169 Microfluidics cell squeezing enables human PBMCs as drivers of antigen-specific CD8 T responses across broad range of antigens for diverse clinical applications

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A183-A183
Author(s):  
Michael Maloney ◽  
Scott Loughhead ◽  
Amritha Ramakrishnan ◽  
Carolyne Smith ◽  
Anita Venkitaraman ◽  
...  
Keyword(s):  
T Cell ◽  
Mononuclear Cells ◽  
Scale Up ◽  
Cell Activity ◽  
Cd8 T Cell ◽  
T Cell Response ◽  
Human Pbmcs ◽  
Class I Mhc ◽  
Wide Range ◽  
The Impact

BackgroundAntigen-specific CD8+ T cell activity is critical for mounting an effective immune response in a wide range of indications, including immune-oncology and infectious diseases.MethodsTo elicit antigen-specific CD8+ T cell activity, we used microfluidics cell squeezing (Cell Squeeze®) to deliver antigens directly to the cytosol of antigen presenting cells (APCs). Direct cytosolic delivery bypasses the need for cross-presentation and efficiently loads antigen into the major histocompatibility complex class I (MHC-I) pathway. The Cell Squeeze® platform is generally agnostic to cell type and material. Therefore, not only does microfluidic squeezing enable cell subsets within human peripheral blood mononuclear cells (PBMCs) to function as unconventional APCs, but it also enables us to efficiently investigate a wide range of antigens including whole protein, peptides, and mRNA. This ‘plug and play’ nature of the platform allows for broad application in multiple disease areas.ResultsIn human cells, we demonstrated that microfluidic squeezing of PBMCs enables effective delivery to the major cell subsets including T cells, B cells, NK cells and monocytes. Delivery of CMV and HPV16 synthetic long peptides (SLPs) resulted in robust in vitro responses of both CD8+ T cell clones and patient-derived memory populations. To broaden the impact of our PBMC-based cell therapy approach, we investigated several other antigens relevant to other disease areas. Additional materials we delivered via squeezing and demonstrated antigen presentation include neoantigens, M1 Influenza mRNA, and pp65 SLP. Cell Squeeze® platform is simple to use and amenable to scale up. We demonstrated that delivery and viability for research scale process (~2 × 106 cells) is equivalent to delivery and viability of PBMCs processed at manufacturing scale (~1 × 109 cells).ConclusionsMicrofluidic cell squeezing of human PBMCs with antigenic material can be tailored to produce APCs that drive robust CD8+ T cell response against targets across multiple disease areas and has been scaled up for clinical use. SQZ-PBMC-HPV are currently under clinical evaluation for treatment of HPV16+ tumors.

scholarly journals Suppression of liver transplant rejection by anti-donor MHC antibodies via□ depletion of donor immunogenic dendritic cells

2020 ◽  
Author(s):  
Hisashi Ueta ◽  
Xue-Dong Xu ◽  
Bin Yu ◽  
Yusuke Kitazawa ◽  
Enqiao Yu ◽  
...  
Keyword(s):  
T Cell ◽  
Liver Transplant ◽  
Transplant Rejection ◽  
Cd8 T Cell ◽  
T Cell Response ◽  
Cellular Damage ◽  
Class I Mhc ◽  
Class Ii Mhc ◽  
Passenger Leukocytes ◽  
Liver Transplant Rejection

