abrogated anti-CD3/CD28 activation of CD8+ T cells as evident by reduced expression of the activation markers CD69 and CD25. Additionally, loss of DNA-PKcs activity inhibited aerobic glycolysis and proliferation of OTI-CD8+ T cells following SIINFEKL peptide stimulation. This caused reduced OTI-CD8+ T cell killing of MC38.OVA tumor cells and expression of IFNγ and the cytotoxic genes perforin and granzyme B. These results were recapitulated in DNA-PKcs-deficient CD8+ T cells isolated from an inducible Cre/DNA-PKcsfl/fl mouse model where loss of DNA-PKcs reduced expression of CD69 and CD25, IFNγ production and altered cellular metabolism. The identification of a function for DNA-PKcs in CD8+ T cell activation highlights the therapeutic potential of DNA-PKcs inhibitors on immune-related diseases. Supported by Center for Pediatric Translational Research NIH COBRE P20GM121293

The ghrelinergic system in zebrafish gonads is suppressed during food unavailability

Ghrelin is an appetite stimulatory peptide that regulates reproduction in vertebrates. The acylation of ghrelin by ghrelin-O-acyltransferase (Goat) is essential for most of its functions. The ghrelinergic system (ghrelin, ghrelin receptor [Ghsr] and Goat) is present in teleost fish ovary and testis. In this research, we determined the abundance of the ghrelinergic system mRNAs in different stages of follicle (oocyte) development in zebrafish ovary. The highest levels of preproghrelin mRNA were observed in the first stage of follicular development (previtellogenesis). Significantly low levels of preproghrelin mRNA were observed in stages 3II, 4 and 5 of the follicle development. Ghsr mRNA abundance remained unchanged at early stages of follicle development and decreased in stage 5 compared to other developmental stages. A significant decrease in preproghrelin and Ghsr mRNAs in the testis, and ghsr mRNA in the ovary of zebrafish deprived of food for 3 days were found. At 7 days food deprivation, preproghrelin, ghsr and goat mRNA in the ovary and preproghrelin and goat in the testis of zebrafish were significantly reduced. These results show that metabolic status modulates gonadal ghrelinergic system in zebrafish and provide support for a role for ghrelin in fish reproduction.

Upregulation of antigen presentation function and inflammation in chondrocytes by induction of proteoglycan aggrecan peptides (P16-31 and P263-280).

A central hallmark of osteoarthritis (OA) is cartilage destruction. Chondrocytes not only control cartilage metabolism, but are capable of immunogenic responses. The role of chondrocytes in the pathogenesis of OA is still unclear. In this study, we aimed to determine the immunological role of chondrocytes in response to proteoglycan aggrecan (PG) peptides. Human chondrocytes were isolated from cartilage of knee OA patients undergoing knee arthroplasty and stimulated with proteoglycan aggrecan peptides in the presence of IFNγ. Antigen presentation markers, co-stimulatory molecules, cytokine production, gene expression and antigen presentation to T cells were evaluated. Our results show that IFNγ was required for the expression of MHC class I and II. However, stimulation with PG peptides P16-31 and P263-280, but not P2379-2394, increased expression level of co-stimulatory molecules (CD80 and CD86) and IL-6, IL-8 and TNFα production. This upregulation was seen in chondrocytes to nearly comparable levels of professional antigen-presenting cells. A similar pattern of gene expression was observed between P16-31 and P263-280 peptide stimulation on chondrocytes and this was different from P2379-2394 peptide treatment. Co-culture with autologous T cells revealed signi cant proliferation of cells when stimulating with the P263-280 peptides. Our study shows that human chondrocytes display unique features of antigen presentation. Their ability to process certain proteoglycan aggrecan peptides, in which these molecules are synthesised by the cartilage themselves render the possibility of a role for "self-antigens" in the immunopathogenesis of OA.

Long-term SARS-CoV-2-specific and cross-reactive cellular immune responses correlate with humoral responses, disease severity, and symptomatology.

