562 THE EFFECT OF IONISING-RADIATION AND THE TUMOUR MICRO-ENVIRONMENT (TME) ON TREATMENT NAIVE ESOPHAGEAL ADENOCARCINOMA PATIENT T CELL FUNCTION AND ACTIVITY.

2020 ◽  
Vol 33 (Supplement_1) ◽  
Author(s):  
N Donlon ◽  
A Sheppard ◽  
M Davern ◽  
C Donohoe ◽  
N Ravi ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Cell Expansion ◽  
Cell Function ◽  
Ionising Radiation ◽  
Nutrient Deprivation ◽  
T Cell Function ◽  
T Cell Expansion ◽  
The Impact

Abstract   There is extensive literature demonstrating CD8+ T cells are essential for initial tumour control following radiation, however, effects are reduced after time due to T cell exhaustion and a lack of release Damage Associated Molecular Patterns (DAMPS) which are essential for anti-tumour immune responses. In vivo, activated T-cells migrate to the tumour site within the field of irradiation, however translational studies on the effects of radiotherapy on T-cell activation, function and activity are lacking. Methods EAC patient (n = 6) PBMCs were isolated by density centrifugation in Ficoll Paque. T cells were activated and were irradiated at 1.8Gy, 3.6Gy bolus dosing and fractionation for 72 hrs. A panel of immune checkpoints, DAMPS, activation markers, and cytokines were assessed by flow cytometry. To determine the effect of the TME on T cells, PBMCs were cultured under conditions of nutrient deprivation (No Glucose & No Glutamine) under conditions of normoxia and hypoxia. We then ran the aforementioned panel by flow cytometry. We also activated PBMCs with immune checkpoint blockers to determine its effects on T cell expansion and survival. Results 3.6Gy induced a significantly higher expression of DAPMS (Fig 1 p < 0.001); Calreticulin and HMGB1, most notably under conditions of nutrient deprivation (p < 0.001). Ionising radiation also resulted in an increase in the expression of cytokines and importantly in the context of targeted therapy, IR at both the conventional 1.8Gy and 3.6Gy induced a higher expression of checkpoints PD-1, PD-L1, TIGIT, and TIM-3 (p < 0.001). Interestingly, when T cells are activated in the presence of ICB (Atezolizumab, Pembrolizumab, Nivolumab), it increases the rate of T cell expansion, and enhances their survival compared to T cell activated only. (p < 0.001). Conclusion This work demonstrates the impact of clinically utilised fractions of radiation, and conditions of the TME on T cell function and activity, with improved T cell expansion and survival in the presence of ICB’s suggesting it may be a feasible combination therapy as an adjunct to radiotherapy.

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 Estrogen Stimulation Differentially Impacts Human Male and Female Antigen-Specific T Cell Anti-Tumor Function and Polyfunctionality

2017 ◽  
Vol 1 (4) ◽  
pp. 1-13 ◽  
Author(s):  
Flor C. Navarro ◽  
Stephanie K. Watkins
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Function ◽  
Tumor Regression ◽  
Cancer Treatments ◽  
T Cell Function ◽  
Antitumor Effector ◽  
Cell Groups ◽  
Development And Differentiation ◽  
The Impact

Sex-specific differences exist in innate and adaptive immune responses and are mediated by hormone signaling. Estrogen is able to differentially modulate the development and differentiation of immune cells, including T cells. However, the effect of estrogen on T cell function, especially at concentrations other than physiological, remains controversial and incompletely understood. Immunotherapy is one of the most promising cancer treatments to date with a high probability of future enhancements. The adoptive transfer of genetically modified T cells can mediate tumor regression but there are still many hurdles to enhancing the proficiency of this treatment. This study demonstrates for the first time that one major aspect to consider for designing potent immunotherapies for cancer is the impact of the patient's sex. Herein, using two different Ag-specific T cell groups, we investigated the effect of sex and estrogen in antitumor effector responses, T helper cytokine secretion, and, importantly, on T cell whole polyfunctionality important for memory T cell development and survival. Major differences were observed in T cell function and polyfunctionality between sexes and on E2 treatment. The findings of this study may be critical to understand the results of immunotherapy on different patients and for the enhancement of immunotherapy for cancer.