Abstract Background We previously found two distinct passenger dendritic cell (DC) subsets in the rat liver that played a central role in the liver transplant rejection. In addition, tolerance-inducing protocol, donor-specific transfusion (DST), triggered systemic polytopical production of depleting alloantibodies to donor class I MHC antigen (DST-antibodies). Methods We examined the role of DST-antibodies in the trafficking of graft DC subsets and the alloresponses in a rat model. We also examined an anti-donor class II MHC (MHCII) antibody that recognizes donor DCs more selectively. Results Preoperative transfer of DST-antibodies and DST pretreatments eliminated all passenger leukocytes, including both DC subsets and depleted the sessile DCs in the graft to ~20% of control. The CD172a +CD11b/c + immunogenic subset was almost abolished. The intrahost direct or semi-direct allorecognition pathway was successfully blocked, leading to a significant suppression of the CD8 + T-cell response in the recipient lymphoid organs and the graft with delayed graft rejection. Anti-donor MHCII antibody had similar effects without temporary graft damage. Although DST pretreatment had a priming effect on the recipient Treg proliferative response, DST-primed sera and the anti-donor MHCII antibody did not. Conclusion DST-antibodies and anti-donor MHCII antibodies could suppress the CD8 + T-cell-mediated liver transplant rejection by depleting donor immunogenic DCs, blocking the direct or semi-direct pathway of allorecognition. Donor MHCII-specific antibodies may be applicable as a selective suppressant of anti-donor immunity for clinical liver transplantation without the cellular damage of donor MHCII – graft cells and recipient cells.

scholarly journals DIFFERENTIAL IMMUNODOMINANCE HIERARCHY OF CD8+ T CELL RESPONSES IN HLA-B*27:05 AND B*27:02-MEDIATED CONTROL OF HIV-1 INFECTION

2017 ◽  
pp. JVI.01685-17
Author(s):  
Emily Adland ◽  
Matilda Hill ◽  
Nora Lavandier ◽  
Anna Csala ◽  
Anne Edwards ◽  
...  
Keyword(s):  
T Cell ◽  
Disease Progression ◽  
Cell Response ◽  
Selection Pressure ◽  
Cell Activity ◽  
T Cell Responses ◽  
Cd8 T Cell ◽  
T Cell Response ◽  
Hiv Disease ◽  
Cell Responses

The well-characterised association between HLA-B*27:05 and protection against HIV disease progression has been linked to immunodominant HLA-B*27:05-restricted CD8+ T-cell responses towards the conserved Gag 263-272 (‘KK10’) and Pol 901-909 ‘KY9’ epitopes. We here studied the impact of the 3 amino acid differences between HLA-B*27:05 and the closely-related HLA-B*27:02 on the HIV-specific CD8+ T-cell response hierarchy and on immune control of HIV. Genetic epidemiological data indicate that both HLA-B*27:02 and HLA-B*27:05 associate with slower disease progression and lower viral loads. The effect of HLA-B*27:02 appears consistently stronger than that of HLA-B*27:05. In contrast to HLA-B*27:05, the immunodominant HIV-specific HLA-B*27:02-restricted CD8+ T-cell response is to a Nef epitope (residues 142-150, ‘VW9’), with Pol-KY9 subdominant and Gag-KK10 further subdominant. This selection was driven by structural differences in the F-pocket, mediated by a polymorphism between these two HLA alleles at position 81. Analysis of autologous virus sequences showed that in HLA-B*27:02-positive subjects all three of these CD8+ T-cell responses impose selection pressure on the virus, whereas in HLA-B*27:05-positive subjects there is no Nef-VW9-mediated selection pressure. These studies demonstrate that HLA-B*27:02 mediates protection against HIV disease progression that is at least as strong or stronger than that mediated by HLA-B*27:05. In combination with the protective Gag-KK10 and Pol-KY9 CD8+ T-cell responses that dominate HIV-specific CD8+ T-cell activity in HLA-B*27:05-positive subjects, a Nef-VW9-specific response is additionally present and immunodominant in HLA-B*27:02-positive subjects, mediated through a polymorphism at residue 81 in the F-pocket, that contributes to selection pressure against HIV.IMPORTANCECD8+ T-cells play a central role in successful control of HIV infection, and have the potential also to mediate the eradication of viral reservoirs of infection. The principal means by which ‘protective’ HLA class I molecules, such as HLA-B*27:05 and HLA-B*57:01, slow HIV disease progression, is believed to be via the particular HIV-specific CD8+ T cell responses restricted by those alleles. We focus here on HLA-B*27:05, one of the best-characterised ‘protective’ HLA molecules, and the closely-related HLA-B*27:02, which differs by only 3 amino acids, and which has not been well-studied in relation to control of HIV infection. We show that HLA-B*27:02 is also protective against HIV disease progression, but the CD8+ T-cell immunodominance hierarchy of HLA-B*27:02 differs strikingly from that of HLA-B*27:05. These findings indicate that the immunodominant HLA-B*27:02-restricted Nef response adds to protection mediated by the Gag and Pol specificities that dominate anti-HIV CD8+ T-cell activity in HLA-B*27:05-positive subjects.

