Ex Vivo Modulation of Donor Cells Results in Enhanced Survival and Reduced Gvhd Mortality

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1884-1884 ◽  
Author(s):  
Lisa Guerrettaz ◽  
Newsha Sahaf ◽  
Leah Mitchell ◽  
Chris Lynn ◽  
Sarah Raynel ◽  
...  
Keyword(s):  
Cell Cycle ◽  
T Cells ◽  
T Cell ◽  
Immune Tolerance ◽  
Peripheral Blood ◽  
Ex Vivo ◽  
T Cell Responses ◽  
Cell Transplant ◽  
Cell Responses ◽  
Mobilized Peripheral Blood

Abstract Allogeneic hematopoietic stem cell transplant (HSCT) represents a potential curative treatment for a number of life-threatening blood malignancies. The utility of this treatment regimen, however, is limited by a number of serious complications including graft versus host disease, which occurs in approximately half of all transplant patients. Standard-of-care for treating acute GvHD has remained unchanged for several decades and consists of high doses of steroids, which are only effective in approximately 35 percent of the cases. Therefore, the reduction of GvHD represents a large unmet medical need, and new approaches are needed to effectively attenuate GvHD. Here we present a fundamentally novel strategy for potentially reducing GVHD - by modulating donor mobilized peripheral blood cells with small molecules prior to HSCT, a programmed mobilized peripheral blood (mPB) allogeneic graft, with reduced T-cell alloreactivity, can be administered as the hematopoietic cell source for HSCT. To this end, we applied our screening platform to identify a combination of small molecule modulators (FT1050, FT4145) that promote the activation of genes implicated in cell cycle, immune tolerance and anti-viral properties of T cells, as well as in the survival, proliferation and engraftment potential of CD34+ cells. Genome-wide expression analysis of the T-cell compartment of mobilized peripheral blood following treatment with FT1050+FT4145 revealed the induction of genes involved in cell cycle (e.g., CCND1, CCNE1), immune tolerance (e.g., ALDH, AREG) and anti-viral properties (e.g., EFNB2). To further assess the therapeutic impact of ex vivo programming with FT1050 and FT4145, a number of T cell assays to assess T cell phenotype and function were conducted on mPB. Overall, ex vivo programming of mPB resulted in reduced allogeneic T cell responses and was accompanied by reduced capacity of modulated T cells to produce Interferon Gamma (IFN-ɣ). Concomitantly, the ability of the modulated T cells to make Interleukin 4 (IL-4) and 10 (IL-10) was enhanced, suggesting a polarization of these cells towards a less inflammatory functional state. This was further evidenced by increased surface expression of an immune-inhibitory molecule, PD1, and reduced expression of the activation markers 41BB and ICOS. We next examined the potential beneficial role of ex vivo programming with FT1050+FT4145 in a major histocompatibility complex (MHC) mis-matched HSCT mouse model. Briefly, lethally irradiated BALB/c mice received bone marrow and splenocytes from C57BL/6 donor mice pulse treated with vehicle or FT1050+FT4145. Significantly less GvHD, as determined by survival, weight loss, GVHD score (diarrhea, inactivity, hunched posture, ruffled fur, eye lesion, snout swelling/skin integrity), cytokine production and histopathology of GvHD target organs was observed in recipients receiving FT1050+FT4145 treated cells as compared to those receiving vehicle treated cells. In addition, we observed increased levels of donor T regulatory cells (Tregs) in secondary lymphoid organs concomitant with decreased levels of circulating IFN-ɣ in recipients receiving FT1050+FT4145 treated cells. Based on the attenuation of alloreactive T-cell responses in these preclinical studies, we believe our findings provide a compelling scientific basis to support the clinical evaluation of ex vivo programmed mobilized peripheral blood in patients undergoing HSCT for the treatment of hematologic malignancies. Disclosures Levin: Fate Therapeutics, Inc: Employment, Equity Ownership. Shoemaker:Fate Therapeutics Inc: Employment.