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.

scholarly journals Iron Supplementation Interferes With Immune Therapy of Murine Mammary Carcinoma by Inhibiting Anti-Tumor T Cell Function

2020 ◽  
Vol 10 ◽  
Author(s):  
Piotr Tymoszuk ◽  
Manfred Nairz ◽  
Natascha Brigo ◽  
Verena Petzer ◽  
Simon Heeke ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Growth ◽  
Iron Supplementation ◽  
Cell Function ◽  
Immune Therapy ◽  
Intravenous Iron ◽  
T Cell Function ◽  
Anti Cancer ◽  
The Impact

Iron is both, an essential compound for many metabolic processes, and iron deficiency can impact on the proliferation of cells including lymphocytes but also tumor cells. On the other hand, excess iron-catalyzed radical formation can induce cellular toxicity which has been previously demonstrated for T cells in hereditary iron overload. Despite these interconnections, little is known on the effects of clinically approved intravenous iron supplements for curing cancer-related anemia, on T cell differentiation, tumor proliferation, anti-tumor T cell responses and, of clinical importance, on efficacy of cancer immunotherapies. Herein, we analyzed the effects of intravenous iron supplementation on T cell function and on the effectiveness of anti-cancer chemotherapy with IL-2/doxorubicin or immunotherapy with checkpoint-inhibitor anti-PD-L1 in C57Bl/6N female mice with implanted E0771 mammary carcinomas. We found that iron application resulted to an increased availability of iron in the tumor microenvironment and stimulation of tumor growth. In parallel, iron application inhibited the activation, expansion and survival of cytotoxic CD8+ T cells and of CD4+ T helper cells type 1 and significantly reduced the efficacy of the investigated anti-cancer treatments. Our results indicate that iron administration has a tumor growth promoting effect and impairs anti-cancer responses of tumor infiltrating T lymphocytes along with a reduced efficacy of anti-cancer therapies. Iron supplementation in cancer patients, especially in those treated with immunotherapies in a curative setting, may be thus used cautiously and prospective studies have to clarify the impact of such intervention on the outcome of patients.

scholarly journals Obesity Attenuates T-Cell Function Which Impacts the Efficacy of T-Cell Based Immunotherapies in Acute Lymphoblastic Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 25-26
Author(s):  
Anthony Ross ◽  
Miyoung Lee ◽  
Jamie Hamilton ◽  
Raira Ank ◽  
Priscilla Do ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Function ◽  
Lymphoblastic Leukemia ◽  
Surface Expression ◽  
Car T Cells ◽  
Secreted Factors ◽  
T Cell Function ◽  
Car T ◽  
The Impact