BackgroundCellular immune memory responses post coronavirus disease 2019 (COVID‐19) have been difficult to assess due to the risks of contaminating the immune response readout with memory responses stemming from previous exposure to endemic coronaviruses. The work herein presents a large‐scale long‐term follow‐up study investigating the correlation between symptomology and cellular immune responses four to five months post seroconversion based on a unique severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2)‐specific peptide pool that contains no overlapping peptides with endemic human coronaviruses.MethodsPeptide stimulated memory T cell responses were assessed with dual interferon‐gamma (IFNγ) and interleukin (IL)‐2 Fluorospot. Serological analyses were performed using a multiplex antigen bead array.ResultsOur work demonstrates that long‐term SARS‐CoV‐2‐specific memory T cell responses feature dual IFNγ and IL‐2 responses, whereas cross‐reactive memory T cell responses primarily generate IFNγ in response to SARS‐CoV‐2 peptide stimulation. T cell responses correlated to long‐term humoral immune responses. Disease severity as well as specific COVID‐19 symptoms correlated with the magnitude of the SARS‐CoV‐2‐specific memory T cell response four to five months post seroconversion.ConclusionUsing a large cohort and a SARS‐CoV‐2‐specific peptide pool we were able to substantiate that initial disease severity and symptoms correlate with the magnitude of the SARS‐CoV‐2‐specific memory T cell responses.

A Survival Prediction for Acute Heart Failure Patients via Web-Based Dynamic Nomogram with Internal Validation: A Prospective Cohort Study.

PurposeThe current study aimed to develop a convenient and accurate prognostic dynamic nomogram model for the risk of all-cause death in acute heart failure (AHF) patients that incorporates clinical characteristics including N-terminal pro-brain natriuretic peptide (NT-pro BNP) and growth stimulation expresses gene 2 protein (ST2).Patients and MethodsWe prospectively studied 537 consecutive AHF patients and derived a clinical prediction model. The least absolute shrinkage and selection operator regression model combined with clinical characteristics were used for dimensional reduction and feature selection. Multivariate Cox proportional hazard analysis and “Dynnom” package were used to build the dynamic nomogram for prediction of 1-,2-,and 5-year overall survival for AHF. With bootstrap validation, the time-dependent concordance index (C-index) and calibration curves were used to assess predictive discrimination and accuracy. The contributions of NT-pro BNP and ST2 to the nomogram were evaluated using integrated discrimination improvement (IDI) and net reclassification improvement (NRI), while decision curve analysis (DCA) was used to assess clinical value.ResultsPatients were randomly divided into derivation (74.9%, n=402) and validation (25.1%, n=135) cohorts. Optimal independent prognostic factors for 1-,2-, and 5-year all-cause mortality were BS-ACMR (B: NT-pro BNP; S: ST2; A: age; C: complete right bundle branch block; M: mean arterial pressure; and R: red cell distribution width >14.5%); these were incorporated into the dynamic nomogram (https://bs-acmr-nom.shinyapps.io/dynnomapp/) with bootstrap validation. The C-indexes in the derivation (0.793) and validation (0.782) cohorts were consistent with comparable performance parameters. The calibration curve showed good agreement between the nomogram-predicted and actual survival. Adding NT-pro BNP and ST2 provided a significant net benefit and improved performance over other less adequate schemes in terms of DCA of survival probability compared to those neglecting either of these two factors.ConclusionThe study constructed a dynamic BS-ACMR nomogram, which is a convenient, practical and effective clinical decision-making tool for providing accurate prognosis in AHF patients.

Higher SARS-CoV-2 Spike Binding Antibody Levels and Neutralization Capacity 6 Months after Heterologous Vaccination with AZD1222 and BNT162b2.

