Donor T Cells Intrinsic Responses to Damps Regulated By Siglec-G-CD24 Axis Mitigate Gvhd but Maintain GVL in Experimental BMT Model

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 229-229 ◽  
Author(s):  
Tomomi Toubai ◽  
Corinne Rossi ◽  
Katherine Oravecz-Wilson ◽  
Nathan Mathewson ◽  
Cynthia Zajac ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Responses ◽  
Innate Immune ◽  
T Cell Subsets ◽  
Activation Markers ◽  
Cell Responses ◽  
Target Organs ◽  
Donor T Cells

Abstract Innate immune receptors like pattern recognition receptors (PRRs) including toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD) like-receptors (NLR) on immune cells play an important role in initiating inflammatory responses to damage- and pathogen- associated molecular patterns (DAMPs and PAMPs) expressed on invading pathogens or released from damaged cells. Although it is well known that DAMPs directly modulate innate immune functions, it is less clear whether DAMPs directly regulate T cell intrinsic function. Members of the sialic acid binding Ig-like lectin (Siglec) family have immunoreceptor tyrosine-based inhibitory motifs (ITIM) or ITIM-like regions in their intracellular domain that negatively regulate immune activation induced by DAMPs. Our previous data suggested that the Siglec- G-CD24 interaction in host APCs plays an important role in the negative regulation of graft-versus host (GVH) responses. However, the T cell autonomous role of Siglec-G in the regulation of T cell responses is not known. Because Siglecs are important negative regulators of immune responses, we tested the hypothesis that the deficiency of Siglec-G in donor T cells would enhance GVHD. To test our hypothesis, we first examined detailed phenotypic analysis of various T cell subsets and activation markers in naïve Siglec-G-/- and wild-type (WT) B6 animals and found similar distribution of naïve, memory, effector and regulatory T cells. In order to examine whether the absence of Siglec-G in donors affects GVHD, WT-BALB/cmice were lethally irradiated (850cGy) and transplanted on day 0 with 5x106 bone marrow and 0.5x106 splenic CD90+ T cells from either syngeneic WT-BALB/c, allogeneic MHC-mismatched WT-B6 or Siglec-G-/- animals. The recipients receiving donor T cells from Siglec-G-/- animals showed a significantly worse survival compared to allogeneic WT-B6 animals (p<0.05). This increased mortality was also associated with more severe GVHD damage in target organs and a higher expansion of activated CD69+, IFN-r+, and IL-17A+ donor T cells in the spleen and target organs. Enhanced GVHD mortality and severity was also observed in MHC mismatched haploidentical matched B6 in to F1models (p<0.05). To explore the mechanism, we tested whether Siglec-G deficiency alters the naïve T cell responses in vitro after allogeneic or non-specific TCR stimulation in the absence of exogenous DAMPs. Interestingly Siglec-G-/- T cells showed similar proliferation in vitro, when compared to WT B6 T cells. In addition, Siglec-G-/- Tregs are equally suppressive in suppression assay and Siglec-G-/- T cells showed severe GVHD even Tregs are depleted in allo-BMT. However, Siglec-G-/- T cells showed a higher proliferation after direct TCR stimulation (CD3/CD28) with addition of DAMP (HMGB-1) when compared to WT T cells in vitro, suggesting direct T cell intrinsic effects. Consistent with this result, allogeneic Siglec-G-/- T cells caused similar mortality compared to WT controls in non-irradiated B6 into F1 model due to the absence of DAMPs from conditioning. To test the critical cellular mechanisms, we examined the function of endogenous Siglec-G ligand, CD24. We utilized BALB/c CD24-/- animals as hosts in same BMT model and found that CD24-/- animals showed an enhanced GVHD mortality and severity when compared to WT animals (p<0.05). To enhance Siglec-G-CD24 axis, we utilized a novel CD24 fusion protein (CD24Fc) in same BMT model and found that CD24 Fc ameliorated GVHD severity and mortality in not only allogeneic WT-B6 animals (p<0.05) but also CD24-/- animals (p<0.05). Next we explored DAMPs regulation by Siglec-G-CD24 axis in GVL. We utilized the same model of CD24Fc treatment but added P815 at the same time of allo-BMT and found that CD24Fc treated animals showed equivalent GVL to non-treated animals, suggesting that regulation of DAMPs with CD24Fc mitigates GVHD with maintaining GVL effect. Collectively our data suggested that the expression of both Siglec-G on donor T cells and CD24 on hosts is critical for controlling GVHD in the context of DAMPs released from conditioning, and represents a novel strategy that CD24Fc can mitigates GVHD with maintaining GVL. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures No relevant conflicts of interest to declare.