The incidence of obesity continues to rise with over 50% of the world's population predicted to be overweight/obese by the year 2030. The global health impact of this trend is concerning given that obesity is a risk factor for developing cancers of varying etiologies. Alarmingly, the survival outcomes for obese patients with cancer are lower than those observed in lean patients. Obesity is characterized by the accumulation of adipocytes which alters drug dynamics and impacts the function of cancer and immune cells in the tumor microenvironment. Obesity-induced immune defects are troubling given the increasing use of immunotherapy in the treatment of malignancies. Here we show that adipocyte-secreted factors upregulate immunosuppressive mechanisms on human B-cell acute lymphoblastic leukemia (B-ALL) cells, attenuate the function of endogenous T-cells, and compromise the efficacy of T-cell based immunotherapies. To study the impact of adiposity on T-cell function, CD4+ and CD8+ T-cells were purified from the spleens of C57BL/6 mice and activated with PMA/Ionomycin for 72 hours in unconditioned media (UCM), bone marrow stromal-cell conditioned media (SCM), and adipocyte-conditioned media (ACM) followed by flow cytometry analysis for surface marker expression, cytokine production, and the induction of cytolytic mediators. Interestingly, T-cells activated in ACM, but not UCM or SCM, showed an attenuated phenotype highlighted by decreased CD44 and PD-1 expression, diminished cytokine production (IFN-γ/TNF-α) and reduced induction of cytolytic mediators (granzyme B/perforin). These observations were also true in obese, relative to lean, patients with B-ALL where we found that T-cells purified from the peripheral blood mononuclear cells (PBMCs) failed to produce significant levels of TNF-α when stimulated with PMA/Ionomycin. In all, these results demonstrate that adipocyte-secreted factors directly compromise the function of endogenous T-cells, which phenocopies T-cell defects observed in obese relative to lean pediatric patients with B-ALL. We next assessed the impact of adiposity on malignant cells by culturing human B-ALL cell lines in the conditioned mediums described above and performed flow cytometric analysis to assess their surface expression of the B-cell lineage antigen CD19 and proteins that modulate immunity. In addition to being a marker for B-cells, CD19 is the primary target of the T-cell based immunotherapies Blinatumomab and CAR T-cells directed against B-ALL cells. Surprisingly, when human B-ALL cells were co-cultured with adipocytes, every cell line tested (n=6) exhibited lower surface CD19 expression with 5 out 6 reaching statistical significance. Furthermore, adipocyte-secreted factors alone were sufficient to reduce CD19 surface levels on B-ALL cells in 2 of the 6 cell lines tested. Human B-ALL cells cultured in ACM, but not UCM or ACM, also upregulated their surface expression of the immunoinhibitory proteins HVEM, PD-L1, and PD-L2. These results demonstrate that adipocytes directly induce the downregulation of CD19 on human B-ALLs and increase their immune evasive capacity. Given these observations, we hypothesized that adipocyte-secreted factors would compromise T-cell-based immunotherapies targeting CD19-expressing B-ALL cells. To this end, primary human T-cells were engineered to express a CD19-directed chimeric antigen receptor (CAR). CAR T-cells and human B-ALL cells were separately pre-treated for 24 hours in UCM, SCM or ACM followed by co-culture for cytolytic analysis using Annexin-V/PI staining. Adipocyte-secreted factors significantly inhibited CAR T-cell mediated killing of CD19-expressing B-ALL cells at 4 hours. In addition to CAR T-cells, we tested the leukemia killing efficacy of the bispecific T-cell engager, Blinatumomab. After 3 days of culture, we found that Blinatumomab significantly increased the killing capacity of endogenous T-cells with 60-80% of B-ALL cells being killed after 3 days of culture in UCM and SCM. In contrast, we found that ACM significantly compromised the efficacy Blinatumomab with only 30% of B-ALL cells being killed over 3 days when co-cultured with human T-cells. Our pre-clinical data highlights the negative impact of an adipose-rich microenvironment on T-cell function and B-ALL immunogenicity, which subsequently compromises the efficacy of multiple classes of immunotherapies targeting CD19. Disclosures No relevant conflicts of interest to declare.

scholarly journals 752 Novel, orally administered HPK1 inhibitors demonstrate anti-tumor efficacy and enhanced immune response

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A786-A786
Author(s):  
Stefan Chmielewski ◽  
Maciej Kujawa ◽  
Eliza Zimolag ◽  
Michal Galezowski ◽  
Andrzej Gondela ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Small Molecule ◽  
Cell Function ◽  
Adaptor Protein ◽  
Negative Regulator ◽  
Tcr Signaling ◽  
T Cell Function ◽  
Tcr Activation

