scholarly journals 764 Expansion with IL-15 increases cytotoxicity of Vγ9Vδ2 T cells and is associated with higher levels of cytotoxic molecules and T-bet

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A812-A812
Author(s):  
Pia Aehnlich ◽  
Per Thor Straten ◽  
Ana Micaela Carnaz Simoes ◽  
Signe Skadborg ◽  
Gitte Olofsson
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Expansion ◽  
Ex Vivo ◽  
Adoptive Cell Therapy ◽  
Hematological Cancers ◽  
Cytotoxic Molecules ◽  
Vγ9vδ2 T Cells ◽  
T Cell Expansion

BackgroundAdoptive cell therapy (ACT) is an approved treatment option for certain hematological cancers and has also shown success for some solid cancers. Still, benefit and eligibility do not extend to all patients. ACT with Vγ9Vδ2 T cells is a promising approach to overcome this hurdle.MethodsIn this study, we explored the effect of different cytokine conditions on the expansion of Vγ9Vδ2 T cells in vitro.ResultsWe could show that Vγ9Vδ2 T cell expansion is feasible with two different cytokine conditions: (a) 1000U/ml interleukin (IL)-2 and (b) 100U/ml IL-2+100U/ml IL-15. We did not observe differences in expansion rate or Vγ9Vδ2 T cell purity between the conditions; however, IL-2/IL-15-expanded Vγ9Vδ2 T cells displayed enhanced cytotoxicity against tumor cells, also in hypoxia. While this increase in killing capacity was not reflected in phenotype, we demonstrated that IL-2/IL-15-expanded Vγ9Vδ2 T cells harbor increased amounts of perforin, granzyme B and granulysin in a resting state and release more upon activation. IL-2/IL-15-expanded Vγ9Vδ2 T cells also showed higher levels of transcription factor T-bet, which could indicate that T-bet and cytotoxic molecule levels confer the increased cytotoxicity.ConclusionsThese results advocate the inclusion of IL-15 into ex vivo Vγ9Vδ2 T cell expansion protocols in future clinical studies.

95 Inhibition of AKT signaling during expansion of TCR-engineered T-cells from patient leukocyte material generates SPEAR T-cells with enhanced functional potential in vitro

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A106-A106
Author(s):  
Katerina Mardilovich ◽  
Lilli Wang ◽  
Rachel Kenneil ◽  
Gareth Betts ◽  
Natalie Bath ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Expansion ◽  
Ex Vivo ◽  
Akt Signaling ◽  
Akt Inhibitor ◽  
Healthy Donors ◽  
Engineered T Cells ◽  
T Cell Expansion

BackgroundT-cells attributes for adoptive cell therapy of patients with advanced cancer can be optimized during ex vivo expansion culture. Autologous TCR-engineered T-cells targeting the MAGE-A4 antigen with Specific Peptide Enhanced Affinity Receptors (SPEAR T-cells) have shown promise in the clinic.1 The highly variable leukocyte material obtained from individual patients during apheresis can present a manufacturing challenge for autologous T-cell therapies. The degree of ex vivo expansion and the functional attributes of the expanded T-cell product impact therapeutic efficacy and can be suboptimal for some patient apheresis material. Both TCR and cytokine growth factor signals used for ex vivo T-cell expansion promote robust activation of AKT (Protein Kinase B) signaling, which drives T-cell activation, proliferation, and terminal differentiation. It is hypothesized that inhibition of AKT signaling during T-cell expansion may uncouple proliferation and terminal differentiation, leading to the generation of less differentiated T-cells that may have functional benefit in vivo.2 3MethodsWe evaluated use of an AKT inhibitor during SPEAR T-cell manufacturing using leukocytes from healthy donors and patients with advanced solid cancers.ResultsAKT inhibition resulted in the generation of a more consistent expansion and phenotype of the final T-cell product. This was observed using two SPEAR T-cell constructs, ADP-A2M4 and ADP-A2M4CD8. Ex vivo SPEAR T-cell expansion in the presence of an AKT inhibitor generated CD8+ T-cells that maintained a less differentiated phenotype (based on CCR7+CD45RA+ and CD62L+ expression). AKT inhibition was associated with enhanced antigen-specific responses of SPEAR T-cells in vitro, including effector cytokine production, target-cell killing, ability to proliferate in response to prolonged antigen-stimulation and maintenance of cytotoxic activity following antigen re-stimulation.ConclusionsWe plan to introduce AKT inhibition into the GMP manufacturing process, and evaluate the efficacy of the resulting products in ongoing clinical studies.AcknowledgementsWe are extremely grateful to the patients, who were previously enrolled in our clinical trials, and healthy donors for their consent for R&D studies. This was a collaborative cross-functional project, and we are grateful for the contributions of the following Scientists: Garth Hamilton, Adel Toth, Abigail Kay, Sophie Badie, Josh Griffiths, Kaushik Sarkar, Anoop Chandran.Ethics ApprovalThe experimental study was conducted in accordance with the principles of the Declaration of Helsinki and the International Conference on Harmonization Good Clinical Practice guidelines and was approved by local authorities. An independent ethics committee or institutional review board approved the clinical protocol at each participating center. All the patients provided written informed consent before study entry.ReferencesHong DS, Van Tine BA, Olszanski AJ, et al, Phase 1 dose escalation and expansion trial to assess safety and efficacy of ADP-A2M4 in advanced solid tumors. J Clin Oncol 2020;38;A102.Klebanoff C, Crompton J, Leonardi A, et al. Inhibition of AKT signaling uncouples T cell differentiation from expansion for receptor-engineered adoptive immunotherapy. JCI Insight 2017;2:e95103.van der Waart A, van de Weem N, Maas F, et al. Inhibition of Akt signaling promotes the generation of superior tumor-reactive T cells for adoptive immunotherapy. Blood. 2014;124;3490-3500