scholarly journals T Cell Monitoring of Chemotherapy in Experimental Rat Tuberculosis

2011 ◽  
Vol 55 (8) ◽  
pp. 3677-3683 ◽  
Author(s):  
Damian Guang Foo ◽  
Hui Chien Tay ◽  
Jie Yee Siew ◽  
Amit Singhal ◽  
Luis Camacho ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Response ◽  
Mononuclear Cells ◽  
Chronic Phase ◽  
Drug Efficacy ◽  
T Cell Response ◽  
Preclinical Development ◽  
Bacterial Killing ◽  
Ifn Γ ◽  
The Impact

ABSTRACTMycobacterium tuberculosisis the causative agent of a pulmonary epidemic that is estimated to infect one-third of the world's population and that has an increased incidence of multidrug resistance. The evaluation of new chemical entities againstM. tuberculosisis hampered by the lack of biological tools to help predict efficacy, from early drug development to clinical trials. As the rat is the animal species of choice in the pharmaceutical industry, we have developed a rat model of acute and chronic phases ofM. tuberculosisinfection for drug efficacy testing. In this model, we have evaluated the impact of tuberculosis drugs on T cell response using the enzyme-linked immunospot assay methodology. Infected rats treated with isoniazid (INH) or rifampin (RIF) responded to therapy, the potency of which was comparable to that seen in the mouse. Peripheral blood mononuclear cells from infected rats produced gamma interferon (IFN-γ) in response to RD-1 antigens, such as the 6-kDa early secretory antigen target (ESAT-6) and the 10-kDa culture filtrate protein (CFP-10). A decrease in IFN-γ spot-forming cells (SFCs) was consistently observed in response to drug treatment. In both the acute- and chronic-phase models, the T cell response was more sensitive to ESAT-6 than to CFP-10. The SFC count in response to ESAT-6 appears to be an indicator of bacterial killing in the rat. Collectively, our data suggest that the ESAT-6 response could be used as a potential surrogate of drug efficacy in the rat and that such a readout could help shorten drug testing during preclinical development.

170 Microfluidics cell squeezing enables potent cellular vaccines in murine models through direct cytosolic loading and direct CD8 T cell priming

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A184-A184
Author(s):  
Emrah Ozay ◽  
Matthew Booty ◽  
Katarina Blagovic ◽  
David Soto ◽  
Olivia Pryor ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Median Survival ◽  
Cd8 T Cells ◽  
Mononuclear Cells ◽  
Cd8 T Cell ◽  
Laboratory Animals ◽  
Peripheral Blood Mononuclear ◽  
Class I Mhc ◽  
Mhc I