Cytotoxic T Lymphocytes (CTL) Specific for CMV, Adenovirus, and EBV Can Be Generated From Naive T Cells for Adoptive Immunotherapy.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 504-504
Author(s):  
Patrick Hanley ◽  
Barbara Savoldo ◽  
Conrad Russell Young Cruz ◽  
Ann M. Leen ◽  
Jeffrey J. Molldrem ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Adoptive Immunotherapy ◽  
Peripheral Blood ◽  
T Cell Responses ◽  
Cell Transplant ◽  
Cell Responses ◽  
Specific Ctls ◽  
Ifn Γ

Abstract Abstract 504 Allogeneic stem cell transplantation is the treatment of choice for patients with high-risk hematologic malignancies. Umbilical cord blood (CB) has emerged as an important source of stem cells for allotransplant patients lacking human leukocyte antigen (HLA)-matched donors–a significant problem for minorities. T cells in UCB grafts are, however, virus-naïve, leading to higher mortality rates due to infections with CMV, EBV, adenovirus (Ad) and other viruses. Peripheral Blood Stem Cell Transplant (PBSCT) from CMV-seronegative (CMVneg) donors to CMV-seropositive (CMVpos) recipients produces a similarly high incidence of CMV infection since donor T cells are naïve to this virus. Adoptive immunotherapy with peripheral blood (PB)-derived CMV/Ad-specific CTL generated from CMVpos donors effectively prevents CMV clinical disease after PBSCT, but this option has not been feasible when the donor cells are naive, irrespective of whether they are sourced from CB or CMV-PBS, since CTL generation from these donors has been unsuccessful. We have now overcome this problem and can routinely generate CMV, Ad and EBV-specific CTLs from CB and CMV specific CTLs from seronegative PBSC. We used an Ad5f35vector carrying the CMVpp65 transgene to transduce CB-derived or PB-derived dendritic cells and stimulate virus-specific CTL in the presence of IL-7, IL-12 and IL-15. This was followed by 2 stimulations with autologous EBV-lymphoblastoid cell lines (LCL) transduced with the same vector. The 9 CB-derived CTL lines we made in this way contained a mean of 87% (range 81-94) CD8+ and 26% (range 12-40) CD4+T cells, and exhibited significant cytotoxicity in 51Cr release assays against CMVpp65, Adhexon, and LCL targets. In IFN-γ ELISPOT assays there was a mean of 209 (range 45-694), 74 (range 0-128), and 157 (range 23-291) SFC following incubation with CMVpp65, Adhexon peptides and LCL, respectively. In addition, we generated CMVpp65, Adhexon, and LCL-specific responses from the peripheral blood of 4 CMVneg adult donors, which produced a mean of 92 (range 50-126), 163 (range 69-293), and 62 (range 37-86) SFC to CMVpp65 respectively. Neither CB- nor CMVneg-derived CTL responded to irrelevant peptides. Of note, the virus-specific T cells that we expanded from both CB and CMVneg donors derived only from T cells with a naive phenotype (CD45RA+/CCR7+). Moreover, both CB and CMVneg-derived CTL recognized ‘unconventional‘ CMV pp65 epitopes, as identified by overlapping pp65 peptide pools and confirmed by IFN-γ ELISPOT as well as multimer analysis (Table 1). In HLA-A2+ subjects, these naive-derived CTLs did not recognize conventional HLA-A2-associated CMV pp65 epitopes such as NLV, suggesting an inherent difference between naïve and memory T cell responses to CMV. In summary, virus-specific responses T cell responses can be obtained even from CB and virus-naive adult donors and may allow prevention and treatment of viral disease in the recipients of these allografts. 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.

scholarly journals Characterisation of CD154+ T cells following ex vivo allergen stimulation illustrates distinct T cell responses to seasonal and perennial allergens in allergic and non-allergic individuals

2013 ◽  
Vol 14 (1) ◽  
pp. 49 ◽  
Author(s):  
Karen A Smith ◽  
Nicola J Gray ◽  
Femi Saleh ◽  
Elizabeth Cheek ◽  
Anthony J Frew ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Ex Vivo ◽  
T Cell Responses ◽  
Cell Responses ◽  
Perennial Allergens

scholarly journals Increased Loss of CCR5+ CD45RA− CD4+ T Cells in CD8+ Lymphocyte-Depleted Simian Immunodeficiency Virus-Infected Rhesus Monkeys

