543 Sensitive quantification and tracking of the active components of a Clonal Neoantigen T cell (cNeT) therapy: From manufacture to peripheral circulation

BackgroundEx-vivo expanded tumour infiltrating lymphocytes (TIL) show promise in delivering durable responses among several solid tumour indications. However, characterising, quantifying and tracking the active component of TIL therapy remains challenging as the expansion process does not distinguish between tumour reactive and bystander T-cells. Achilles Therapeutics has developed ATL001, a patient-specific TIL-based product, manufactured using the VELOS™ process that specifically targets clonal neoantigens present in all tumour cells within a patient. Two Phase I/IIa clinical trials of ATL001 are ongoing in patients with advanced Non-Small Cell Lung Cancer, CHIRON (NCT04032847), and metastatic or recurrent melanoma, THETIS (NCT03997474). Extensive product characterisation and immune-monitoring are performed through Achilles’ manufacturing and translational science programme. This enables precise quantification and characterisation of the active component of this therapy – Clonal Neoantigen T cells (cNeT) – during manufacture and following patient administration, offering unique insight into the mechanism of action of ATL001 and aiding the development of next generation processes.MethodsATL001 was manufactured using procured tumour and matched whole blood from 8 patients enrolled in the THETIS (n=5) and CHIRON (n=3) clinical trials. Following administration of ATL001, peripheral blood samples were collected up to week 6. The active component of the product was detected via re-stimulation with clonal neoantigen peptide pools and evaluation of IFN-γ and/or TNF-α production. Deconvolution of individual reactivities was achieved via ELISPOT assays. Immune reconstitution was evaluated by flow cytometry. cNeT expansion was evaluated by restimulation of isolated PBMCs with peptide pools and individual peptide reactivities (ELISPOT).ResultsThe median age was 57 (range 30 – 71) and 6/8 patients were male. The median number of previous lines of systemic anti-cancer treatment at the time of ATL001 dosing was 2.5 (range 1 – 5). Proportion of cNeT in manufactured products ranged from 0.20% - 77.43% (mean 26.78%) and unique single peptide reactivities were observed in 7 of 8 products (range 0 – 28, mean 8.6). Post-dosing, cNeTs were detected in 5/8 patients and cNeT expansion was observed in 3/5 patients.ConclusionsThese data underscore our ability to sensitively detect, quantify and track the patient-specific cNeT component of ATL001 – during manufacture and post dosing. As the dataset matures, these metrics of detection and expansion will be correlated with product, clinical and genomic characteristics to determine variables associated with peripheral cNeT dynamics and clinical response.ReferencesNCT04032847, NCT03997474Ethics ApprovalThe first 8 patients described have all been located within the UK and both trials (CHIRON and THETIS) have been approved by the UK MHRA (among other international bodies, e.g FDA). Additionally, these trials have been approved by local ethics boards at active sites within the UK. Patient‘s are fully informed by provided materials and investigators prior to consenting to enrol into either ATL001 trial.

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

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.248 ◽

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

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Depletion of Alloreactive T Cells after Haploidentical HSCT: Comparison of Outcomes for Ex Vivo Versus In Vivo Treatment Strategies

Blood ◽

10.1182/blood-2018-99-118857 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3474-3474

Author(s):

Steven Devine ◽

Stephan Mielke ◽

Eduardo Olavarria ◽

Bert Tuk ◽

Kees Meewisse ◽

...

Keyword(s):