scholarly journals Optimizing interleukin-2 concentration, seeding density and bead-to-cell ratio of T-cell expansion for adoptive immunotherapy

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Sasan Ghaffari ◽  
Monireh Torabi-Rahvar ◽  
Sajjad Aghayan ◽  
Zahra Jabbarpour ◽  
Kobra Moradzadeh ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Growth Kinetics ◽  
Cell Expansion ◽  
Mononuclear Cells ◽  
Adoptive Cell Therapy ◽  
Interleukin 2 ◽  
Seeding Density ◽  
Healthy Donors ◽  
T Cell Expansion

Abstract Background The successful ex vivo expansion of T-cells in great numbers is the cornerstone of adoptive cell therapy. We aimed to achieve the most optimal T-cell expansion condition by comparing the expansion of T-cells at various seeding densities, IL-2 concentrations, and bead-to-cell ratios. we first expanded the peripheral blood mononuclear cells (PBMCs) of a healthy donor at a range of 20 to 500 IU/mL IL-2 concentrations, 125 × 103 to 1.5 × 106 cell/mL, and 1:10 to 10:1 B:C (Bead-to-cell) ratios and compared the results. We then expanded the PBMC of three healthy donors using the optimized conditions and examined the growth kinetics. On day 28, CD3, CD4, and CD8 expression of the cell populations were analyzed by flow cytometry. Results T-cells of the first donor showed greater expansion results in IL-2 concentrations higher than 50 IU/mL compared to 20 IU/mL (P = 0.02). A seeding density of 250 × 103 cell/mL was superior to higher or lower densities in expanding T-cells (P = 0.025). Also, we witnessed a direct correlation between the B:C ratio and T-cell expansion, in which, in 5:1 and 10:1 B:C ratios T-cell significantly expanded more than lower B:C ratios. The results of PBMC expansions of three healthy donors were similar in growth kinetics. In the optimized condition, 96–98% of the lymphocyte population expressed CD3. While the majority of these cells expressed CD8, the mean expression of CD4 in the donors was 19.3, 16.5, and 20.4%. Conclusions Our methodology demonstrates an optimized culture condition for the production of large quantities of polyclonal T-cells, which could be useful for future clinical and research studies.

scholarly journals Locally produced C5a binds to T cell–expressed C5aR to enhance effector T-cell expansion by limiting antigen-induced apoptosis

Blood ◽  
2008 ◽  
Vol 112 (5) ◽  
pp. 1759-1766 ◽  
Author(s):  
Peter N. Lalli ◽  
Michael G. Strainic ◽  
Min Yang ◽  
Feng Lin ◽  
M. Edward Medof ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Apoptosis ◽  
Cell Expansion ◽  
Immune Cell ◽  
Regulatory Protein ◽  
T Cell Apoptosis ◽  
T Cell Expansion