2008 ◽  
Vol 82 (11) ◽  
pp. 5618-5630 ◽  
Author(s):  
Ronald S. Veazey ◽  
Paula M. Acierno ◽  
Kimberly J. McEvers ◽  
Susanne H. C. Baumeister ◽  
Gabriel J. Foster ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Lymph Nodes ◽  
Simian Immunodeficiency Virus ◽  
Peripheral Blood ◽  
T Cell Responses ◽  
Lymphocyte Depletion ◽  
Cell Responses ◽  
Immunodeficiency Virus ◽  
Siv Infection

ABSTRACT Previously we have shown that CD8+ T cells are critical for containment of simian immunodeficiency virus (SIV) viremia and that rapid and profound depletion of CD4+ T cells occurs in the intestinal tract of acutely infected macaques. To determine the impact of SIV-specific CD8+ T-cell responses on the magnitude of the CD4+ T-cell depletion, we investigated the effect of CD8+ lymphocyte depletion during primary SIV infection on CD4+ T-cell subsets and function in peripheral blood, lymph nodes, and intestinal tissues. In peripheral blood, CD8+ lymphocyte-depletion changed the dynamics of CD4+ T-cell loss, resulting in a more pronounced loss 2 weeks after infection, followed by a temporal rebound approximately 2 months after infection, when absolute numbers of CD4+ T cells were restored to baseline levels. These CD4+ T cells showed a markedly skewed phenotype, however, as there were decreased levels of memory cells in CD8+ lymphocyte-depleted macaques compared to controls. In intestinal tissues and lymph nodes, we observed a significantly higher loss of CCR5+ CD45RA− CD4+ T cells in CD8+ lymphocyte-depleted macaques than in controls, suggesting that these SIV-targeted CD4+ T cells were eliminated more efficiently in CD8+ lymphocyte-depleted animals. Also, CD8+ lymphocyte depletion significantly affected the ability to generate SIV Gag-specific CD4+ T-cell responses and neutralizing antibodies. These results reemphasize that SIV-specific CD8+ T-cell responses are absolutely critical to initiate at least partial control of SIV infection.

scholarly journals 248 Empiric profiling of peripheral T cell recall responses to tumor mutanomes versus in silico predictions in NSCLC patients undergoing pembrolizumab treatment ± chemotherapy

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A268-A268
Author(s):  
Madison Milaszewski ◽  
James Loizeaux ◽  
Emily Tjon ◽  
Crystal Cabral ◽  
Tulin Dadali ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Combination Therapy ◽  
In Silico ◽  
Ex Vivo ◽  
T Cell Responses ◽  
Patient Specific ◽  
In Silico Prediction ◽  
Cell Responses ◽  
In Silico Predictions