Within a year after the emergence of SARS-CoV-2, several vaccines had been developed, clinically evaluated, proven to be efficacious in preventing symptomatic disease, and licensed for global use. The remaining questions about the vaccines concern the duration of protection offered by vaccination and its efficacy against variants of concern. Therefore, we set out to analyze the humoral and cellular immune responses 6 months into homologous and heterologous prime-boost vaccinations. We recruited 190 health care workers and measured their anti-spike IgG levels, their neutralizing capacities against the Wuhan-Hu-1 strain and the Delta variant using a surrogate viral neutralization test, and their IFNγ-responses towards SARS-CoV-2-derived spike peptides. We here show that IFNγ secretion in response to peptide stimulation was significantly enhanced in all three vaccination groups and comparable in magnitude. In contrast, the heterologous prime-boost regimen using AZD1222 and BNT162b2 yielded the highest anti-spike IgG levels, which were 3–4.5 times more than the levels resulting from homologous AZD1222 and BNT162b2 vaccination, respectively. Likewise, the neutralizing capacity against both the wild type as well as the Delta receptor binding domains was significantly higher following the heterologous prime-boost regimen. In conclusion, our results suggest that mixing different SARS-CoV-2 vaccines might lead to more efficacious and longer-lasting humoral protection against breakthrough infections.

Mitochondrial Dysfunction Associates With Acute T Lymphocytopenia and Impaired Functionality in COVID-19 Patients.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection results in rapid T lymphocytopenia and functional impairment of T cells. The underlying mechanism, however, remains incompletely understood. In this study, we focused on characterizing the phenotype and kinetics of T-cell subsets with mitochondrial dysfunction (MD) by multicolor flow cytometry and investigating the association between MD and T-cell functionality. While 73.9% of study subjects displayed clinical lymphocytopenia upon hospital admission, a significant reduction of CD4 or CD8 T-cell frequency was found in all asymptomatic, symptomatic, and convalescent cases. CD4 and CD8 T cells with increased MD were found in both asymptomatic and symptomatic patients within the first week of symptom onset. Lower proportion of memory CD8 T cell with MD was found in severe patients than in mild ones at the stage of disease progression. Critically, the frequency of T cells with MD in symptomatic patients was preferentially associated with CD4 T-cell loss and CD8 T-cell hyperactivation, respectively. Patients bearing effector memory CD4 and CD8 T cells with the phenotype of high MD exhibited poorer T-cell responses upon either phorbol 12-myristate-13-acetate (PMA)/ionomycin or SARS-CoV-2 peptide stimulation than those with low MD. Our findings demonstrated an MD-associated mechanism underlying SARS-CoV-2-induced T lymphocytopenia and functional impairment during the acute phase of infection.

Key interactions in the trimolecular complex consisting of the rheumatoid arthritis-associated DRB1*04:01 molecule, the major glycosylated collagen II peptide and the T-cell receptor

ObjectivesRheumatoid arthritis (RA) is an autoimmune disease strongly associated with the major histocompatibility complex (MHC) class II allele DRB1*04:01, which encodes a protein that binds self-peptides for presentation to T...

Function and Mechanism of RGD in Bone and Cartilage Tissue Engineering.

Bone and cartilage injury is common, tissue engineered scaffolds are potential means to repair. Because most of the scaffold materials used in bone and cartilage tissue engineering are bio-inert, it is necessary to increase the cellular adhesion ability of during tissue engineering reconstruction. The Arginine - Glycine - Aspartic acid (Arg-Gly-Asp, RGD) peptide family is considered as a specific recognition site for the integrin receptors. Integrin receptors are key regulators of cell-cell and cell-extracellular microenvironment communication. Therefore, the RGD polypeptide families are considered as suitable candidates for treatment of a variety of diseases and for the regeneration of various tissues and organs. Many scaffold material for tissue engineering and has been approved by US Food and Drug Administration (FDA) for human using. The application of RGD peptides in bone and cartilage tissue engineering was reported seldom. Only a few reviews have summarized the applications of RGD peptide with alloy, bone cements, and PCL in bone tissue engineering. Herein, we summarize the application progress of RGD in bone and cartilage tissue engineering, discuss the effects of structure, sequence, concentration, mechanical stimulation, physicochemical stimulation, and time stimulation of RGD peptide on cells differentiation, and introduce the mechanism of RGD peptide through integrin in the field of bone and cartilage tissue engineering.