Abstract Our recent studies have shown that immune cell–produced complement provides costimulatory and survival signals to naive CD4+ T cells. Whether these signals are similarly required during effector cell expansion and what molecular pathways link locally produced complement to T-cell survival were not clarified. To address this, we stimulated monoclonal and polyclonal T cells in vitro and in vivo with antigen-presenting cells (APCs) deficient in the complement regulatory protein, decay accelerating factor (DAF), and/or the complement component C3. We found that T-cell expansion induced by DAF-deficient APCs was augmented with diminished T-cell apoptosis, whereas T-cell expansion induced by C3−/− APCs was reduced because of enhanced T-cell apoptosis. These effects were traced to locally produced C5a, which through binding to T cell–expressed C5aR, enhanced expression of Bcl-2 and prevented Fas up-regulation. The results show that C5aR signal transduction in T cells is important to allow optimal T-cell expansion, as well as to maintain naive cell viability, and does so by suppressing programmed cell death.

scholarly journals Clinical Predictors of T Cell Fitness for CAR T Cell Manufacturing and Efficacy in Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1886-1886 ◽  
Author(s):  
Ehren Dancy ◽  
Alfred L. Garfall ◽  
Adam D. Cohen ◽  
Joseph A Fraietta ◽  
Megan Davis ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Cell Expansion ◽  
Research Funding ◽  
Advisory Committees ◽  
Car T Cell ◽  
Car T ◽  
T Cell Expansion