BackgroundEffective immune checkpoint blockade (ICB) treatment is dependent on T-cell recognition of patient-specific mutations (neoantigens). Empirical identification of neoantigens ex vivo has revealed shortcomings of in silico predictions.1 To better understand the impact of ICB treatment on T cell responses and differences between in silico and in vitro methods, neoantigen-specific T cell responses were evaluated in patients with non-small cell lung cancer undergoing first-line therapy with pembrolizumab ± chemotherapy.MethodsTumor and whole blood samples were collected from 14 patients prior to and after immunotherapy; seven each in monotherapy and combination therapy cohorts. The ex vivo ATLAS™ platform was used to profile neoantigen-specific T-cell responses. Patient-specific tumor mutations identified by next-generation sequencing (NGS) were expressed individually as ATLAS clones, processed patient-specific autologous antigen presenting cells, and presented to their T cells in vitro. ATLAS-verified antigens were compared with epitope predictions made using algorithms.ResultsOn average, 150 (range 37–339) non-synonymous mutations were identified. Pre-treatment, ATLAS identified T cell responses to a median of 15% (9–25%) of mutations, with nearly equal proportions of neoantigens (8%, 5–15%) and Inhibigens™, targets of suppressive T cell responses (8%, 3–13%). The combination therapy cohort had more confirmed neoantigens (46, 20–103) than the monotherapy cohort (7, 6–79). After treatment, the median ratio of CD4:CD8 T cells doubled in the monotherapy but not combination cohort (1.2 to 2.4 v. 1.6 to 1.3). Upon non-specific stimulation, T cells from patients on combination therapy expanded poorly relative to monotherapy (24 v. 65-fold, p = 0.014); no significant differences were observed pre-treatment (22 v. 18-fold, p = 0.1578). Post-treatment, the median number of CD8 neoantigens increased in the combination therapy cohort (11 to 15) but in monotherapy were mostly unchanged (6 to 7). Across timepoints, 36% of ATLAS-identified responses overlapped. In silico analysis resulted in 1,895 predicted epitopes among 961 total mutations; among those, 30% were confirmed with ATLAS, although nearly half were Inhibigens, which could not be predicted. Moreover, 50% of confirmed neoantigens were missed by in silico prediction.ConclusionsMonotherapy and combination therapy had differential effects on CD4:CD8 T cell ratios and their non-specific expansion. A greater proportion of neoantigens was identified than previously reported in studies employing in silico predictions prior to empirical verification.2 Overlap between confirmed antigens and in silico prediction was observed, but in silico prediction continued to have a large false negative rate and could not characterize Inhibigens.AcknowledgementsWe would like to acknowledge and thank the patients and their families for participating in this study.ReferencesLam H, McNeil LK, Starobinets H, DeVault VL, Cohen RB, Twardowski P, Johnson ML, Gillison ML, Stein MN, Vaishampayan UN, DeCillis AP, Foti JJ, Vemulapalli V, Tjon E, Ferber K, DeOliveira DB, Broom W, Agnihotri P, Jaffee EM, Wong KK, Drake CG, Carroll PM, Davis TA, Flechtner JB. An empirical antigen selection method identifies neoantigens that either elicit broad antitumor T-cell responses or drive tumor growth. Cancer Discov 2021;11(3):696–713. doi: 10.1158/2159- 8290.CD-20-0377. Epub 2021 January 27. PMID: 33504579. Rosenberg SA. Immersion in the search for effective cancer immunotherapies. Mol Med 27,63(2021). https://doi.org/10.1186/s10020-021-00321-3

Long term results from a phase 1 trial of GEN-009, a personalized neoantigen vaccine, combined with PD-1 inhibition in advanced solid tumors.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. 2613-2613
Author(s):  
Maura L. Gillison ◽  
Mark M. Awad ◽  
Przemyslaw Twardowski ◽  
Ammar Sukari ◽  
Melissa Lynne Johnson ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Solid Tumors ◽  
Salvage Therapy ◽  
Ex Vivo ◽  
T Cell Responses ◽  
Long Term Results ◽  
Advanced Solid Tumors ◽  
Cell Responses