Early Administration of Partially HLA Matched Third Party Virus-Specific T-Cells in Conjunction with Antiviral Treatment for Initial Viral Infection after Allogeneic Stem Cell Transplant Is Safe and Leads to High Rates of Viral Control

Introduction:Reactivation of viruses such as cytomegalovirus (CMV) or Epstein Barr virus (EBV) after allogeneic hemopoietic stem cell transplant (aHSCT) is associated with increased non relapse mortality and a requirement for antivirals with mainly hemopoietic and renal toxicities that can further compromise transplant outcomes and increase health care costs. The use of 3 rd party virus specific T cells (VSTs) has been effective in treating recurrent and refractory viral reactivation after transplant and leads to rapid restoration of viral immunity. We investigated whether early administration of 3 rd party VSTs together with antiviral therapy could safely enhance immune recovery and improve viral control in patients requiring treatment for their initial CMV and EBV infection after aHSCT.Methods:We performed a single arm phase 1 clinical trial in which aHSCT patients requiring treatment for their first CMV or EBV reactivation (or EBV driven malignancy) received infusions of partially HLA matched 3 rd party VSTs within 7 days of commencing standard antiviral treatment. Patients were required to have a viral copy number of at least 1,000 copies/mL for CMV, 10,000 copies/mL of blood for EBV, or proven tissue infection irrespective of copy number for treatment initiation. T cell products were expanded from G-CSF stimulated aphereses from normal donors following peptide stimulation and CD137 magnetic bead selection. T cells were cultured for up to 12 days before specificity testing and cryopreservation. Patients were eligible to receive up to 4 doses of VSTs at 4 week intervals with products selected with a minimum of 1 of 6 HLA matches at HLA-A, -B and -DRB1 with antiviral activity demonstrated through the shared HLA molecule. The primary endpoint of the study was infusion safety.Results:Thirty aHSCT patients were treated with 1-4 VST infusions (27 CMV, 3 EBV) commencing at a median of 4 days after initiation of antiviral treatment. 27 patients were transplanted for hematological malignancies, 3 for immune deficiencies. Conditioning was myeloablative in 12 patients and the majority of patients (22/30) received in vivo T cell depletion. 7/27 CMV seropositive recipients were transplanted from CMV seronegative donors. A total of 41 infusions were given, most frequently targeting antigens presented through shared HLA molecules A2 and/or B7. All infusions were administered at a cell dose of 2 x 10 7/m 2. VST products were CD3 + (median 97.9%, range 96.2 - 99.4%), with median percentage of CD3 +CD8 + 85.8% (range 23.2 - 95.5%). There was one infusion related adverse event consisting of fever that resolved rapidly after admission and antibiotics. Overall viral response rate was 100% with a complete response rate of 94% (Figure 1). Of the 28 patients who achieved a CR (after either 1 or 2 infusions), 22 remained virus PCR negative (n = 8) or below the limit of quantitation (n = 14) for the duration of follow up. 3 patients had brief episodes of quantifiable reactivation not requiring additional therapy and 3 patients required a second infusion following initial CR. All remained PCR negative after their 2 nd CR. All 3 patients treated for EBV PTLD achieved sustained CR. Overall rates of acute and chronic GVHD post-infusion were 33% (10/30) and 20% (6/30) respectively (grade IIII/IV aGVHD 10%, severe cGVHD 7%). VST infusion was associated with a reduction in activation and inhibitory marker expression on CD4 + and CD8 + lymphocytes within the first 30 days and recovery of CD8 + (and more slowly CD4 +) CD45RA -CD62L - effector memory cells. Within the first 100 days after infusion there was an increase in interferon-γ responsiveness of blood lymphocytes. CMV and EBV specific tetramer positive T cells were detected comprising up to 13% of CD8 + cells for up to 6 months post infusion. At 1 year post transplant, non relapse mortality was 10%, cumulative incidence of relapse was 7% and overall survival was 87%.Conclusion:The combination of traditional antiviral treatment and early administration of 3 rd party VSTs is safe and achieves high rates of viral control without evidence of increased acute or chronic GVHD and with evidence of enhanced immune responses to viral antigens. 1 year overall survival was high with low rates of non relapse mortality and relapse. These encouraging results require confirmation in a prospective randomized study comparing best available therapy with best available therapy combined with early VST administration. [Display omitted] DisclosuresRitchie: Novartis: Honoraria; BMS: Research Funding; Takeda: Research Funding; CRISPR Therapeutics: Research Funding; Amgen Inc: Honoraria, Research Funding; CSL: Honoraria.