Clinical Trials ◽

T Cells ◽

Single Dose ◽

T Cell ◽

Ex Vivo ◽

Chronic Gvhd ◽

Weighted Average ◽

Alloreactive T Cells ◽

Relapse Rates

Abstract Background: The use of haploidentical allogeneic hematopoietic stem cell transplantation (haplo-HSCT) has increased owing to therapeutic advances that have mitigated the main barriers such as high incidence of graft-versus-host disease (GVHD) and non-relapse mortality (NRM). This is primarily attributable to the elimination of alloreactive T cells in either the patient or the graft. Such T-cell depletion can be performed in vivo early after T-cell-replete haplo-HSCT using post-transplant cyclophosphamide (PTCy). Alternatively, T-cell-depleted haplo-HSCT can be supplemented with T-lymphocytes that are depleted ex vivo of their alloreactive component in the form of ATIR101 (Kiadis Pharma). Patient exposure to PTCy to eliminate donor alloreactive cells can be performed easily and at low cost but may cause patient toxicity and increase relapse rates, and it requires post-transplant immune suppression. Although ATIR101 requires cell manufacturing and is more expensive, it limits toxicity to the patient, enables haplo-HSCT without the use of immunosuppressants, and may reduce relapse rates. Both strategies are promising, but no attempt has yet been made to compare clinical results in similar patient populations to delineate key features of alloreactive T-cell depletion performed either ex vivo or in vivo. Methods: Data from published retrospective studies (single-site or registry data) were analyzed to assess clinical outcomes of haplo-HSCT plus PTCy. The 1-year outcomes from these studies were compared with results from a pooled analysis of 2 phase II clinical trials of a single dose of ATIR101 (N=37, all patients with AML/MDS/ALL [CR-AIR-007, CR-AIR-008]). Studies in which PTCy was used in patient populations with >50% AML/MDS/ALL were identified (Ciurea 2015, Piemontese 2017, Solomon 2012, Ciurea 2012, Devillier 2015, Di Stasi 2014, Esquirol 2016, Sugita 2015). The 1-year rates of relapse, relapse-related mortality (RRM), NRM, GVHD, and overall survival (OS) for the ATIR101 clinical trials were compared with the weighted average of these outcomes for the identified studies. OS is known to correlate with disease risk index (DRI; Armand 2014); therefore, publications reporting both OS and DRI (McCurdy 2017, Ciurea 2015, Devillier 2015, Sugita 2015) were identified to compare OS. Differences in DRI between PTCy and ATIR101 study populations were adjusted according to the relationship between DRI and OS. Finally, PTCy studies reporting GVHD-free and relapse-free survival (GRFS) were identified (Solh 2016, McCurdy 2017, Santoro 2017). There is a clinically relevant and statistically significant correlation between GRFS and DRI, so 1-year GRFS rates from the 2 studies reporting DRI status (Solh 2016, McCurdy 2017) were also normalized according to the DRI profile in the ATIR101 clinical trials to allow comparison. Results: The weighted average of PTCy (N=571) outcomes in populations with >50% AML/MDS/ALL vs ATIR101 patient outcomes were: 29% vs 8% for relapse; 18% vs 8% for RRM; 22% vs 33% for NRM; 5% vs 5% for acute GVHD grade III/IV; 24% vs 3% for chronic GVHD; and 60% vs 58% for OS. The OS in DRI-adjusted studies for PTCy (N=561) was similar to that in ATIR101 clinical trials (63% vs 58%, respectively). The GRFS-reporting studies included a total of 708 patients (Sohl 2016, N=128; McCurdy 2017, N=372; Santoro 2017, N=208); 1-year GRFS rates for PTCy in these studies were 33% (95% CI: 25-41), 45% (95% CI: 40-50), and 33% (average), respectively. In the 2 studies reporting DRI (N=500), the DRI profile was more favorable than in the ATIR101 studies and the 1-year GRFS rates normalized in line with the ATIR101 studies were reduced to 30% (Sohl 2016) and 40% (McCurdy 2017). In patients intended to receive a single dose of ATIR101 after haplo-HSCT, Kaplan-Meier estimate of 1-year GRFS was 53% (95% CI 39-72) (Table 1). Conclusion: This is not a head-to-head comparison, so data should be interpreted with caution. However, in these cross-study analyses, first insights into a potential advantage of ex vivo (ATIR101) over in vivo (PTCy) depletion of alloreactive T cells is suggested, including but not limited to rates of relapse, chronic GVHD, and GRFS. A large, phase III, randomized control trial is thus underway to assess the relative safety and efficacy of ATIR101 after T-cell-depleted haplo-HSCT versus PTCy after T-cell-replete haplo-HSCT (CR-AIR-009 HATCY; NCT02999854). Disclosures Devine: Kiadis Pharma: Consultancy. Mielke:Kiadis Pharma: Other: Travel grants, Research Funding. Tuk:Kiadis Pharma: Consultancy. Meewisse:Kiadis Pharma: Employment. Sandler:Kiadis Pharma: Employment. Roy:University of Montreal: Patents & Royalties: Author on patent; Kiadis Pharma: Other: Travel support; Hopital Maisonneuve Rosemont: Patents & Royalties: Author on patent.