2613 Background: GEN-009 is an adjuvanted personalized cancer vaccine containing up to 20 neoantigens selected by ATLAS, an ex vivo bioassay screening autologous T cells for immune responses against both neoantigens as well as Inhibigens. Inhibigen-specific T cells suppress immunity and have been shown to accelerate tumor progression in mice and are avoided in GEN-009. In cohort A, all patients immunized in the adjuvant setting with GEN-009 monotherapy developed immune responses. Nearly all (99%) of selected peptides were immunogenic: ex vivo CD4+ and CD8+ fluorospot responses specific for 51% and 41% of immunized peptides, respectively. Seven of 8 patients continue without progression with a median follow up of 18 months. Methods: GEN-009 is being evaluated in patients (pts) with advanced cancer who received standard-of-care (SOC) PD-1 inhibitor as monotherapy or in combination therapy during vaccine manufacturing. Five vaccine doses were administered over 24 weeks in combination with a PD-1 CPI. Patients who progressed prior to vaccination received alternative salvage therapy followed by GEN-009 in combination. Peripheral T cell responses were measured by fluorospot assays in ex vivo and in vitro stimulation. Results: 15 pts received GEN-009 in combination with a PD-1 inhibitor; 1 patient received GEN-009 monotherapy. Median number of neoantigens per vaccine was 14 (5-18). GEN-009-related adverse events were limited to vaccine injection site reactions and mild myalgias or fatigue. Longitudinal evaluation of ex vivo T cell responses revealed that sequential vaccination with GEN-009 had an overall additive effect on the robustness of IFNγ secretion and responses were persistent for at least 6 months in some patients. Epitope spread was detected in CPI sensitive patients, but not in CPI refractory patients receiving salvage therapy. Three patients who responded to PD-1 inhibition followed by disease stabilization then demonstrated further reduction after GEN-009 vaccination that could represent vaccine effect. Eight of 9 CPI responsive patients are progression-free from 3 to 10 months after first vaccine dose. Four of 7 CPI refractory patients have experienced unexpected prolonged stable disease after vaccination of up to 8 months after vaccination. 2 of 2 patients with available samples lost all evidence of circulating tumor DNA including non-targeted neoantigens. Conclusions: Vaccination with GEN-009 in combination with anti-PD-1 CPI in patients with advanced solid tumors shows little additive toxicity. Preliminary data demonstrate induction of broad neoantigen-specific immune responses and epitope spreading in the presence of PD-1 CPI. Broad immunity against tumor specific targets and encouraging patient outcomes support further study. Clinical trial information: NCT03633110.

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.

Ex-Vivo Characterization of Polyclonal Memory CD8+ T-Cell Responses to PRAME-Specific Peptides in Patients with Acute Leukemia and Chronic Myeloid Leukemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2910-2910
Author(s):  
Katayoun Rezvani ◽  
Agnes S. M. Yong ◽  
Abdul Tawab ◽  
Behnam Jafarpour ◽  
Rhoda Eniafe ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Myeloid Leukemia ◽  
Ex Vivo ◽  
T Cell Responses ◽  
Cd8 T Cell ◽  
High Avidity ◽  
Cell Responses ◽  
Ifn Γ

Abstract PRAME (Preferentially expressed antigen of melanoma) is aberrantly expressed in hematological malignancies and may be a useful target for immunotherapy in leukemia. We studied CD8+ T-cell responses to four HLA-A*0201-restricted PRAME-derived epitopes (PRA100, PRA142, PRA300, PRA425) in HLA-A*0201-positive patients with acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML) and healthy donors, using PRA300/HLA-A*0201 tetramer staining, intracellular cytokine (IC) assay and ex-vivo and cultured ELISPOT analysis. CD8+ T-cells recognizing PRAME peptides were detected directly ex-vivo in 4/10 ALL, 6/10 AML, 3/10 CML patients and 3/10 donors. The frequency of PRAME-specific CD8+ T-cells was greater in patients with AML, CML and ALL than in healthy controls. All peptides were immunogenic in patients, whilst PRA300 was the only immunogenic peptide in donors. High PRAME expression in patient peripheral blood mononuclear cells was associated with responses to two or more PRAME epitopes (4/7 vs. 0/23 in individuals with low PRAME expression, P = 0.001), suggesting a PRAME-driven T-cell response. In 2 patients studied PRA300/HLA-A*0201+ CD8+T-cells were found to be a mixture of effector and central memory phenotypes. To determine the functional avidity of the PRAME T-cell response, the response of CD8+ T-cells to stimulation with 2 concentrations of peptide was measured by IC-IFN-γ staining. High-avidity CD8+ T-cells were defined as those capable of producing IFN-γ in response to the lower concentration of peptide (0.1μM), while low-avidity CD8+ T-cells were those that only produced IFN-γ in response to the higher concentration of peptide (10 μM). Both high and low-avidity CD8+ T-cell responses could be detected for all peptides tested (median 1.05, 0.90, 0.52, 0.40 high/lowavidity ratios for PRA100, PRA142, PRA300 and PRA425 respectively). In patients with high PRAME expression (>0.001 PRAME/ABL) low-avidity CD8+ T-cell responses to PRAME peptides were more prominent than high-avidity responses, suggesting selective deletion of high-avidity T-cells. In contrast, in some patients with levels <0.001 PRAME/ABL, we could detect the presence of high-avidity CD8+ T-cell responses to PRAME. PRAME-specific CD8+ T-cells were further characterized by IC staining for IL-2, IL-4 and IL-10 production and CD107a mobilization (as a marker of cytotoxicity). Following stimulation with the relevant PRAME peptide, there was no significant production of IL-2, IL-4 or IL-10, suggesting a Tc1 effector response but no significant CD107a mobilization was detected despite significant CD107a mobilization in the same patient in response to CMVpp65495. This finding suggests that patients with leukemia have a selective functional impairment of PRAME-specific CD8+ T-cells, consistent with PRAME-specific T cell exhaustion. However, PRAME-specific T-cells were readily expanded in the presence of cytokines in short-term cultures in-vitro to produce IFN-γ, suggesting that it may be possible to improve the functional capacity of PRAME-specific T-cells for therapeutic purposes. These results provide evidence for spontaneous T-cell reactivity against multiple epitopes of PRAME in ALL, AML and CML and support the usefulness of PRAME as a target for immunotherapy in leukemia. The predominance of low-avidity PRAME-specific CD8+ T-cells suggests that achievement of a state of minimal residual disease may be required prior to peptide vaccination to augment T-cell immune surveillance.