Immune Profiling of Responses to Influenza Vaccination in Patients with Myeloproliferative Neoplasms

▪Myeloproliferative neoplasms (MPNs) are clonal stem cell neoplasms that include polycythemia vera (PV), essential thrombocythemia (ET), and myelofibrosis (MF). MPN patients are immunocompromised due to immune dysregulation from clonal hematopoiesis, heightened inflammation, and immunosuppressive treatments. Infection is a leading cause of morbidity and mortality in MPNs, and as a result annual influenza vaccination is a critical component of care. However, efficacy of the influenza vaccine in MPN patients is unclear. This study sought to identify differences between MPN patients and healthy donors in their immune response to influenza vaccination and subsequent in vitro stimulation.The Massachusetts General Hospital (MGH) maintains a tissue bank of peripheral blood and bone marrow samples collected in consented patients with hematologic malignancy, including MPNs. The tissue bank was interrogated for MPN patients with peripheral blood samples banked 1-6 months after yearly trivalent/quadrivalent influenza vaccination. A total of 13 MPN patients were included, with a median age of 70 (32-86). Five patients had MF, 3 patients had PV, 4 patients had ET, and 1 patient had pre-fibrotic MF. Five patients were on treatment with ruxolitinib, 4 patients were on hydroxyurea (HU), and 4 patients were without treatment. Peripheral blood mononuclear cells (PBMCs) from 11 healthy donors 3-6 months post-vaccination, with a median age of 50 (28-56), were sourced commercially as controls.Cryopreserved samples were thawed and PBMC samples divided equally prior to incubation for 18 hours with peptides derived from Influenza A or sham. Subsequently samples were labeled with a 41-antibody panel and analyzed by mass cytometry. Manual gating together with FlowSOM clustering followed by EdgeR using T-test and FDR to correct for multiple comparisons were used to identify differentially abundant immune cell clusters between cohorts and treatment conditions.Without stimulation, MPN patients were decreased in B cell and memory CD4+ T cell populations as compared to healthy donors (p<0.05), in agreement with a previous report. MPN patients also exhibited an increase in a subset of mature B cells (p<0.05) and lineage negative CD123+ cells (p<0.05) as compared to controls. Stimulation included in this study aimed to expand on these distinctions and identified that baseline differences were retained with additional increases in CD4+ and CD8+ T cell responses in healthy donors as compared to MPN patients. Conversely, stimulation increased the frequency of inflammatory monocytic cells in MPN patients compared to healthy controls (p<0.05).In healthy donors stimulation induced several alterations in, most notably an increase in CD4+ effector memory cells (p<0.03), as compared to matched unstimulated controls. In contrast, comparison of pre- and post-stimulation within the MPN cohort revealed no significant differences in immune cell populations apart from a decrease in naïve CD4+ T cells (p<0.05). Patients receiving HU were enriched for naïve CD4+ and effector CD8+ T cells as compared to patients receiving ruxolitinib (p<0.05); however, these differences were diminished following stimulation. Within MPN patients, pre-stimulation samples in MF patients were characterized by increased abundance of activated monocyte/macrophage clusters and a lineage-negative CD123+ population; there were no significant differences in post-stimulation samples between MF and ET/PV/pre-fibrotic MF patients.We evaluated immunologic responses to influenza vaccination, with and without peptide stimulation, in MPN patients and compared to healthy controls. Overall, MPN patients demonstrated less robust B- and T-cell responses and a muted response to stimulation as compared to healthy controls, which may indicate reduced cellular responses following influenza vaccination. Ongoing analysis will interrogate specific immune populations and subsets in detail to refine current observations and will evaluate associations between immunologic cell clusters with serologic responses and additional patient characteristics. The results of our study highlight important alterations in the immune response to influenza vaccination and muted response to stimulation in MPN patients and will inform a future prospective study of vaccine responsiveness and immune dysfunction in patients with MPNs. [Display omitted] DisclosuresReeves: PR Fludigm: Honoraria; Immunoscape: Consultancy. Hobbs: Merck: Research Funding; Incyte Corporation: Research Funding; AbbVie.: Consultancy; Novartis: Consultancy; Celgene/Bristol Myers Squibb: Consultancy; Constellation Pharmaceuticals: Consultancy, Research Funding; Bayer: Research Funding.