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Receptor-gated IL-2 delivery by an anti-human IL-2 antibody activates regulatory T cells in three different species

Science Translational Medicine ◽

10.1126/scitranslmed.abb9283 ◽

2020 ◽

Vol 12 (574) ◽

pp. eabb9283 ◽

Cited By ~ 1

Author(s):

Ufuk Karakus ◽

Dilara Sahin ◽

Peer R. E. Mittl ◽

Petra Mooij ◽

Gerrit Koopman ◽

...

Keyword(s):

Clinical Trials ◽

T Cells ◽

Intracellular Signaling ◽

Metabolic Diseases ◽

Ex Vivo ◽

Rhesus Macaques ◽

Interleukin 2 ◽

Great Promise ◽

A Cell

Stimulation of regulatory T (Treg) cells holds great promise for the treatment of autoimmune, chronic inflammatory, and certain metabolic diseases. Recent clinical trials with low-dose interleukin-2 (IL-2) to expand Treg cells led to beneficial results in autoimmunity, but IL-2 immunotherapy can activate both Treg cells and pathogenic T cells. Use of IL-2 receptor α (IL-2Rα, CD25)–biased IL-2/anti–IL-2 antibody complexes improves IL-2 selectivity for Treg cells; however, the mechanism of action of such IL-2 complexes is incompletely understood, thus hampering their translation into clinical trials. Using a cell-based and dynamic IL-2R platform, we identified a particular anti-human IL-2 antibody, termed UFKA-20. When bound to UFKA-20, IL-2 failed to stimulate cells expressing IL-2Rβ (CD122) and IL-2Rγ (CD132), unless these cells also expressed high amounts of CD25. CD25 allowed IL-2/UFKA-20 complexes to bind, and binding to CD25 in the presence of CD122 and CD132 was followed by rapid dissociation of UFKA-20 from IL-2, delivery of IL-2 to CD122 and CD132, and intracellular signaling. IL-2/UFKA-20 complexes efficiently and preferentially stimulated CD4+ Treg cells in freshly isolated human T cells ex vivo and in mice and rhesus macaques in vivo. The crystal structure of the IL-2/UFKA-20 complex demonstrated that UFKA-20 interfered with IL-2 binding to CD122 and, to a lesser extent, also CD25. Together, we translated CD25-biased IL-2 complexes from mice to nonhuman primates and extended our mechanistic understanding of how CD25-biasing anti-human IL-2 antibodies work, which paves the way to clinical trials of CD25-biased IL-2 complexes.

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A rapid diagnostic test for human regulatory T-cell function to enable regulatory T-cell therapy

Blood ◽

10.1182/blood-2011-09-380048 ◽

2012 ◽

Vol 119 (8) ◽

pp. e57-e66 ◽

Cited By ~ 62

Author(s):

James B. Canavan ◽

Behdad Afzali ◽

Cristiano Scottà ◽

Henrieta Fazekasova ◽

Francis C. Edozie ◽

...

Keyword(s):

Clinical Trials ◽

T Cells ◽

T Cell ◽

Cell Therapy ◽

Autoimmune Diseases ◽

Diagnostic Test ◽

Rapid Diagnostic Test ◽

Regulatory T Cell ◽

Ex Vivo ◽

Treg Function

Abstract Regulatory T cells (CD4+CD25hiCD127loFOXP3+ T cells [Tregs]) are a population of lymphocytes involved in the maintenance of self-tolerance. Abnormalities in function or number of Tregs are a feature of autoimmune diseases in humans. The ability to expand functional Tregs ex vivo makes them ideal candidates for autologous cell therapy to treat human autoimmune diseases and to induce tolerance to transplants. Current tests of Treg function typically take up to 120 hours, a kinetic disadvantage as clinical trials of Tregs will be critically dependent on the availability of rapid diagnostic tests before infusion into humans. Here we evaluate a 7-hour flow cytometric assay for assessing Treg function, using suppression of the activation markers CD69 and CD154 on responder T cells (CD4+CD25− [Tresp]), compared with traditional assays involving inhibition of CFSE dilution and cytokine production. In both freshly isolated and ex vivo expanded Tregs, we describe excellent correlation with gold standard suppressor cell assays. We propose that the kinetic advantage of the new assay may place it as the preferred rapid diagnostic test for the evaluation of Treg function in forthcoming clinical trials of cell therapy, enabling the translation of the large body of preclinical data into potentially useful treatments for human diseases.