scholarly journals Pparα Ablation Suppresses T Cell Responses and Anti-Tumor Immunity By Compromising the Antigen-Presenting Properties of Tumor-Associated Macrophages

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 438-438
Author(s):  
Anthos Christofides ◽  
Carol Cao ◽  
Qi Wang ◽  
Natalia M Tijaro-Ovalle ◽  
Eirini Konstantinidou ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Suppressor Cells ◽  
T Cell Responses ◽  
Innate Immune ◽  
Myeloid Derived Suppressor Cells ◽  
Cell Responses

Abstract Peroxisome proliferator activated receptors (PPARs) are transcription factors that belong to nuclear hormone superfamily, with three distinct types identified: PPARapha (PPARα), PPARgamma (PPARγ), and PPARbeta/delta (PPARβ/δ). PPARs possess a critical role in the regulation of lipid metabolism, and thus play critical roles in the differentiation and fate of immune cells. PPARα is involved in lipid and carbohydrate metabolism and PPARα agonists, such as fibrates, have been used for the treatment of hypertriglyceridemia and cardiovascular diseases. PPARα has an anti-inflammatory role during infection, and similar to PPARγ, affects the polarization of macrophages. In acute myelogenous leukemia (AML), PPARα mutations correlate with chemoresistance, poor treatment outcomes and unfavorable prognosis. In experimental tumor models, it has been proposed that PPARα agonists might enhance anti-tumor T cell responses during PD-1 blocking immunotherapy. To dissect the mechanistic role of PPARα in tumor immunity, we used mice with global deletion of PPARα and examined tumor growth and profile of the immunological landscape, using various syngeneic tumor models. Significantly larger B16-F10 melanoma and MC-17 fibrosarcoma tumors were observed in PPARα KO mice compared with wild-type control, suggesting that PPARα deletion attenuated the immunological response against cancer. To dissect the role of PPARα in key populations of the innate and adaptive immune system involved in anti-tumor responses, we analyzed the immunological landscape of tumor, tumor draining lymph nodes (TDLN) and spleen, 14-16 days after tumor implantation. Assessment of CD4 + and CD8 + T cells, CD11b +F4/80 + tumor-associated macrophages (TAMs), CD11b +Ly6C hiLy6G - monocytic myeloid derived suppressor cells (M-MDSC), and CD11b +Ly6C loLy6G + polymorphonuclear myeloid derived suppressor cells (PMN-MDSC), by using flow cytometry, showed no quantitative differences between the two experimental groups. Functionally, MDSC from PPARα KO and WT mice showed comparable immunosuppressive properties as determined by suppression assay using splenocytes from OTI transgenic mice. However, PPARα KO TAMs demonstrated a less activated state, as determined by the lower expression levels of MHC-II that is critical for antigen presentation, and CD86 that is critical for T cell costimulation and prevention of T cell anergy and exhaustion. In agreement with these properties of TAMs, CD4 + T cells from TDLN of PPARα KO mice had diminished expression of activation markers, including PD-1, PD-L1 and ICOS, and numerically decreased central memory-like CD4 + T cells (T CM), compared to control tumor bearing mice. Furthermore, CD69, an emerging marker of T cell exhaustion, was significantly upregulated in CD4 + and CD8 + T cells from the TDLN of PPARα KO mice. To determine whether PPARα ablation altered the cell intrinsic properties of myeloid cells and/or T cells resulting in impaired anti-tumor function, we examined in vitro responses of isolated populations. In response to activation via TCR/CD3 and CD28, PPARα deficient T cells had no significant differences in expansion and cytokine production compared to control. In contrast, PPARα deficient Ly6C + monocytes isolated from the bone marrow displayed diminished responses to TLR-mediated signaling as determined by production of IL-6 and TNFα. Our in vitro and in vivo findings reveal a dominant role of PPARα in regulating the fate of innate immune cells thereby altering T cell responses and anti-tumor function. Our findings have implications for the development of new therapeutic approaches to enhance innate immune cell function for the improvement of cancer immunotherapy. Disclosures No relevant conflicts of interest to declare.

scholarly journals 413 GEN-009, a personalized neoantigen vaccine, elicits robust immune responses in individuals with advanced or metastatic solid tumors

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A438-A438
Author(s):  
Mara Shainheit ◽  
Devin Champagne ◽  
Gabriella Santone ◽  
Syukri Shukor ◽  
Ece Bicak ◽  
...  
Keyword(s):  
Clinical Trial ◽  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Solid Tumors ◽  
Ex Vivo ◽  
T Cell Responses ◽  
Checkpoint Blockade ◽  
T Cell Subsets ◽  
Cell Responses