A Novel Humanized Murine Model to Identify Neoantigen-Specific T Cells in CBFA2T3-GLIS2 Positive Acute Megakaryoblastic Leukemia

CBFA2T3-GLIS2 is an oncogenic fusion gene that acts as the driver mutation in ~18% of pediatric patients with acute megakaryoblastic leukemia (AMKL) that do not have Down syndrome. CBFA2T3-GLIS2 driven leukemia carries the worst prognosis among all pediatric acute myeloid (AML) subtypes, with event free and overall survival probabilities of 8% and 14% respectively. Chemotherapy and stem cell transplantation fail to cure this disease, thus innovative treatment approaches are needed. While monoclonal antibody and chimeric antigen receptor T-cell (CAR-T) therapies have proven successful in acute lymphoblastic leukemia (ALL), similar successes haven't been realized in AML due to the lack of targetable antigens that eradicate leukemia cells while minimizing off target toxicities such as depletion of normal myeloid cells which can lead to prolonged neutropenia. An alternative is T cell receptor engineered T cell (TCR-T) immunotherapy which use heterodimers consisting of alpha and beta peptide chains to recognize polypeptide fragments presented by MHC molecules on the tumor cells. An advantage of this approach is the ability to recognize intracellular tumor specific and tumor associated antigen fragments in addition to extracellular proteins which results in a wider range of targets. To investigate the immunogenicity of CBFA2T3-GLIS2 positive AMKL cells, we established a humanized patient derived xenograft (PDX) murine model by conditioning 15 immunodeficient NSG-SGM3 mice with 200 rads 24 hours prior to transplantation of 3.5 million CBFA2T3-GLIS2 positive PDX cells derived from an individual female patient. After 20 days of engraftment, 2.5 million HLA class I matched PBMCs from an unrelated female donor were introduced. Experimental mice (engrafted with both PDX cells and PBMCs), leukemia only control mice, and PBMC only control mice were sacrificed at days 30, 35, and 40. Flow cytometry analysis at day 40 showed dramatic reduction of leukemic blasts (huCD45lo, huCD34+) when exposed to PBMCs compared to the leukemia only control (1.48% and 93.7%, respectively). Circulating human Granzyme A was elevated at day 40 in the PDX mice with PBMCs compared to the PBMC only control (9665.4 pg/ml vs. 789.7 pg/ml, p<0.001), indicating a potential leukemia-directed cytokine lymphocyte response. Single cell TCR repertoire analysis of CD8+ T cells from day 40 revealed a decrease in alpha beta TCR diversity when exposed to PDX cells compared to the PBMC only control, consistent with a clonal expansion of neoantigen-directed TCRs. Single TCR expressing stable cell lines have been generated for the top expanded clones. In ongoing experiments, we are utilizing these cell lines to assess leukemia clearance in our model and measuring specificity and functionality for the putative neoantigens through tetramer staining and peptide stimulation assays, respectively. For the latter studies, we have taken two approaches for the identification of potential neoantigens that may be directing this observed clonal expansion. First, to interrogate potential neoantigens spanning the fusion junction we used the algorithm from NetMHCcons which identified two peptides with intermediate binding affinity (defined by an IC50 of 150 - 500 nM). Second, we utilized immunopeptidomics to identify HLA class I presented peptides on the surface of the PDX cells. One of our top significant hits was a peptide that corresponds to the 5‘ untranslated region (UTR) of bone morphogenetic protein 2 (BMP2). This peptide fragment was not present in the human Uniprot database, suggesting the 5‘ UTR is not expressed in normal tissues. Although its role in leukemia is not well described, BMP2 is aberrantly upregulated in CBFA2T3-GLIS2 positive AMKL and required for serial replating in colony formation unit assays; therefore, this peptide is of great interest as a candidate leukemia-associated antigen. In summary, our novel humanized PDX model sheds light on the immunogenicity of CBFA2T3-GLIS2 mediated AMKL and the potential for TCR-T immunotherapy in the setting of minimal residual disease for this chemotherapy resistant malignancy. Studies are ongoing to identify the TCR clone(s) responsible for the anti-leukemic response seen in our model and the leukemia-associated antigens that are mediating this response. DisclosuresGruber: Kura Oncology: Consultancy.