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Clinical trial reporting performance of thirty UK universities on ClinicalTrials.gov—evaluation of a new tracking tool for the US clinical trial registry

Trials ◽

10.1186/s13063-021-05330-5 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Sarai Mirjam Keestra ◽

Florence Rodgers ◽

Daphne Lenz ◽

Rhiannon Osborne ◽

Till Bruckner ◽

...

Keyword(s):

Clinical Trial ◽

Clinical Trials ◽

Median Number ◽

World Health ◽

Trial Registry ◽

Clinical Trial Registry ◽

Link Type ◽

The Us ◽

Health Organization ◽

The Uk

AbstractClinical trial transparency forms the foundation of evidence-based medicine, and trial sponsors, especially publicly funded institutions such as universities, have an ethical and scientific responsibility to make the results of clinical trials publicly available in a timely fashion. We assessed whether the thirty UK universities receiving the most Medical Research Council funding in 2017–2018 complied with World Health Organization best practices for clinical trial reporting on the US Clinical Trial Registry (ClinicalTrials.gov). Firstly, we developed and evaluated a novel automated tracking tool (clinical-trials-tracker.com) for clinical trials registered on ClinicalTrials.gov. This tracker identifies the number of due trials (whose completion lies more than 395 days in the past) that have not reported results on the registry and can now be used for all sponsors. Secondly, we used the tracker to determine the number of due clinical trials sponsored by the selected UK universities in October 2020. Thirdly, using the FDAAA Trials Tracker, we identified trials sponsored by these universities that are not complying with reporting requirements under the Food and Drug Administration Amendments Act 2007. Finally, we quantified the average and median number of days between primary completion date and results posting. In October 2020, the universities included in our study were sponsoring 1634 due trials, only 1.6% (n = 26) of which had reported results within a year of completion. 89.8% (n = 1468) of trials remained unreported, and 8.6% (n = 140) of trials reported results late. We also identified 687 trials that contained inconsistent data, suggesting that UK universities often fail to update their data adequately on ClinicalTrials.gov. The mean reporting delay after primary completion for trials that posted results was 981 days, the median 728 days. Only four trials by UK universities violated the FDAAA 2007. We suggest a number of reasons for the poor reporting performance of UK universities on ClinicalTrials.gov: (i) efforts to improve clinical trial reporting in the UK have to date focused on the European clinical trial registry (EU CTR), (ii) the absence of a tracking tool for timely reporting on ClinicalTrials.gov has limited the visibility of institutions’ reporting performance on the US registry and (iii) there is currently a lack of repercussions for UK sponsors who fail to report results on ClinicalTrials.gov which should be addressed in the future.

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A Phase 1 Clinical Trial of Personalized Adoptive Cellular Therapy Targeting Myelodysplastic Syndrome (MDS) Stem Cell Neoantigens (PACTN)

Blood ◽

10.1182/blood-2018-99-118834 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4373-4373

Author(s):

Valentina Ferrari ◽

Tiffany N Tanaka ◽

Alison Tarke ◽

Hanna Fields ◽

Luca Ferrari ◽

...

Keyword(s):