Abstract Introduction: The optimal clinical setting and cell product characteristics for chimeric antigen receptor (CAR) T cell therapy in multiple myeloma (MM) are uncertain. In CLL patients treated with anti-CD19 CAR T cells (CART19), prevalence of an early memory (early-mem) T cell phenotype (CD27+ CD45RO- CD8+) at time of leukapheresis was predictive of clinical response independently of other patient- or disease-specific factors and was associated with enhanced capacity for in vitro T cell expansion and CD19-responsive activation (Fraietta et al. Nat Med 2018). T cell fitness is therefore a major determinant of response to CAR T cell therapy. In an accompanying abstract (Cohen et al.), we report that higher percentage of early-mem T cells and CD4/CD8 ratio within the leukapheresis product are associated with favorable clinical response to anti-BCMA CAR T cells (CART-BCMA) in relapsed/refractory MM. Here, we compare leukapheresis samples from MM patients obtained at completion of induction therapy (post-ind) with those obtained in relapsed/refractory (rel/ref) patients for frequency of early-mem T cells, CD4/CD8 ratio, and in vitro T cell expansion. Methods: Cryopreserved leukapheresis samples were analyzed for the percentage of early-mem T cells and CD4/CD8 ratio by flow cytometry and in vitro expansion kinetics during anti-CD3/anti-CD28 bead stimulation. Post-ind samples were obtained between 2007 and 2014 from previously reported MM trials in which ex-vivo-expanded autologous T cells were infused post-ASCT to facilitate immune reconstitution (NCT01245673, NCT01426828, NCT00046852); rel/ref samples were from MM patients treated in a phase-one study of CART-BCMA (NCT02546167). Results: The post-ind cohort includes 38 patients with median age 55y (range 41-68) and prior exposure to lenalidomide (22), bortezomib (21), dexamethasone (38), cyclophosphamide (8), vincristine (2), thalidomide (8), and doxorubicin (4); median time from first systemic therapy to leukapheresis was 152 days (range 53-1886) with a median of 1 prior line of therapy (range 1-4). The rel/ref cohort included 25 patients with median age 58y (range 44-75), median 7 prior lines of therapy (range 3-13), and previously exposed to lenalidomide (25), bortezomib (25), pomalidomide (23), carfilzomib/oprozomib (24), daratumumab (19), cyclophosphamide (25), autologous SCT (23), allogeneic SCT (1), and anti-PD1 (7). Median marrow plasma cell content at leukapheresis was lower in the post-ind cohort (12.5%, range 0-80, n=37) compared to the rel/ref cohort (65%, range 0-95%). Percentage of early-mem T cells was higher in the post-ind vs rel/ref cohort (median 43.9% vs 29.0%, p=0.001, left figure). Likewise, CD4/CD8 ratio was higher in the post-ind vs rel/ref cohort (median 2.6 vs 0.87, p<0.0001, mid figure). Magnitude of in vitro T cell expansion during manufacturing (measured as population doublings by day 9, or PDL9), which correlated with response to CART19 in CLL, was higher in post-ind vs rel/ref cohort (median PDL9 5.3 vs 4.5, p=0.0008, right figure). Pooling data from both cohorts, PDL9 correlated with both early-mem T cell percentage (Spearman's rho 0.38, multiplicity adjusted p=0.01) and CD4/CD8 ratio (Spearman's rho 0.42, multiplicity adjusted p=0.005). Within the post-ind cohort, there was no significant association between early-mem T cell percentage and time since MM diagnosis, duration of therapy, exposure to specific therapies (including cyclophosphamide, bortezomib, or lenalidomide), or bone marrow plasma cell content at time of apheresis. However, in the post-ind cohort, there was a trend of toward lower percentage early-mem phenotype (29% vs 49%, p=0.07) and lower CD4/CD8 ratio (median 1.4 vs 2.7, p=0.04) among patients who required >2 lines of therapy prior to apheresis (n=3) compared to the rest of the cohort (n=35). Conclusion: In MM patients, frequency of the early-mem T cell phenotype, a functionally validated biomarker of fitness for CAR T cell manufacturing, was significantly higher in leukapheresis products obtained after induction therapy compared to the relapsed/refractory setting, as was CD4/CD8 ratio and magnitude of in vitro T cell expansion. This result suggests that CAR T cells for MM would yield better clinical responses at early points in the disease course, at periods of relatively low disease burden and before exposure to multiple lines of therapy. Figure. Figure. Disclosures Garfall: Novartis: Research Funding; Kite Pharma: Consultancy; Amgen: Research Funding; Bioinvent: Research Funding. Cohen:GlaxoSmithKline: Consultancy, Research Funding; Kite Pharma: Consultancy; Oncopeptides: Consultancy; Celgene: Consultancy; Novartis: Research Funding; Poseida Therapeutics, Inc.: Research Funding; Bristol Meyers Squibb: Consultancy, Research Funding; Janssen: Consultancy; Seattle Genetics: Consultancy. Fraietta:Novartis: Patents & Royalties: WO/2015/157252, WO/2016/164580, WO/2017/049166. Davis:Novartis Institutes for Biomedical Research, Inc.: Patents & Royalties. Levine:CRC Oncology: Consultancy; Brammer Bio: Consultancy; Cure Genetics: Consultancy; Incysus: Consultancy; Novartis: Consultancy, Patents & Royalties, Research Funding; Tmunity Therapeutics: Equity Ownership, Research Funding. Siegel:Novartis: Research Funding. Stadtmauer:Janssen: Consultancy; Amgen: Consultancy; Takeda: Consultancy; Celgene: Consultancy; AbbVie, Inc: Research Funding. Vogl:Karyopharm Therapeutics: Consultancy. Milone:Novartis: Patents & Royalties. June:Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Immune Design: Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding; Celldex: Consultancy, Membership on an entity's Board of Directors or advisory committees; Immune Design: Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding. Melenhorst:Novartis: Patents & Royalties, Research Funding; Incyte: Research Funding; Tmunity: Research Funding; Shanghai UNICAR Therapy, Inc: Consultancy; CASI Pharmaceuticals: Consultancy.

scholarly journals Synergy of IL-21 and IL-15 in regulating CD8+ T cell expansion and function

2005 ◽  
Vol 201 (1) ◽  
pp. 139-148 ◽  
Author(s):  
Rong Zeng ◽  
Rosanne Spolski ◽  
Steven E. Finkelstein ◽  
SangKon Oh ◽  
Panu E. Kovanen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cell Expansion ◽  
Tumor Regression ◽  
Severe Combined Immunodeficiency ◽  
Cd8 T Cell ◽  
T Cell Expansion