Immunogenicity of Covid-19 Vaccination in Subjects with Myelodysplastic Syndromes

Myelodysplastic syndromes (MDS) represent a spectrum of clonal bone marrow neoplasms from low risk disease through to those transforming into acute myeloid leukaemia. The COVID-19 pandemic has presented a great risk to those with hematological malignancies who are at higher risk of severe disease and death than the general population. Previous studies looking at the immune response to influenza vaccination in those with MDS had shown promising results, with immune responses not differing from those of healthy family members. Whilst some data exist to reassure the MDS community that majority of patients show seroconversion following Covid-19 vaccination, little data exists on their neutralizing capacity or post vaccination T-cell responses in this cohort. In addition, the majority of patients in these studies received BNT162b2 and there is little published data on vaccine response to the ChAdOx1 nCoV-19 vaccine. We have investigated the humoral and T-cell response of 39 patients with MDS two to four weeks following Covid-19 booster vaccination with BNT162b2 or ChAdOx1 nCoV-19 through the SOAP study (Sars-cov-2 fOr cAncer Patients, IRAS project ID:282337).Plasma and PBMCs from MDS cases and healthy controls have been collected, and are being assessed for both humoral and cellular responses to SARS_CoV_2, the alpha (B.1.1.7) and delta (B.1.617.2) variants. Humoral responses will be assessed using ELISA (peptide binding) and functional viral neutralization assays. Cellular responses will be assessed using IFNy ELISPOT and flow cytometry (CD25 and CD69 expression) after 24h peptide stimulation. All data at time point 1 (2 - 4 weeks following booster vaccination) have been collected and will subsequently be collected at 6 months and 12 months post-vaccination. We also report on the safety data for these vaccines within this patient population.Of this cohort 64% were male with a median age of 65 years (range 21-84). 54% received vaccination with ChAdOx1 nCoV-19 and 44% received BNT162b2 (2% unrecorded). The vaccines were well tolerated with no serious adverse events to date. The mean interval between doses was 70.7 days (range 50 - 90 days). 71% of the cohort were receiving no disease modifying therapy at the time of vaccination, half of whom were receiving supportive therapy and the other half no intervention for their MDS. Of those receiving disease modifying therapy; 5 were receiving azacitidine, (1 in conjunction with low-dose cytarabine) and 3 ciclosporin.We will report the largest study of the humoral and T-cell mediated response to the Covid-19 vaccine in MDS patients to date. This will include cellular response to the delta variant and immunogenicity of both the BNT162b2 and ChAdOx1 nCoV-19 vaccines. Given the vulnerability of these patients to severe disease, investigating the immune response to the vaccines begins to build an evidence base for advising MDS patients on their ongoing risk of infection during the pandemic and going forward. The SOAP study will reassess the immune response at 6 and 12 months post-vaccination to continue to investigate post-vaccine immunity in this cohort. DisclosuresKulasekararaj: F. Hoffmann-La Roche Ltd.: Consultancy, Honoraria, Speakers Bureau; Apellis: Consultancy; Akari: Consultancy, Honoraria, Speakers Bureau; Biocryst: Consultancy, Honoraria, Speakers Bureau; Achilleon: Consultancy, Honoraria, Speakers Bureau; Alexion: Consultancy, Honoraria, Speakers Bureau; Ra Pharma: Consultancy, Honoraria, Speakers Bureau; Amgen: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Alexion, AstraZeneca Rare Disease Inc.: Consultancy, Honoraria, Other: Travel support. Patten: JANSSEN: Honoraria; NOVARTIS: Honoraria; GILEAD SCIENCES: Honoraria, Research Funding; ROCHE: Research Funding; ASTRA ZENECA: Honoraria; ABBVIE: Honoraria.