BackgroundHematopoietic progenitor kinase 1 (HPK1, MAP4K1) is emerging as a well-renowned, druggable target for T cell-based immunotherapies. HPK1 is a member of the serine/threonine MAP4K family, predominantly expressed in hematopoietic cell lineages and shown to be a negative regulator of the T cell receptor (TCR) signaling pathway. Upon TCR activation, HPK1 is recruited to the proximity of the cell membrane and phosphorylates an adaptor protein SLP-76 at the Ser376 residue which, in turn, abrogates TCR signaling. Other studies point to a potential role of HPK1 in T cell exhaustion as well as in functional re-programming of regulatory T cells. Moreover, mounting evidence suggest that HPK1 kinase activity suppresses the immune functions of a wide range of other immune cell subsets like B cells and dendritic cells. Taken together, these observations support small-molecule HPK1 inhibitors as an attractive modality in cancer immunotherapy either as single agents or in combination with immune checkpoint inhibitors.MethodsActivity of compounds against HPK1 and selected off- and anti-targets was assessed in biochemical assays. Phosphorylation of SLP-76 was measured either by flow cytometry or TR-FRET. Jurkat and primary T cells were activated and cultured in the presence of tested compounds and immunosuppressive agents. Impact on TCR selectivity and T cell function was measured by AlphaLISA and flow cytometry. Target engagement was measured in splenocytes of mice administered orally with tested compounds followed by IP injection of aCD3 antibody. Anti-tumor efficacy of HPK1 inhibitors was assessed in a syngeneic tumor model.ResultsRyvu's proprietary small molecule HPK1 inhibitors exhibit sub-nanomolar activity against human and mouse HPK1 proteins and good selectivity against other TCR pathway kinases. Tested compounds efficiently block phosphorylation of SLP-76 upon TCR engagement. TCR selectivity of Ryvu's inhibitors, measured as a ratio between CD69 and pSer376 SLP-76 inhibition, is on par or superior to reference molecules. Tested compounds are not only able to overcome PGE-2 induced resistance following TCR activation in human PBMCs, inducing elevated IL-2 release but also affect T cell function in co-culture assay. Developed molecules have favorable PK profiles, allowing for sustained target coverage in proposed dosing regimens and demonstrate efficacy in a mammary carcinoma syngeneic model.ConclusionsRyvu has developed potent and selective HPK1 inhibitors with favorable PK and PD profiles, whose activity in vitro translates to in vivo efficacy. Further preclinical work is warranted to select a lead candidate for IND-enabling studies and subsequently clinical studies across a variety of solid tumors.

Rapid Generation of Antigen-Specific T Cells for Pre-Clinical and Clinical Applications Using a Novel Mini Cell Bioreactor

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 208-208
Author(s):  
Juan F Vera ◽  
Lara Brenner ◽  
Ann M. Leen ◽  
Helen E. Heslop ◽  
Gianpietro Dotti ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Cytotoxic T Lymphocytes ◽  
Cell Expansion ◽  
Culture Conditions ◽  
Antigen Specificity ◽  
Permeable Membrane ◽  
T Cell Expansion ◽  
The Impact

Abstract Although flasks, bags, or rocking bioreactors can readily expand T lymphocytes after non-specific stimulation, the requirements for antigen-driven expansion of cytotoxic T lymphocytes (CTLs) are more rigorous. Antigen-specific T cells proliferate optimally only in the 2 mL wells of 24-well plates and cannot reproducibly be adapted to growth in flasks or bags. Hence, preparation of antigen-specific T cells for adoptive immunotherapy of malignancies is extremely time-consuming, requiring between 4wks and 3mths to produce sufficient cells for therapeutic purposes, and expensive (media + plastics + cytokines + man hours). The extensive manipulation required during the culturing process increases the risk of contamination. In combination, these problems obstruct the broader clinical application of antigen-specific T cells. Antigen-specific T cell growth is limited by gas exchange, nutrients and waste buildup. Bioreactors developed to provide these requirements tend to be complex, involving mechanical rocking or stirring and continuous perfusion, which increases the expense of the procedure and limits the number of products to the number of mechanical devices that can be housed and maintained. We have now explored the use of a new static mini Cell Bioreactor for antigen-specific T cell expansion. This device is essentially a flask with a gas permeable membrane supported by a plastic lattice as its base. The O2/CO2 exchange from the base allows large volumes of media to be added thereby reducing nutrient limitations and waste build-up, and consequently the manipulation required to sustain cell expansion. We tested two different sizes of Cell Bioreactor, 10 cm2 and 100 cm2 that hold a maximum of 40mL and 2000mL of media, respectively. We were able to generate and expand Epstein-Barr virus antigen-specific cytotoxic T lymphocytes (EBVCTLs) from normal donors by coculturing antigen presenting cells (APC) (1.4E+05 × cm2) with established EBV-CTL (4.3E+03 × cm2) at an optimized cell density and stimulator: responder ratio (32:1). These culture conditions induced accelerated CTL expansion (42.5 fold ±14.8 vs 3.4 fold ±1.2 within 7 days) without media change. Manipulation was restricted to cytokine addition every 3–4 days and to LCL stimulation on a weekly basis. A single 100cm2 bioreactor could produce up to 800E+06 antigen-specific T cells, which would have required approximately 320 wells in 24 well plates (&gt;13 plates) under standard culture conditions. The CD4:CD8 T cell ratio and phenotype of the Cell Bioreactor-expanded CTLs was similar to those expanded using the conventional method (CD27 48% vs 52.4%, CD28 65.2% vs 62.2%, CD62L 53.15% vs 54.5%, CD45RO 58.1% vs 55.7%, and CD45RA 51.1% vs 54.9%). Antigen specificity, as evaluated by tetramer analysis and IFN-g ELIspot assay demonstrated no significant differences between CTL expanded by each process. Finally, cytolytic function was confirmed using a standard chromium release assay where both sources of CTL had high specific killing of the autologous EBV-transformed LCL targets (85%±12% vs 77%±19%) and minimal killing of allogeneic targets (22%±9% vs 15%±12). In summary, we have successfully utilized the new mini Cell Bioreactor technology to induce optimal in vitro antigen-specific T cell expansion with minimal handling. Future work will evaluate the impact of the accelerated expansion on differentiation and memory markers. This new system is suited to the clinical grade expansion of other cell types including suspension cell lines, and mitogen-activated T cells, as well as T cell blasts engrafted with chimeric antigen receptors.