Interleukin (IL)-21 is the most recently recognized of the cytokines that share the common cytokine receptor γ chain (γc), which is mutated in humans with X-linked severe combined immunodeficiency. We now report that IL-21 synergistically acts with IL-15 to potently promote the proliferation of both memory (CD44high) and naive (CD44low) phenotype CD8+ T cells and augment interferon-γ production in vitro. IL-21 also cooperated, albeit more weakly, with IL-7, but not with IL-2. Correspondingly, the expansion and cytotoxicity of CD8+ T cells were impaired in IL-21R−/− mice. Moreover, in vivo administration of IL-21 in combination with IL-15 boosted antigen-specific CD8+ T cell numbers and resulted in a cooperative effect on tumor regression, with apparent cures of large, established B16 melanomas. Thus, our studies reveal that IL-21 potently regulates CD8+ T cell expansion and effector function, primarily in a synergistic context with IL-15.

scholarly journals Combined PD-L1 and TIM-3 blockade improves the expansion of fit human CD8+ antigen-specific T cells for adoptive immunotherapy

2021 ◽  
Author(s):  
Shirin Lak ◽  
Valérie Janelle ◽  
Anissa Djedid ◽  
Gabrielle Boudreau ◽  
Ann Brasey ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Checkpoint ◽  
Cell Expansion ◽  
Ex Vivo ◽  
Immune Checkpoint Blockade ◽  
Checkpoint Blockade ◽  
Immune Checkpoints ◽  
T Cell Expansion ◽  
And Function

AbstractBackgroundThe stimulation and expansion of antigen-specific T cells ex vivo enables the targeting of a multitude of cancer antigens. However, clinical scale T-cell expansion from rare precursors requires repeated stimulations ex vivo leading to T-cell terminal effector differentiation and exhaustion that adversely impact therapeutic potential. We leveraged immune checkpoint blockade relevant to antigen-specific CD8+ human T cells to improve the expansion and function of T cells targeting clinically relevant antigens.MethodsA clinically-compliant protocol relying on peptide-pulsed monocyte-derived dendritic cells and cytokines was used to expand antigen-specific CD8+ targeting the oncogenic Epstein-Barr virus (EBV) and the tumor associated antigen (TAA) Wilms Tumor 1 (WT1) protein. The effects of antibody-mediated blockade of immune checkpoints applied to the cultures (T-cell expansion, phenotypes and function) were assessed at various time points. Genomic studies including single cell RNA (scRNA) sequencing and T-cell receptor sequencing were performed on EBV-specific T cells to inform about the impact of immune checkpoint blockade on the clonal distribution and gene expression of the expanded T cells.ResultsSeveral immune checkpoints were expressed early by ex vivo expanded antigen-specific CD8+ T cells, including PD-1 and TIM-3 with co-expression matching evidence of T-cell dysfunction as the cultures progressed. The introduction of anti-PD-L1 (expressed by the dendritic cells) and anti-TIM-3 antibodies in combination (but not individually) to the culture led to markedly improved antigen-specific T-cell expansion based on cell counts, fluorescent multimer staining and functional tests. This was not associated with evidence of T-cell dysfunction when compared to T cells expanded without immune checkpoint blockade. Genomics studies largely confirmed these results, showing that double blockade does not impart specific transcriptional programs or patterns on TCR repertoires. However, our data indicate that combined blockade may nonetheless alter gene expression in a minority of clonotypes and have donor-specific impacts.ConclusionsThe manufacturing of antigen-specific CD8+ T cells can be improved in terms of yield and functionality using blockade of TIM-3 and the PD-L1/PD-1 axis in combination. Overcoming the deleterious effects of multiple antigenic stimulations through PD-L1/TIM-3 blockade is a readily applicable approach for several adoptive-immunotherapy strategies.

scholarly journals Clinically-relevant T cell expansion protocols activate distinct cellular metabolic programs and phenotypes

2021 ◽  
Author(s):  
Sarah MacPherson ◽  
Sarah Keyes ◽  
Marisa K Kilgour ◽  
Julian Smazynski ◽  
Jessica Sudderth ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Expansion ◽  
Cell Function ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Metabolic Phenotypes ◽  
T Cell Function ◽  
T Cell Expansion ◽  
And Function

Ex vivo expansion conditions used to generate T cells for immunotherapy are thought to adopt metabolic phenotypes that impede therapeutic efficacy in vivo. The comparison of five different culture media used for clinical T cell expansion revealed unique optima based on different output variables including proliferation, differentiation, function, activation and mitochondrial phenotypes. T cells adapted their metabolism to match their media expansion condition as shown by glucose and glutamine uptake, and patterns of glucose isotope labeling. However, adoption of these metabolic phenotypes was uncoupled to T cell function. Furthermore, T cell products cultured in ascites from ovarian cancer patients displayed suppressed mitochondrial activity and function irrespective of the ex vivo expansion media. In one case, culturing in ascites resulted in increased glucose uptake which was insufficient to rescue T cell function. Thus, ex vivo T cell expansion conditions have profound impacts on metabolism and function.