Clinical Trial ◽

T Cells ◽

T Cell ◽

Myelodysplastic Syndrome ◽

Ex Vivo ◽

Phase 1 ◽

Patient Specific ◽

Primary Study ◽

Disease Response ◽

Phase 1 Clinical Trial

Abstract Background: There are few therapeutic options for higher risk patients with myelodysplastic syndrome (MDS) who fail standard therapy, and their 2-year survival rate is approximately 15%. Here we report on a recently initiated collaborative (industry-academia) first-in-human phase 1 clinical trial to assess the safety and tolerability of a novel form of adoptive T cell immunotherapy for such patients that targets patient and disease-specific, mutation-derived neoantigens. This experimental therapy is based on the concept that a) cancer is caused by somatic mutations that may generate novel immunogenic proteins (ie, neopeptides and possible neoantigens), b) that the adaptive immune system can be trained ex vivo to recognize neopeptides as neoantigens and c) that infusion of culture-expanded, neoantigen-immunized autologous T cells may be safe and therapeutically effective. Methods: This is an open-label, phase 1, 3+3 dose escalation trial with 3 cell doses (0.3, 1, and 3.0 × 107nucleated cells/kg) in cohorts of 3 patients each (see www.clinicaltrials.gov, NCT-03258359). Eligible subjects are 18 years of age or older and will have Intermediate, High, or Very High risk MDS by the revised International Prognostic Scoring System, with at least one cytopenia, and will have failed or relapsed after 6 cycles of standard hypomethylating therapy or declined such therapy, an ECOG status 0-2, and adequate organ function. Each patient's MDS-related mutations are identified and autologous T cells are immunized ex vivo with peptides corresponding to the mutated protein(s), then expanded and suitability-tested for experimental infusion (referred to as PACTN). Importantly, the T cells must demonstrate neoantigen specificity and must kill autologous MDS stem-progenitor cells prior to qualification for infusion. Each eligible subject receives a single infusion of autologous PACTN followed by intensive monitoring for adverse events (AEs) for 4 weeks and periodic monitoring for 1 year. The primary study end-point (EP) is assessment of dose-limiting toxicity (DLT) and maximum tolerated PACTN dose (MTD). Secondary EPs include disease response 1 month after PACTN infusion, overall and progression-free survival at 6 and 12 months, and assessment of the peak abundance and persistence of the infused T cells in peripheral blood. Exploratory EPs include an assessment of the effect of PACTN infusion on the allele frequencies of the targeted and non-targeted MDS mutations in blood and marrow leukocytes. Results: At this time, two subjects have been infused with PACTN in the first dose cohort. Neither subject had an infusion reaction, severe AE, or DLT after follow-up for 2-3 months, nor has a disease response occurred in these subjects. Of interest, the infused PACTN product in the first subject showed 59% clonal dominance by a single T cell receptor (TCR) clone that was present at only 0.002% in patient's blood prior to T cell immunization with neoantigen related peptides. Multiple additional expanded TCR clones were also identified in the infused PACTN product. The presence of a dominant TCR clone in the PACTN product enabled the assessment of the in vivo abundance and persistence of the clone after PACTN infusion. The dominant clone expanded between day 1 and day+4 after PACTN infusion to a peak frequency of 0.13%, representing a 64-fold expansion of this TCR compared with the pre-infusion sample of blood leukocytes, then decreased to 0.09% by day +8. The clone was also demonstrated in bone marrow on day +15 at a frequency of 0.03%, representing a 20-fold expansion of this TCR clone compared with the pre-infusion marrow sample. Similar studies on the second subject are in progress, and will be continued in future subjects as the clinical trial continues. Finally, our studies show that it has been possible to effectively immunize autologous T cells to patient-specific neoantigens in all patients studied with MDS (n=4) and also all patients with AML (n=3) studied to date. Conclusion: The early results of this clinical trial support the feasibility and safety of this novel approach to adoptive T cell mediated immunotherapy for patients with higher-risk MDS and encourages continuation of the trial in the higher dose level cohorts. Disclosures Ferrari: Persimmune, Inc.: Employment. Tarke:Persimmune, Inc.: Employment. Fields:Persimmune, Inc.: Employment. Ferrari:Persimmune, Inc.: Employment. Ni:Persimmune, Inc.: Employment. Ferrari:Persimmune, Inc.: Employment. Warner:Persimmune, Inc.: Employment. Jochelson:PersImmune, Inc.: Consultancy. Bejar:Celgene: Consultancy, Honoraria; AbbVie/Genentech: Consultancy, Honoraria; Takeda: Research Funding; Genoptix: Consultancy; Modus Outcomes: Consultancy; Foundation Medicine: Consultancy; Astex/Otsuka: Consultancy, Honoraria. Vitiello:Persimmune, Inc.: Employment. Lane:PersImmune, Inc.: Employment.