Imiquimod: A TLR-7 agonist as adjuvant for a recombinant protein cancer vaccine

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 8545-8545
Author(s):  
S. Adams ◽  
D. O'Neill ◽  
D. Nonaka ◽  
O. Manches ◽  
L. Chiriboga ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Ex Vivo ◽  
Intracellular Cytokine Staining ◽  
T Cell Responses ◽  
Protein Vaccine ◽  
Imiquimod Cream ◽  
Antigen Uptake ◽  
Cell Responses ◽  
Cell Immunity

8545 Purpose: This clinical trial evaluates the safety and adjuvant activity of imiquimod, a toll-like receptor (TLR)-7 agonist, when given with a NY-ESO-1 protein vaccine. Imiquimod, by locally activating and recruiting dendritic cells (DCs) into the skin, is expected to stimulate antigen uptake by DCs, induce maturation and migration to draining lymph nodes, and to induce antigen-specific T and B cell immunity. Methods: Pilot study; 9 patients with resected stage 2B-3C malignant melanoma. Four 21 day cycles consisted of topical imiquimod cream (250 mg) on days 1–5 and id. injected NY-ESO-1 protein (100 mcg) into the site on day 3. Blood was drawn at several time points for immune monitoring; skin punch biopsies were obtained from control, imiquimod and vaccination sites 48 hours after the last vaccination. Results: The regimen was tolerated well, all patients completed four vaccinations. AEs were mild and transient and included injection site reactions (8/9 patients), fatigue (4/9 patients) and fever (2/9 patients). Significant levels of antigen-specific CD4+ or CD8+ T cell responses were not detected in ex-vivo ELISPOT assays. However, intracellular cytokine staining assays after in vitro pre-stimulation indicated that 6 of 8 subjects developed NY-ESO-1 CD4+ T cell responses. Humoral immunity was manifest by the induction of anti-NY-ESO-1 antibodies in 7/9 patients post-vaccination. Histochemistry of skin sections showed significant dermal mononuclear cell infiltrates in Imiquimod treated skin, whereas none were seen in untreated skin (p<0.01). IHC revealed markedly increased numbers of CD3+ (T-cells), CD68+ (macrophages/monocytes), CD123+ (plasmacytoid DCs) and DC-LAMP+ (mature myeloid DCs) immune cells in Imiquimod treated skin when compared with control skin of the same patients (p<0.05). Conclusion: Imiquimod, a topical immune response modifier, generated clear inflammatory infiltrates in the dermis, with significant increases in antigen-presenting cells and T cells. Imiquimod was well tolerated when used as an adjuvant to an NY-ESO-1 protein vaccine. Systemic immunity of both humoral and cellular types was induced in the majority of patients; however, responses were weak and the vaccine combination needs to be optimized in future studies. No significant financial relationships to disclose.