scholarly journals Optimization of Culture Media for T Cell Expansion: T Cell Expansion Is a Bottle Neck in Adoptive T Cell Therapy

2021 ◽  
Author(s):  
Ilnaz Rahimmanesh ◽  
Hossein Khanahmad
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Cell Expansion ◽  
Culture Media ◽  
Adoptive Cell Therapy ◽  
Interleukin 2 ◽  
Adoptive T Cell Therapy ◽  
T Cell Therapy ◽  
T Cell Expansion

Abstract Adoptive T cell therapy is a promising treatment strategy for cancer immunotherapy. The methods used for the expansion of high numbers of T cells are essential steps for adoptive cell therapy. In this study, we evaluated the expansion, proliferation, activation, and anti-tumor response of T lymphocytes, in presence of different concentrations of interleukin-2, phytohemagglutinin, and insulin. Our results showed that supplemented culture media with an optimized concentration of phytohemagglutinin and interleukin-2 increased total fold expansion of T cells up to 500-fold with about 90% cell viability over 7 days. The quantitative assessment of Ki-67 in expanded T cells showed a significant elevation of this proliferation marker. In addition, the proportion of CD4+ and CD8+ cells were evaluated using flow cytometry, and data showed that both cells were present in the expanded population. Finally, we assessed the activation and tumor cytotoxicity of expanded T cells against target cells. Overexpression of CD107a, as a functional marker of T cell degranulation on expanded T cells and their ability to induce cell death in tumor cells, was observed in the co-cultured experiment. Based on these data we have developed a cost-effective and rapid method to support the efficient expansion of T cells for adoptive cell therapy.

scholarly journals Costimulation of γδTCR and TLR7/8 promotes Vδ2 T-cell antitumor activity by modulating mTOR pathway and APC function

2021 ◽  
Vol 9 (12) ◽  
pp. e003339
Author(s):  
Huaishan Wang ◽  
Hui Chen ◽  
Shujing Liu ◽  
Jie Zhang ◽  
Hezhe Lu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Expansion ◽  
Γδ T Cells ◽  
Mtor Pathway ◽  
Two Dimensional ◽  
Γδ T Cell ◽  
T Cell Expansion ◽  
Antigen Presenting

BackgroundGamma delta (γδ) T cells are attractive effector cells for cancer immunotherapy. Vδ2 T cells expanded by zoledronic acid (ZOL) are the most commonly used γδ T cells for adoptive cell therapy. However, adoptive transfer of the expanded Vδ2 T cells has limited clinical efficacy.MethodsWe developed a costimulation method for expansion of Vδ2 T cells in PBMCs by activating γδ T-cell receptor (γδTCR) and Toll-like receptor (TLR) 7/8 using isopentenyl pyrophosphate (IPP) and resiquimod, respectively, and tested the functional markers and antitumoral effects in vitro two-dimensional two-dimensional and three-dimensional spheroid models and in vivo models. Single-cell sequencing dataset analysis and reverse-phase protein array were employed for mechanistic studies.ResultsWe find that Vδ2 T cells expanded by IPP plus resiquimod showed significantly increased cytotoxicity to tumor cells with lower programmed cell death protein 1 (PD-1) expression than Vδ2 T cells expanded by IPP or ZOL. Mechanistically, the costimulation enhanced the activation of the phosphatidylinositol 3-kinase (PI3K)–protein kinase B (PKB/Akt)–the mammalian target of rapamycin (mTOR) pathway and the TLR7/8–MyD88 pathway. Resiquimod stimulated Vδ2 T-cell expansion in both antigen presenting cell dependent and independent manners. In addition, resiquimod decreased the number of adherent inhibitory antigen-presenting cells (APCs) and suppressed the inhibitory function of APCs by decreasing PD-L1 and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) expression in these cells during in vitro Vδ2 T-cell expansion. Finally, we showed that human Vδ2 T cells can be expanded from PBMCs and spleen of humanized NSG mice using IPP plus resiquimod or ZOL, demonstrating that humanized mice are a promising preclinical model for studying human γδ T-cell development and function.ConclusionsVδ2 T cells expanded by IPP and resiquimod demonstrate improved anti-tumor function and have the potential to increase the efficacy of γδ T cell-based therapies.