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An easy and reliable whole blood freezing method for flow cytometry immunophenotyping and functional analyses

10.1101/2020.08.12.248450 ◽

2020 ◽

Author(s):

Cecile Braudeau ◽

Nina Salabert-Le Guen ◽

Chevreuil Justine ◽

Rimbert Marie ◽

Jerome C. Martin ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

Whole Blood ◽

Mononuclear Cells ◽

Ex Vivo ◽

Immune Monitoring ◽

Fresh Blood ◽

Peripheral Blood Mononuclear ◽

Blood Samples ◽

Whole Blood Samples

ABSTRACTBackgroundImmune profiling by flow cytometry is not always possible on fresh blood samples due to time and/or transport constraints. Besides, the cryopreservation of peripheral blood mononuclear cells (PBMC) requires on-site specialized lab facilities, thus severely restricting the extent by which blood immune monitoring can be applied to multicenter clinical studies. These major limitations can be addressed through the development of simplified whole blood freezing methods.MethodsIn this report, we describe an optimized easy protocol for rapid whole blood freezing with the CryoStor® CS10 solution. Using flow cytometry, we compared cellular viability and composition on cryopreserved whole blood samples to matched fresh blood, as well as fresh and frozen PBMC.ResultsThough partial loss of neutrophils was observed, leucocyte viability was routinely >75% and we verified the preservation of viable T cells, NK cells, monocytes, dendritic cells and eosinophils in frequencies similar to those observed in fresh samples. A moderate decrease in B cell frequencies was observed. Importantly, we validated the possibility to analyze major intracellular markers, such as FOXP3 and Helios in regulatory T cells. Finally, we demonstrated good functional preservation of CS10-cryopreserved cells through the analysis of intracellular cytokine production in ex vivo stimulated T cells (IFNg, IL-4, IL-17A,) and monocytes (IL-1b, IL-6, TNFa).ConclusionsIn conclusion, our protocol provides a robust method to apply reliable immune monitoring studies to cryopreserved whole blood samples, hence offering new important opportunities for the design of future multicenter clinical trials.

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Maraviroc as a potential HIV-1 latency-reversing agent in cell line models and ex vivo CD4 T cells

Journal of General Virology ◽

10.1099/jgv.0.001499 ◽

2020 ◽

Author(s):

Ilaria Vicenti ◽

Filippo Dragoni ◽

Martina Monti ◽

Claudia Maria Trombetta ◽

Alessia Giannini ◽

...

Keyword(s):

T Cells ◽

Cell Line ◽

Cd4 T Cells ◽

Ex Vivo ◽

Reversal Potential ◽

Patient Specific ◽

Luciferase Expression ◽

Target Cells ◽

Reversal Effect ◽

Hiv 1

Recent studies have suggested that the CCR5 antagonist maraviroc (MVC) may exert an HIV-1 latency reversal effect. This study aimed at defining MVC-mediated induction of HIV-1 in three cell line latency models and in ex vivo CD4 T cells from six patients with suppressed viraemia. HIV-1 induction was evaluated in TZM-bl cells by measuring HIV-1 LTR-driven luciferase expression, and in ACH-2 and U1 latently infected cell lines by measuring cell-free (CFR) and cell-associated (CAR) HIV-1 RNA by qPCR. NF-κB p65 was quantified in nuclear extracts by immunodetection. In ex vivo CD4 T cells, CAR, CFR and cell-associated DNA (CAD) were quantified at baseline and 1–7–14 days post-induction (T1, T7, T14). At T7 and T14, the infectivity of the CD4 T cells co-cultured with MOLT-4/CCR5 target cells was evaluated in the TZM-bl assay (TZA). Results were expressed as fold activation (FA) with respect to untreated cells. No LTR activation was observed in TZM-bl cells at any MVC concentration. NF-κB activation was only modestly upregulated (1.6±0.4) in TZM-bl cells with 5 µM MVC. Significant FA of HIV-1 expression was only detected at 80 µM MVC, namely on HIV-1 CFR in U1 (3.1±0.9; P=0.034) and ACH-2 cells (3.9±1.4; P=0.037). CFR was only weakly stimulated at 20 µM in ACH-2 (1.7±1.0 FA) cells and at 5 µM in U1 cells (1.9±0.5 FA). Although no consistent pattern of MVC-mediated activation was observed in ex vivo experiments, substantial FA values were detected sparsely on individual samples with different parameters. Notably, in one sample, MVC stimulated all parameters at T7 (2.3±0.2 CAD, 6.8±3.7 CAR, 18.7±16.7 CFR, 7.3±0.2 TZA). In conclusion, MVC variably induces HIV-1 production in some cell line models not previously used to test its latency reversal potential. In ex vivo CD4 T cells, MVC may exert patient-specific HIV-1 induction; however, clinically relevant patterns, if any, remain to be defined.