BackgroundThe presentation of sufficient antigen on major histocompatibility complex class I (MHC-I) is essential to prime CD8+ T cells.MethodsTo achieve efficient MHC-I presentation, we used microfluidics cell squeezing (Cell Squeeze®) to deliver antigens directly to the cytosol of antigen presenting cells (APCs), bypassing the need for cross-presentation. In addition to facilitating priming by professional APCs, this approach enables lymphocytic subsets within peripheral blood mononuclear cells (PBMCs) to function as unconventional APCs in mouse preclinical models.ResultsWe demonstrated that microfluidic cell squeezing delivers cargo to major cell populations within splenocytes (T cells, B cells, NK cells, and monocytes) and that protein, peptide, or mRNA antigens are rapidly processed and presented. In vivo, squeezed splenocytes directly presented antigen to CD8+ T cells. In the TC-1 tumor model for HPV+ cancers, squeezed splenocytes completely protect mice when administered prophylactically, protecting 15/15 animals from primary challenge and 11/15 animals from tumor re-challenge. Following therapeutic administration, squeezed splenocytes significantly improved median survival time to 56 days from 28 days, as observed with untreated controls. Immunization can also be combined with chemotherapy to further enhance therapeutic efficacy, improving median survival to over 100 days compared to 81 days with SQZ monotherapy or 32 days with chemotherapy alone. When tumor infiltrating lymphocytes (TILs) were analyzed following therapeutic immunization, squeezed splenocyte immunization elicited a significant influx of antigen specific CD8+ T cells: with SQZ treatment, ~87% of tumor-infiltrating CD8 T cells were antigen-specific, as measured by an E7-tetramer stain, while only ~33.6% and ~1.15% of infiltrating CD8 T cells were specific for E7 with subcutaneous peptide vaccination and no treatment, respectively.ConclusionsThrough the direct cytosolic delivery of antigen, we have engineered unfractionated PBMCs to function as potent APCs. This strategy generates potent antigen-specific CD8+ T cell responses in mouse models. Taken together, these findings support the potential of SQZ-PBMCs as an effective antigen-specific vaccination strategy against cancer. SQZ-PBMC-HPV is currently under clinical evaluation for HPV16+ tumor indications.Ethics ApprovalAll methods were performed in accordance with relevant guidelines and regulations; Animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) at SQZ Biotechnologies, using the recommendations from the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and the Office of Laboratory Animal Welfare. All activities were also conducted in accordance with Public Health Service (PHS) Policy on Humane Use and Care of Laboratory Animals.

scholarly journals Why Are CD8 T Cell Epitopes of Human Influenza A Virus Conserved?

2019 ◽  
Vol 93 (6) ◽  
Author(s):  
Zheng-Rong Tiger Li ◽  
Veronika I. Zarnitsyna ◽  
Anice C. Lowen ◽  
Daniel Weissman ◽  
Katia Koelle ◽  
...  
Keyword(s):  
T Cell ◽  
Influenza A Virus ◽  
Influenza A ◽  
Cd8 T Cell ◽  
Influenza Vaccines ◽  
T Cell Epitopes ◽  
Class I Mhc ◽  
Mhc I ◽  
Escape Variant ◽  
Wide Range

ABSTRACTThe high degree of conservation of CD8 T cell epitopes of influenza A virus (IAV) may allow for the development of T cell-inducing vaccines that provide protection across different strains and subtypes. This conservation is not fully explained by functional constraint, since an additional mutation(s) can compensate for the replicative fitness loss of IAV escape variants. Here, we propose three additional mechanisms that contribute to the conservation of CD8 T cell epitopes of IAV. First, influenza-specific CD8 T cells may protect predominantly against severe pathology rather than infection and may have only a modest effect on transmission. Second, polymorphism of the human major histocompatibility complex class I (MHC-I) gene restricts the advantage of an escape variant to only a small fraction of the human population who carry the relevant MHC-I alleles. Finally, infection with CD8 T cell escape variants may result in a compensatory increase in the responses to other epitopes of IAV. We use a combination of population genetics and epidemiological models to examine how the interplay between these mechanisms affects the rate of invasion of IAV escape variants. We conclude that for a wide range of biologically reasonable parameters, the invasion of an escape variant virus will be slow, with a timescale of a decade or more. The results suggest T cell-inducing vaccines do not engender the rapid evolution of IAV. Finally, we identify key parameters whose measurement will allow for more accurate quantification of the long-term effectiveness and impact of universal T cell-inducing influenza vaccines.IMPORTANCEUniversal influenza vaccines against the conserved epitopes of influenza A virus have been proposed to minimize the burden of seasonal outbreaks and prepare for the pandemics. However, it is not clear how rapidly T cell-inducing vaccines will select for viruses that escape these T cell responses. Our mathematical models explore the factors that contribute to the conservation of CD8 T cell epitopes and how rapidly the virus will evolve in response to T cell-inducing vaccines. We identify the key biological parameters to be measured and questions that need to be addressed in future studies.