IL-7 along with Optimized CD3/CD28 Artificial APC Beads Promotes Ex Vivo Expansion of Cord Blood T Cells towards Adoptive Immunotherapy.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2992-2992
Author(s):  
Craig C. Davis ◽  
Melissa Mazur ◽  
Paul Szabolcs
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cord Blood ◽  
Cell Expansion ◽  
Culture Media ◽  
Ex Vivo ◽  
High Rate ◽  
Post Transplant ◽  
T Cell Expansion ◽  
The Impact

Abstract Background: Viral infections are the leading cause of non-relapse mortality after unrelated umbilical cord blood transplants (UCBT). Post-transplant lymphopenia, lack of established antiviral memory, and lack of cytotoxic function among infused UCB lymphocytes are jointly responsible for ineffective immunity. We have previously demonstrated ex vivo T cell expansion and maturation starting from a very small fraction (<5%) of the graft that could be employed post transplant as donor leukocyte infusion (DLI). Naïve UCB T cells proliferate when stimulated with IL-2 + CD3/CD28 co-stimulatory beads that function as artificial antigen presenting cells (APCs). However, high rate of apoptosis (>10%) has hindered T cell expansion. We hypothesized that the presence of cytokines essential for T cell homeostasis, in particular IL-7 may aid expansion and survival, beads with different densities of CD3/CD28 antibodies may impact UCB T cell expansion. Research Design: From frozen/thawed specimens we tested 2 different sources of CD3/CD28 artificial beads (historical control beads vs ClinExVivo®, (Invitrogen Corporation), ± IL-7. Methods: Thawed UCB samples were centrifuged over Ficoll gradient. T cells were positively selected with EasySep®, (StemCell Technologies) and were incubated in gas permeable bags with CD3/CD28 beads in 5% serum replete media + 100u/ml IL-2 ± 10ng/ml of IL7. Media & cytokines were replenished to maintain a concentration of ∼0.75 ×106 cells/ ml. Automated cell count, trypan blue viability assessed overall cell growth and viability at each feeding . ‘Lyse no wash’ FACS staining with Trucount® beads quantified viable CD3+ T cells. 4-color FACS was employed to characterize the evolution of surface and intracellular immunophenotype. Cytotoxicity profile of the day 0 and D12-14 progeny was tested by Europium release assay (Delfia® assay) against a Burkitt’s lymphoma cell line (IM9). Two-tailed paired Student t-tests analyzed the impact of the experimental variables. Results: At the end of 12–14 day expansion periods, analyzing all cytokine conditions, we could demonstrate an average of 43-fold viable CD3+ T cell expansion using control APC beads (n=8), while T cells on ClinExVivo® beads expanded on average 94-fold (p=0.11, n=11). Addition of IL7 to the culture media afforded significantly better expansion with the control bead condition (mean 43 vs 26 fold, p=0.02). IL7 also enhanced T cell expansion with ClinExVivo® beads (mean 147 fold vs 49 fold, though statistically NS). There were significantly more viable T cells generated with ClinExVivo® beads when all timepoints were analyzed, irrespective of the absence (p=0.017) or presence (p=0.05) of IL7. We also analyzed the mechanism how IL7 may potentiate overall T cell expansion. T cells entering cell cycle was enhanced (though not significantly) as demonstrated with intracellular Ki-67 staining (65% T cells in cycle with IL7 versus 52% without, p=0.2). IL7’s overall salutary effect on total expansion may be best explained by simultaneously reduced T cell apoptosis as quantified by activated ic Caspase-3 detection (9.3% versus 7.6% without vs with IL7, respectively, p=0.08) regardless of the source of beads. Granzyme A, B, perforin expression was enhanced comparably, while cytotoxicity was absent against IM9 cells regardless of ± IL-7. Conclusions: These results demonstrate significant expansion of UCB T cell that is further improved by utilizing ClinExVivo® clinical grade APC beads and IL7. These results pave the way to donor-derived UCB T cell expansion suitable for DLI to boost immunity.

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