BackgroundATLASTM is a cell-based bioassay that utilizes a cancer patient‘s own monocyte-derived dendritic cells and CD4+ and CD8+ T cells to screen their mutanome and identify neoantigens that elicit robust anti-tumor T cell responses, as well as, deleterious InhibigensTM.1 GEN-009, a personalized vaccine comprised of 4–20 ATLAS-identified neoantigens combined with Hiltonol®, harnesses the power of neoantigen-specific T cells to treat individuals with solid tumors. The safety and efficacy of GEN-009 is being assessed in a phase 1/2a clinical trial (NCT03633110).MethodsA cohort of 15 adults with solid tumors were enrolled in the study. During the screening period, patients received standard of care PD-1-based immunotherapies appropriate for their tumor type. Subsequently, patients were immunized with GEN-009 with additional doses administered at 3, 6, 12, and 24 weeks. Peripheral blood mononuclear cells (PBMCs) were collected at baseline, pre-vaccination (D1), as well as 29, 50, 92, and 176 days post first dose. Vaccine-induced immunogenicity and persistence were assessed by quantifying neoantigen-specific T cell responses in ex vivo and in vitro stimulation dual-analyte fluorospot assays. Polyfunctionality of neoantigen-specific T cells was evaluated by intracellular cytokine staining. Additionally, potential correlations between the ATLAS-identified profile and vaccine-induced immunogenicity were assessed.ResultsGEN-009 augmented T cell responses in 100% of evaluated patients, attributable to vaccine and not checkpoint blockade. Furthermore, neoantigen-induced secretion of IFNγ and/or TNFα by PBMCs, CD4+, and CD8+ T cells was observed in all patients. Responses were primarily from polyfunctional TEM cells and detectable in both CD4+ and CD8+ T cell subsets. Some patients had evidence of epitope spreading. Unique response patterns were observed for each patient with no apparent relationship between tumor types and time to emergence, magnitude or persistence of response. Ex vivo vaccine-induced immune responses were observed as early as 1 month, and in some cases, persisted for 176 days. Clinical efficacy possibly attributable to GEN-009 was observed in several patients, but no correlation has yet been identified with neoantigen number or magnitude of immune response.ConclusionsATLAS empirically identifies stimulatory neoantigens using the patient‘s own immune cells. GEN-009, which is comprised of personalized, ATLAS-identified neoantigens, elicits early, long-lasting and polyfunctional neoantigen-specific CD4+ and CD8+ T cell responses in individuals with advanced cancer. Several patients achieved clinical responses that were possibly attributable to vaccine; efforts are underway to explore T cell correlates of protection. These data support that GEN-009, in combination with checkpoint blockade, represents a unique approach to treat solid tumors.AcknowledgementsWe are grateful to the patients and their families who consented to participate in the GEN-009-101 clinical trial.Trial RegistrationNCT03633110Ethics ApprovalThis study was approved by Western Institutional Review Board, approval number 1-1078861-1. All subjects contributing samples provided signed individual informed consent.ReferenceDeVault V, Starobinets H, Adhikari S, Singh S, Rinaldi S, Classon B, Flechtner J, Lam H. Inhibigens, personal neoantigens that drive suppressive T cell responses, abrogate protection of therapeutic anti-tumor vaccines. J. Immunol 2020; 204(1 Supplement):91.15.

BK Polyomavirus–Specific CD8 T-Cell Expansion In Vitro Using 27mer Peptide Antigens for Developing Adoptive T-Cell Transfer and Vaccination

2020 ◽  
Author(s):  
Maud Wilhelm ◽  
Amandeep Kaur ◽  
Marion Wernli ◽  
Hans H Hirsch
Keyword(s):  
T Cells ◽  
T Cell ◽  
Kidney Transplant ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
T Cell Responses ◽  
Cd8 T Cell ◽  
Cell Responses ◽  
Bk Polyomavirus