scholarly journals The T-Cell Receptor Repertoire Predicts Interim-PET in Patients with DLBCL Treated with R-CHOP: An Observational Study from a Prospective Clinical Trial

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 825-825
Author(s):  
Mohamed Shanavas ◽  
Mark Hertzberg ◽  
Rodney J Hicks ◽  
John F Seymour ◽  
Joshua W.D. Tobin ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Peripheral Blood ◽  
T Cell Responses ◽  
Cell Receptor ◽  
Variable Degree ◽  
Cell Responses ◽  
Interim Pet ◽  
Tcr Repertoire

Abstract T-cell infiltration of the tumor microenvironment (TME) in DLBCL is a key determinant of response to chemo-immunotherapy (Keane, Lancet Haem 2015). We have previously shown that greater diversity of the T-cell receptor (TCR) repertoire within the TME is correlated with improved survival following R-CHOP in DLBCL (Keane, CCR 2017). There are limited data on the impact of the intratumoral TCR repertoire on interim-PET (iPET), the relationship between intratumoral and circulating TCRs, and on dynamic changes of the TCR during therapy. In this study, we interrogated the TCR repertoire in a subset of DLBCL patients treated on the prospective Australasian Leukaemia Lymphoma Group NHL21 study (Hertzberg, Haematologica 2017), in which all patients had 4x RCHOP prior to iPET risk stratification. The CDR3 region of TCRβ chain underwent high-throughput unbiased TCRβ sequencing (Adaptive Biotechnologies). Metrics included: productive templates (total functional T-cells), productive rearrangements (functional T-cells with distinct specificity), productive clonality (repertoire unevenness due to clonal expansions), and maximal frequency clones (% most dominant single clone). Matched intratumoral diagnostic samples, blood at pre-therapy and post-cycle 4 (at the time of iPET) were tested. 42 patients (enriched for iPET+ cases) had sufficient material for testing. Median age was 55 (range 22-69) years and 72% were males. IPI was low/intermediate/high in 13/63/25% respectively. Cell of origin (COO) by Lymph 2CX method (nanoString) was ABC in 30%, and GCB in 44%. 40% were iPET+. In tissue, there was a median of 4652 productive templates, translating into 2998 productive rearrangements identified. Notably, the clonal repertoire of intratumoral TCRs in iPET+ patients was larger than iPET-ve patients (productive clonality 8.1 vs 5.1 x10-2, p=0.04), whereas the numbers of functional T-cells did not vary between groups. Comparing the tumor with the blood samples showed a high, but variable, degree of overlap between peripheral blood and the TME - TCR repertoire. Median number of top 100 tumor tissue clones shared in peripheral blood was 53.5 (range, 1-97) in pre-therapy and 39.5 (range, 0-93) in post-therapy blood, indicating that the both the circulation and the tumor likely contribute to immune-surveillance. In pre-therapy blood, the median productive templates and productive rearrangements were 44,950 (range, 6,003-273,765) and 29,090 (range, 5,190-152,706), and the median clonality was 8.5 (1.46-45.3) x 10-2. There were no differences between iPET+ and iPET-ve patients in these parameters. However, there was a marked change in T-cell composition between time points. Interestingly, in iPET-ve patients clonality measures were increased, with productive clonality 9.4 vs 14.4 x10-2, p=0.03; and % maximum productive frequency 3.39 vs 5.89, p=0.04. These findings demonstrate that the intratumoral TCR repertoire, and sequential blood sampling provide important information on outcome in DLBCL treated with RCHOP. A highly clonal T-cell repertoire in the TME was associated with iPET positivity after 4 cycles of R-CHOP. In line with findings in solid cancers treated with checkpoint blockade, development of clonal responses in peripheral blood was associated with iPET negativity. These findings indicate that clones expanded during therapy may be important in tumor clearance but that highly clonal T-cell responses in the tumor at diagnosis may hinder expansion of other T-cell responses to neoantigens. The circulating TCR composition is representative of the TME. These findings will assist the rationale design and therapeutic monitoring of novel immuno-therapeutic strategies. Disclosures No relevant conflicts of interest to declare.

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