MLL Histone Methyltransferase Restrains Effector Differentiation and PD-1 Expression in Human CD8+ T Cells While Promoting Their Proliferation

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 863-863
Author(s):  
Janaki Purushe ◽  
Hongxin Sun ◽  
Shan He ◽  
Yali Dou ◽  
Yi Zhang
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Conflicts Of Interest ◽  
Flow Cytometric Analysis ◽  
Specific Effect ◽  
Cd8 T Cell ◽  
T Cell Response ◽  
Ifn Γ ◽  
The Impact

Abstract Adoptive cellular therapy (ACT) for cancer requires amplification and persistence of tumor-specific T cells. To completely eliminate malignant tumor cells, infused tumor-reactive T cells must retain the capacity to expand over weeks and months in order to produce sufficient effector cells. However, T-cell potency can be blunted by excessive differentiation and upregulation of PD-1, which mediates exhaustion and impairs their proliferative capacity. Histone methylation is thought to be central in directing transcriptional programs important for effector proliferation, survival and differentiation; however, the epigenetic regulator(s) of this process are not well characterized. We report that the histone methyltransferases Mixed Lineage Leukemia 1 (MLL1) and MLL4, which catalyze trimethylation of histone H3 at lysine 4 (H3K4me3), play important roles in restraining effector differentiation and promoting proliferation of activated human CD8+ T cells. Upon T cell receptor (TCR) activation, human CD8+ T cells upregulated MLL1 and MLL4, however, down-regulated the global level of H3K4me3. To assess the specific effect of MLL1 in CD8+ T cell differentiation, we produced lentivirus encoding short hairpin RNA (shRNA) specific to MLL1 or MLL4. Knockdown of either protein increased the frequency of IFN-γ-producing cells by 50% to 100%, with silencing of MLL1 having the more potent effect. Pharmacological inhibition with MI-2-2, which simultaneously inhibits the menin-MLL1 and menin-MLL4 interactions, increased the frequency of IFN-γ + CD8+ T cells three-fold and reduced cell proliferation from 94% to 64%. Using a second MLL1 inhibitor MM-401, which affects MLL1 specifically, we confirmed that inhibiting MLL1 resulted in a two-to-four-fold increase in the expression of numerous effector molecule transcripts, including IFNG, TNFA, PRF1, FASL and GZMB. Our results suggest that while both MLL1 and MLL4 are important for proliferation of activated CD8+ T cells, MLL1 potently restrains effector differentiation. T-BET and EOMES are two transcription factors critical for mediating effector differentiation. We found that inhibition of MLL1 in cultured, TCR-activated CD8+ T cells using either MI-2-2 or MM-401 led to a significant increase in expression of EOMES, but had no significant effect on T-BET expression. Flow cytometric analysis showed that silencing MLL1 also increased the expression of EOMES protein in activated CD8+ T cells. Interestingly, MLL1 knockdown impaired subsequent persistence of ex vivo expanding CD8+ T cells, which was associated with a substantial increase of CD45RO+CCR7- short-lived effector CD8+ T cells. Remarkably, knockdown of MLL1 in proliferating CD8+ T cells led to their upregulation of PD-1. Taken together, these data suggest that MLL1 may play an important role in restraining precocious effector differentiation and exhuastion in CD8+ T cells. Future studies will investigating the impact of both MLL1 and MLL4 in regulating the CAR CD8+ T cell response in vivo and in vitro. Results from these experiments will allow us to identify epigenetic mechanisms that regulate the generation and persistence of antitumor effector and memory CD8+ T cells. Disclosures No relevant conflicts of interest to declare.