Abstract Background BK polyomavirus (BKPyV) remains a significant cause of premature kidney transplant failure. In the absence of effective antivirals, current treatments rely on reducing immunosuppression to regain immune control over BKPyV replication. Increasing BKPyV-specific CD8 T cells correlate with clearance of BKPyV DNAemia in kidney transplant patients. We characterized a novel approach for expanding BKPyV-specific CD8 T cells in vitro using 27mer-long synthetic BKPyV peptides, different types of antigen-presenting cells, and CD4 T cells. Methods Langerhans cells and immature or mature monocyte-derived dendritic cells (Mo-DCs) were generated from peripheral blood mononuclear cells of healthy blood donors, pulsed with synthetic peptide pools consisting of 36 overlapping 27mers (27mP) or 180 15mers (15mP). BKPyV-specific CD8 T-cell responses were assessed by cytokine release assays using 15mP or immunodominant 9mers. Results BKPyV-specific CD8 T cells expanded using 27mP and required mature Mo-DCs (P = .0312) and CD4 T cells (P = .0156) for highest responses. The resulting BKPyV-specific CD8 T cells proliferated, secreted multiple cytokines including interferon γ and tumor necrosis factor α, and were functional (CD107a+/PD1–) and cytotoxic. Conclusions Synthetic 27mP permit expanding BKPyV-specific CD8 T-cell responses when pulsing mature Mo-DCs in presence of CD4 T cells, suggesting novel and safe approaches to vaccination and adoptive T-cell therapies for patients before and after kidney transplantation.

130 Immunogenic potential of chimeric antigen receptor (CAR)-engineered T cells expressing inducible nuclease-deactivated SpCas9 (dCas9)

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A143-A143
Author(s):  
Dharmeshkumar Patel ◽  
Dharmeshkumar Patel ◽  
Angshumala Goswami ◽  
Vitaly Balan ◽  
Zhifen Yang ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Responses ◽  
Cd8 T Cell ◽  
Peptide Sequence ◽  
T Cell Epitopes ◽  
Cell Responses ◽  
Car T Cells ◽  
Car T

BackgroundThe application of CRISPR-Cas9 for personalized medicine is potentially revolutionary for the treatment of several diseases including cancer. However, the bacterial origin of the Cas9 protein raises concerns about immunogenicity. Recent ELISA-based assays detected antibodies against Cas9 from Streptococcus pyogenes (SpCas9) and Staphylococcus aureus (SaCas9) in 5–10% of sera from 343 normal healthy individuals.1,2 SpCas9-specific memory CD8 T cell responses were not demonstrated in those individuals. To date, there are no conclusive studies assessing whether CRISPR-Cas9-modified CAR-T could raise CD8 T cell-mediated immunogenicity in humans. Refuge CAR-T cell platform employs an inducible, non-gene editing, nuclease deactivated Cas9 (dCas9) to modulate gene expression in response to external stimuli such as antigen-dependent CAR signaling to suppress PD-1 expression.MethodsIn the present study, we analyzed two putative HLA-A*02:01 and two HLA-B*07:02-associated SpCas9 T cell epitopes. The candidate epitopes were derived from a prediction algorithm that incorporates T cell receptor contact residue hydrophobicity and HLA binding affinity. We engaged in-vitro sensitization (IVS) assay to identify immunogenic potential of dCas9 peptides.ResultsAutologous IVS assay of T cells in two healthy donor PBMCs identified CD8-T cell responses after two rounds of stimulation against only one HLA-A*02:01-associated Cas9 peptide (sequence NLIALSLGL) P1– while the other candidate epitopes did not elicit any response. Dextramer analysis demonstrated that 15% of CD8+ T cells were specific for P1 and ~11% of CD8+ cells produced INFG upon challenge with P1-loaded T2 cells.ConclusionsOur in-vitro sensitization assay was able to demonstrate that dCas9 epitope P1 is immunogenic and may elicit adaptive immune response against gene edited CAR-T cells. Endogenous processing and presentation of P1 and other putative epitopes by Refuge CAR-T cells are currently being analyzed.AcknowledgementsRefuge Biotechnologies Inc. Menlo Park, California, 94025Trial RegistrationN/AEthics ApprovalN/AConsentN/AReferencesSimhadri VL, McGill J, McMahon S, Wang J, Jiang H, Sauna ZE. Prevalence of Pre-existing Antibodies to CRISPR-Associated Nuclease Cas9 in the USA Population. Mol Ther Methods Clin Dev 2018;10:105–112. Published 2018 Jun 15. doi:10.1016/j.omtm.2018.06.006Ferdosi SR, Ewaisha R, Moghadam F, et al. Multifunctional CRISPR-Cas9 with engineered immunosilenced human T cell epitopes. Nat Commun2019;10(1):1842. Published 2019 Apr 23. doi:10.1038/s41467-019-09693-x