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Abstract LB-329: Defining rgulatory T-cells as CD4+CD25hiCD127- or as CD4+CD25hiFoxP3+ in immune monitoring of cancer vaccine clinical trials

10.1158/1538-7445.am10-lb-329 ◽

2010 ◽

Author(s):

Guy T. Clifton ◽

Ritesh Patil ◽

Kevin Clive ◽

Josh Tyler ◽

Athina Zacharia ◽

...

Keyword(s):

Clinical Trials ◽

T Cells ◽

Cancer Vaccine ◽

Immune Monitoring

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Human Langerhans-Type Dendritic Cells Break Tolerance against the Tumor Antigen, WT1, by a Largely IL-15-Dependent Mechanism.

Blood ◽

10.1182/blood.v112.11.1554.1554 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1554-1554

Author(s):

Emanuela Romano ◽

Rosa Barreira da Silva ◽

Sharanya Chandrasekar ◽

Christian Münz ◽

Glenn Heller ◽

...

Keyword(s):

T Cells ◽

Dendritic Cells ◽

Cell Line ◽

Nk Cell ◽

Wilms Tumor ◽

Ex Vivo ◽

Tumor Cell Line ◽

Patient Specific ◽

Specific Lysis ◽

Self Differentiation

Abstract The Wilms’ tumor 1 (WT1) gene encodes a transcription factor essential for cell growth and development, but overexpression in many leukemias, myelodysplastic syndromes, and solid tumors correlates with poor prognosis. The WT1 protein could therefore serve as a therapeutic target, if tolerance could be broken against this self-differentiation, tumor-associated antigen (TAA). Vaccination protocols using peptide-pulsed dendritic cells (DCs) to induce anti-tumor responses present only defined epitopes restricted to certain human leukocyte antigens (HLA). In contrast, vaccination with mRNA-electroporated DCs supports processing and presentation of multiple TAA epitopes tailored to patient-specific HLA. Human Langerhans-type dendritic cells (LCs) derived from CD34+ hematopoietic progenitors are superior to other conventional DC subtypes, like monocyte-derived DCs (moDCs), in producing IL-15 to generate MHC-restricted, Ag-specific cytolytic T lymphocytes (CTL). Mature LCs transcribe 3 to 5-fold higher amounts of IL-15R-alpha mRNA and express the highest levels of IL-15R-alpha protein compared with other conventional DCs as measured by real-time RT-PCR, Western blot, and immunofluorescence microscopy (P<0.001 to 0.05, n=3–4 independent expts). Anti-IL-15R-alpha blockade also significantly reduces but does not completely eliminate the capacity of LCs to stimulate CTL. This indicates that LCs have a greater capacity than moDCs for presenting IL-15 in trans to stimulate lytic T cell responses. We therefore compared the ability of LCs and moDCs electroporated with WT1 mRNA to break tolerance against this self-differentiation TAA by generating CTLs against WT1+ tumor cells. After a single 7-day round of stimulation at T cell:LC ratio of only 10:1 to 30:1 and in the absence of exogenous IL-15, T cells stimulated by WT-1-electroporated LCs lyse 85–95% of a WT-1+ tumor cell line. MoDCs are completely incapable of generating WT-1-specific CTL under comparable conditions and require exogenous IL-15 10ng/ml to generate 70–80% specific lysis of WT-1+ targets. Lysis of a WT-1- HLA-A*0201+ target cell line or NK cell-sensitive LCL721.221 targets is at background levels (<5%) in all cases. Most importantly, WT-1-electoporated LCs also induce 63% lysis (+/− 11 SEM) of primary WT1+ HLA-A*0201+ blasts from a patient with AML after 7 days’ coculture at a T cell:LC ratio of 10:1 without exogenous IL-15. These results demonstrate a major role for the IL-15R-alpha-IL-15 complex on LCs in expanding anti-tumor cytolytic T cells and breaking tolerance against self-differentiation Ags like WT1. These data also support the use of IL-15-producing LCs in active immunization or expansion of T cells ex vivo for adoptive immunotherapy.

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