scholarly journals Broad-Based Influenza-Specific CD8+ T Cell Response without the Typical Immunodominance Hierarchy and Its Potential Implication

Viruses ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1080
Author(s):  
Miaojuan Huang ◽  
Rong Xu ◽  
Cristina Triffon ◽  
Nicole Mifsud ◽  
Weisan Chen
Keyword(s):  
T Cell ◽  
Cell Response ◽  
Influenza A ◽  
Epitope Prediction ◽  
Cd8 T Cell ◽  
T Cell Response ◽  
Potential Implication ◽  
Allelic Differences ◽  
Screen Approach ◽  
The Impact

Syngeneic murine systems have pre-fixed MHC, making them an imperfect model for investigating the impact of MHC polymorphism on immunodominance in influenza A virus (IAV) infections. To date, there are few studies focusing on MHC allelic differences and its impact on immunodominance even though it is well documented that an individual’s HLA plays a significant role in determining immunodominance hierarchy. Here, we describe a broad-based CD8+ T cell response in a healthy individual to IAV infection rather than a typical immunodominance hierarchy. We used a systematic antigen screen approach combined with epitope prediction to study such a broad CD8+ T cell response to IAV infection. We show CD8+ T cell responses to nine IAV proteins and identify their minimal epitope sequences. These epitopes are restricted to HLA-B*44:03, HLA-A*24:02 and HLA-A*33:03 and seven out of the nine epitopes are novel (NP319–330#, M1124–134, M27–15, NA337–346, PB239–49, HA445–453 and NS1195–203). Additionally, most of these novel epitopes are highly conserved among H1N1 and H3N2 strains that circulated in Australia and other parts of the world.

scholarly journals T-cell mediated immunity after AZD1222 vaccination: A polyfunctional spike-specific Th1 response with a diverse TCR repertoire

2021 ◽  
Author(s):  
Phillip A. Swanson ◽  
Marcelino Padilla ◽  
Wesley Hoyland ◽  
Kelly McGlinchey ◽  
Paul A. Fields ◽  
...  
Keyword(s):  
T Cell ◽  
Mononuclear Cells ◽  
T Cell Responses ◽  
Cd8 T Cell ◽  
T Cell Response ◽  
Spike Protein ◽  
Cell Mediated Immunity ◽  
Peripheral Blood Mononuclear ◽  
Adult Age ◽  
Cell Responses

AZD1222 (ChAdOx1 nCoV-19), a replication-deficient simian adenovirus-vectored vaccine, has demonstrated safety, efficacy, and immunogenicity against coronavirus disease 2019 (COVID-19) in clinical trials and real-world studies. We characterized CD4+ and CD8+ T-cell responses induced by AZD1222 vaccination in peripheral blood mononuclear cells (PBMCs) from 280 unique vaccine recipients aged 18-85 years who enrolled in the phase 2/3 COV002 trial. Total spike-specific CD4+ T cell helper type 1 (Th1) and CD8+ T-cell responses were significantly increased in AZD1222-vaccinated adults of all ages following two doses of AZD1222. CD4+ Th2 responses following AZD1222 vaccination were not detected. Furthermore, AZD1222-specific Th1 and CD8+ T cells both displayed a high degree of polyfunctionality in all adult age groups. T-cell receptor (TCR) β ; sequences from vaccinated participants mapped against TCR sequences known to react to SARS-CoV-2 revealed substantial breadth and depth across the SARS-CoV-2 spike protein for the AZD1222-induced CD4+ and CD8+ T-cell responses. Overall, AZD1222 vaccination induced a robust, polyfunctional Th1-dominated T-cell response, with broad CD4+ and CD8+ T-cell coverage across the SARS-CoV-2 spike protein.

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