Roscovitine Prevents TCR-Mediated T Cell Clonal Expansion In-Vitro and Protects Against GvHD In-Vivo.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 318-318 ◽  
Author(s):  
Lequn Li ◽  
Hui Wang ◽  
Vassiliki A. Boussiotis
Keyword(s):  
Cell Cycle ◽  
T Cells ◽  
T Cell ◽  
Bone Marrow Cells ◽  
T Cell Responses ◽  
Clonal Expansion ◽  
Control Group ◽  
Cell Responses

Abstract Cell cycle re-entry of quiescent T lymphocytes is required for generation of productive T cell responses. Cyclin-dependent kinases (cdk), particularly cdk2, have an essential role in cell cycle re-entry. Cdk2 promotes phosphorylation of Rb and related pocket proteins thereby reversing their ability to sequester E2F transcription factors. Besides Rb, cdk2 phosphorylates Smad2 and Smad3. Smad3 inhibits cell cycle progression from G1 to S phase, and impaired phosphorylation on the cdk-mediated sites renders it more effective in executing this function. In contrast, cdk-mediated phosphorylation of Smad3 reduces Smad3 transcriptional activity and antiproliferative function. Recently, we determined that induction of T cell tolerance resulted in impaired cdk2 activity, leading to reduced levels of Smad3 phosphorylation on cdk-specific sites and increased Smad3 antiproliferative function due to upregulation of p15. We hypothesized that pharmacologic inhibition of cdk2 during antigen-mediated T cell stimulation might provide an effective strategy to control T cell expansion and induce tolerance. (R)-roscovitine (CYC202) is a potent inhibitor of cdk2-cyclin E, which in higher concentrations also inhibits other cdk-cyclin complexes including cdk7, cdk9 and cdk5. It is currently in clinical trials as anticancer drug and recently was shown to induce long-lasting arrest of murine polycystic kidney disease. We examined the effect of roscovitine on T cell responses in vitro and in vivo. We stimulated C57BL/6 T cells with anti-CD3-plus-anti-CD28 mAbs, DO11.10 TCR-transgenic T cells with OVA peptide or C57BL/6 T cells with MHC disparate Balb/c splenocytes. Addition of roscovitine in these cultures resulted in blockade of cell proliferation without induction of apoptosis. Biochemical analysis revealed that roscovitine prevented phosphorylation of cdk2, downregulation of p27, phosphorylation of Rb and synthesis of cyclin A, suggesting an effective G1/S cell cycle block. To determine whether roscovitine could also inhibit clonal expansion of activated T cells in vivo, we employed a mouse model of GvHD. Recipient (C57BL/6 x DBA/2) F1 mice were lethally irradiated and were subsequently infused with bone marrow cells and splenocytes, as source of allogeneic T cells, from parental C57BL/6 donors. Roscovitine or vehicle-control was given at the time of allogeneic BMT and on a trice-weekly basis thereafter for a total of three weeks. Administration of roscovitine protected against acute GvHD resulting in a median survival of 49 days in the roscovitine-treated group compared to 24 days in the control group (p=0.005), and significantly less weight loss. Importantly, roscovitine treatment had no adverse effects on engraftment, resulting in full donor chimerism in the treated mice. To examine whether tolerance had been induced by in vivo treatment with roscovitine, we examined in vitro rechallenge responses. While control C57BL/6 T cells exhibited robust responses when stimulated with (C57BL/6 x DBA/2) F1 splenocytes, responses of T cells isolated from roscovitine-treated recipients against (C57BL/6 x DBA/2) F1 splenocytes were abrogated. These results indicate that roscovitine has direct effects on preventing TCR-mediated clonal expansion in vitro and in vivo and may provide a novel therapeutic approach for control of GvHD.

Suppression of CTL Responses against AAV-Capsid Epitopes by Peptide-Induced Regulatory T Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 377-377 ◽  
Author(s):  
Daniel J Hui ◽  
Gary C Pien ◽  
Etiena Basner-Tschakarjan ◽  
Federico Mingozzi ◽  
Jonathan D Finn ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Cd8 T Cells ◽  
Transgene Expression ◽  
T Cell Responses ◽  
Effector Cells ◽  
Cell Responses ◽  
Ctl Activity

Abstract Abstract 377 Hemophilia B represents a promising model for the development of adeno-associated viral (AAV) vectors-based gene therapeutics. In the first clinical trial for AAV serotype 2 mediated gene transfer of Factor IX (F.IX) to the liver of severe hemophilia B subjects, transgene expression was short-lived with a gradual decline of F.IX levels. The loss of transgene expression was accompanied by a transient transaminitis, which we hypothesized to be the result of the reactivation of a pool of capsid-specific memory CD8+ T cells originated from a previous exposure to wild-type AAV. These results were unanticipated since previous work in small and large animal models showed that AAV administration is uneventful, allowing prolonged expression of F.IX transgene at therapeutic levels. We developed an in vitro cytotoxicity assay using a human hepatocyte cell line expressing HLA-B*0702, a common MHC class I allele for which the AAV capsid immunodominant epitope VPQYGYLTL was identified. Using this model, we demonstrated that HLA-matched AAV-specific effector CD8+ T cells were able to lyse target hepatocytes transduced with AAV-2. We now use this in vitro model of CTL killing of AAV-transduced hepatocytes to demonstrate the efficacy of a novel strategy to circumvent undesirable immune response through the engagement of regulatory T cells. A recently characterized MHC Class II-restricted T cell epitope (Tregitope) in the Fc fragment of IgG has been shown to induce regulatory T cells in vitro and in vivo (Blood, 2008; 112: 3303-3311). AAV-specific HLA-B*0702 effector cells expanded in the presence of a human Tregitope peptide resulted in 79% to 89% inhibition of cytotoxic activity against peptide-pulsed and AAV-transduced target cells, respectively. These results were confirmed using PBMCs from 5 different donors. A similar degree of inhibition of CTL activity was observed for the HLA allele A*0101, which binds to the AAV-derived epitope SADNNNSEY; co-culture of effector cells with the Tregitope inhibited CTL-mediated killing by 60%. Interestingly, the same Tregitope efficiently mediated suppression of CTL activity in subjects carrying different HLA alleles, indicating a high level of promiscuity of Tregitope binding. Staining for the regulatory T cell markers CD4, CD25, and FoxP3 supported the hypothesis that Tregitopes suppress T cell responses by expanding regulatory T cells; 62.2% of the CD4+ population stained positive for CD25 and FoxP3 in PBMCs expanded against AAV epitopes in the presence of Tregitope, compared with PBMCs expanded against an AAV epitope alone (3.63%), or against an AAV epitope and an irrelevant control peptide (1.94%). Polyfunctional analysis for markers for T cell activation showed that CD8+ T cells incubated in the presence of Tregitope had an approximately 5-fold decrease in production of IL-2 and IFN-γand a 2-fold reduction in TNF-α production, indicating levels of activation close to naïve CD8+ T cells. We further characterized the mechanism of action of Tregitopes by showing that Tregitopes are required at the time of CD8+ T cell priming, as CTL activity of AAV-expanded CD8+ T cells against transduced hepatocytes was not inhibited by the CD4+ T cell fraction of PBMC expanded separately in vitro with Tregitopes only. We conclude that the use of Tregitopes represents a promising strategy for antigen-specific, Treg-mediated modulation of capsid-specific T cell responses. Disclosures: Martin: EpiVax: Employment. De Groot:EpiVax, Inc.: Employment, Equity Ownership.

Sipuleucel-T (sip-T) induced lytic CD8+ T cell responses to target antigens in men with hormone-sensitive and castration-resistant prostate cancer (CRPC).

2017 ◽  
Vol 35 (7_suppl) ◽  
pp. 162-162
Author(s):  
Emmanuel S. Antonarakis ◽  
David I. Quinn ◽  
Adam S. Kibel ◽  
Daniel Peter Petrylak ◽  
Tuyen Vu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Cd8 T Cells ◽  
Mononuclear Cells ◽  
T Cell Responses ◽  
Cell Responses ◽  
Hormone Sensitive ◽  
Ctl Activity

162 Background: Sip-T is an FDA-approved immunotherapy for patients (pts) with asymptomatic or minimally symptomatic metastatic CRPC. Sip-T is manufactured from autologous peripheral blood mononuclear cells cultured with the immunogen PA2024, a fusion antigen of prostatic acid phosphatase (PAP) conjugated to granulocyte macrophage colony-stimulating factor. After sip-T, antibody and T cell responses to PA2024 and/or PAP correlate with improved survival. To further elucidate the mechanism of sip-T–induced immune responses, we evaluated the proliferative and lytic ability of PA2024- and PAP-specific CD8+ T cells. Methods: Mononuclear blood cells were labeled with the membrane dye carboxyfluorescein succinimidyl ester (CFSE) and cultured with PA2024 or PAP. In vitro proliferative and lytic CD8+ (cytotoxic T lymphocyte [CTL]) T cell responses to these antigens were evaluated by flow cytometry. For proliferation, progressive dilution of CFSE was measured. For CTL activity, the loss of intracellular granzyme B (GzB), indicating exocytosis of this apoptosis-mediating enzyme, was assessed. Samples were from 2 sip-T clinical trials STAND (NCT01431391) and STRIDE (NCT01981122), hormone-sensitive and CRPC pts, respectively. Results: Six wk after sip-T administration, CD8+ PAP- and PA2024-specific responses were observed (n=14 pts assessed). The magnitude of PA2024-specific CD8+ proliferative responses was greater than that for PAP-specific responses. CD8+ T cells from a subset of pts who exhibited PA2024- and/or PAP-specific proliferative responses were assessed for lytic ability. After in vitro antigen stimulation, CTL activity in all evaluated samples (n=14, PA2024; n=13, PAP) was demonstrated by a significant decrease (p<0.05) in intracellular GzB relative to a no-antigen control. Conclusions: Sip-T induced CD8+ CTL proliferation against the target antigens PAP and PA2024. Moreover, antigen-specific CTL activity provides the first direct evidence that sip-T can induce tumor cell lysis. These antigen-specific CD8+ lytic abilities were observed within 6 wk following sip-T, suggesting rapidly generated immune responses. Clinical trial information: NCT01431391; NCT01981122.

scholarly journals Development of a System To Study CD4+-T-Cell Responses to Transgenic Ovalbumin-Expressing Toxoplasma gondii during Toxoplasmosis

2004 ◽  
Vol 72 (12) ◽  
pp. 7240-7246 ◽  
Author(s):  
Marion Pepper ◽  
Florence Dzierszinski ◽  
Amy Crawford ◽  
Christopher A. Hunter ◽  
David Roos
Keyword(s):  
T Cells ◽  
T Cell ◽  
Toxoplasma Gondii ◽  
T Cell Responses ◽  
Cell Receptor ◽  
In Vivo Studies ◽  
Cell Responses ◽  
Ifn Γ

ABSTRACT The study of the immune response to Toxoplasma gondii has provided numerous insights into the role of T cells in resistance to intracellular infections. However, the complexity of this eukaryote pathogen has made it difficult to characterize immunodominant epitopes that would allow the identification of T cells with a known specificity for parasite antigens. As a consequence, analysis of T-cell responses to T. gondii has been based on characterization of the percentage of T cells that express an activated phenotype during infection and on the ability of these cells to produce cytokines in response to complex mixtures of parasite antigens. In order to study specific CD4+ T cells responses to T. gondii, recombinant parasites that express a truncated ovalbumin (OVA) protein, in either a cytosolic or a secreted form, were engineered. In vitro and in vivo studies reveal that transgenic parasites expressing secreted OVA are able to stimulate T-cell receptor-transgenic OVA-specific CD4+ T cells to proliferate, express an activated phenotype, and produce gamma interferon (IFN-γ). Furthermore, the adoptive transfer of OVA-specific T cells into IFN-γ−/− mice provided enhanced protection against infection with the OVA-transgenic (but not parental) parasites. Together, these studies establish the utility of this transgenic system to study CD4+-T-cell responses during toxoplasmosis.

scholarly journals The Effects of Trained Innate Immunity on T Cell Responses; Clinical Implications and Knowledge Gaps for Future Research

2021 ◽  
Vol 12 ◽  
Author(s):  
Dearbhla M. Murphy ◽  
Kingston H. G. Mills ◽  
Sharee A. Basdeo
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Cells ◽  
T Cell Responses ◽  
Innate Immune ◽  
Metabolic Reprogramming ◽  
Innate Immune Cells ◽  
Peripheral Tissues ◽  
Trained Immunity ◽  
Cell Responses

The burgeoning field of innate immune training, also called trained immunity, has given immunologists new insights into the role of innate responses in protection against infection and in modulating inflammation. Moreover, it has led to a paradigm shift in the way we think about immune memory and the interplay between innate and adaptive immune systems in conferring immunity against pathogens. Trained immunity is the term used to describe the medium-term epigenetic and metabolic reprogramming of innate immune cells in peripheral tissues or in the bone marrow stem cell niche. It is elicited by an initial challenge, followed by a significant period of rest that results in an altered response to a subsequent, unrelated challenge. Trained immunity can be associated with increased production of proinflammatory mediators, such as IL-1β, TNF and IL-6, and increased expression of markers on innate immune cells associated with antigen presentation to T cells. The microenvironment created by trained innate immune cells during the secondary challenge may have profound effects on T cell responses, such as altering the differentiation, polarisation and function of T cell subtypes, including Th17 cells. In addition, the Th1 cytokine IFN-γ plays a critical role in establishing trained immunity. In this review, we discuss the evidence that trained immunity impacts on or can be impacted by T cells. Understanding the interplay between innate immune training and how it effects adaptive immunity will give insights into how this phenomenon may affect the development or progression of disease and how it could be exploited for therapeutic interventions or to enhance vaccine efficacy.

scholarly journals NLRP6 in Donor T Cells Separately Regulates CD4 and CD8 Mediated Graft-Versus-Host Disease in Experimental Murine BMT

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1926-1926
Author(s):  
Masahiro Suto ◽  
Eri Matsuki ◽  
Erika Sekiguchi ◽  
Hiroya Tamaki ◽  
Isao Tawara ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Research Funding ◽  
Pharmaceutical Research ◽  
T Cell Responses ◽  
Inflammasome Activation ◽  
Activated T Cells ◽  
Cell Responses ◽  
Donor T Cells

NLRP6 (NOD-like receptor family pyrin domain containing 6) is an important inflammasome component and is highly expressed in intestinal epithelial and in immune cells. NLRP6 mediated inflammasome activation plays a critical role in response to intestinal infection and preventing dysbiosis of gut microbiota through the secretion of IL-18 and mucus. However, we recently found that NLRP6 plays a pathogenic role in GVHD that is independent of microbial dysbiosis, which is in contrast to its well-appreciated microbiome-dependent protective role in intestinal colitis and tumorigenesis. Interestingly, we also found that activated T cells increased NLRP6 expression, but the T cell autonomousrole of NLRP6 in regulating T cell responses is unknown. Because NLRP6 is an important regulator of GVH responses, we tested the hypothesis that NLRP6 deficiency in donor T cells would ameliorate GVHD. To test our hypothesis, we first performed adetailed phenotypic analysis of various T cell subsets and activation markers in naïve NLRP6-/-and wild-type (WT) B6 animals and found a similar distribution of naïve, memory, effector and regulatory T cells. In order to examine whether the absence of NLRP6 in donors affects GVHD, WT-BALB/canimals were lethally irradiated (700cGy) and transplanted on day 0 with 5x106bone marrow and 1.0x106 splenic CD90+T cells from either syngeneic WT-BALB/c, allogeneic MHC-mismatched WT-B6 or NLRP6-/-animals. Contrary to our hypothesis, the recipients receiving donor T cells from NLRP6-/-animals showed a significantly worse survival compared to allogeneic WT-B6 animals (p<0.05). GVHD mortality and severity were also increased in an MHC mismatched B6 into B10.BR model, and in an MHC mismatched haploidentical B6 into F1model (p<0.05). In contrast, GVHD severity and mortality were similar in an MHC matched multiple minor antigen mismatched B6 into C3H.sw model. We hypothesized that GVHD severity and mortality was similar in the B6 into C3H.sw model because NLRP6 regulates CD4+ and CD8+ T cell responses, differently. In order to test this, we transplanted C3H.sw recipients as above except we infused either 1x106CD4+ or CD8+ T cells from B6-WT or NLRP6-/-animals. GVHD severity and mortality (P<0.05) were enhanced only when NLRP6-/-CD4+ T cells transplanted. We confirmed enhanced GVHD mortality and severity mediated by donor NLRP6-/-CD4+ T cells in a second MHC-mismatched GVHD model, B6 into BALB/c (p<0.05). To explore how NLRP6 effects T cell responses independent ofinflammasome activation, we tested naïve T cell proliferation in vitro after allogeneic or non-specific TCR stimulation by anti-CD3 and CD28 antibody and found that NLRP6-/-CD4+ but not CD8+T cells proliferated more than WT-B6 CD4+ or CD8+ T cells, respectively, following either stimulus. Furthermore, allogeneicNLRP6-/-T cells also caused greater mortality compared to WT allogenic T cells in a non-irradiated B6 into F1 model, which lacks inflammasome activation associated with conditioning induced DAMPs and PAMPs. Microarray analysis of activated T cells from NLRP6-/-animals showed higher expression of IL-2 and IFN-γ than WT B6 T cells, and we observed no effect of NLRP6 in a Treg suppression assay. These data suggest that NLRP6 regulates CD4+ T cell- mediated immune responses and that NLRP6 in donor T cells is critical for controlling CD4+ T cell mediated GVHD. The effect of NLRP6 on T cell mediated GVL is currently under investigation. Disclosures Tawara: Kyowa Hakko Kirin: Honoraria, Research Funding; Ono Pharmaceutical: Research Funding; Astellas Pharma: Research Funding. Ishizawa:Otsuka Pharmaceutical: Research Funding; Pfizer: Research Funding; Novartis: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau.

Sign in / Sign up

Export Citation Format

Share Document