A Randomized Trial to Evaluate the Impact of Cytokines on Dendritic Cell, T-Cell and T-Cell Function When Mobilizing Normal Donors for Allogeneic Progenitor Cell Transplant: An Interim Analysis.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2854-2854
Author(s):  
Sagar Lonial ◽  
Claire Torre ◽  
Michelle Hicks ◽  
Stephanie Mcmillan ◽  
Amelia A. Langston ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Cell Function ◽  
T Cell Proliferation ◽  
Gm Csf ◽  
T Cell Function ◽  
Hla Dr ◽  
The Impact ◽  
Stem Cell Collection

Abstract Introduction:Optimal cellular immunity following allogeneic HPC transplant represents a balance between the induction of sufficient anti-tumor immunity to eradicate residual cancer cells without the induction of life-threatening GvHD. Dendritic cells are potent APCs with the ability to regulate immune responses. Our group has previously reported that increased numbers of donor DC2 result in inferior EFS following allo BMT (Waller et al, Blood 2001), and that myeloid cytokines used for mobilization modulate the DC content of the auto graft (Lonial et al, BBMT in press). The current trial was designed to evaluate the impact of different cytokine combinations on DC content and T-cell function in normal donors mobilized with either G-CSF or the combination of G-CSF + GM-CSF. Methods: 32 normal donors were randomized to mobilization with G-CSF (7.5 mcg/kg BID) or the combination of GM-CSF (7.5 mcg/kg qAM) + G-CSF (7.5 mcg/kg qPM) until completion of the stem cell collection. Side effects between the 2 regimens were documented using a questionnaire filled out by the donors within 2 weeks of stem cell collection. DC, T-cell, and other cell subsets were measured from the graft using flow cytometry. T-cell function was evaluated by measuring T-cell proliferation in response to PMA, Con A, PHA, and PWM. Cytokines (IL2, IL4, IL10,IL12, TNF, and INF) secreted in response to antigens were measured by ELISA. DC1 (myeloid DC) were defined as Lin-/HLA-DR+/CD11c+/CD123- while DC2 (lymphoid DC) were defined as Lin-/HLA-DR+/CD11c-/CD123+. Results: 28 patients have been successfully collected to date (G-CSF n=15, GM+G-CSF n=13). No donor has failed to mobilize in either group. Among the 15 donors mobilized with G-CSF alone, 5 required multiple days of apheresis as compared with 1 of 13 donors who received GM+G-CSF who required multiple days of apheresis (p=0.06). There was no difference in baseline values of T-cells or DC subsets in the peripheral blood prior to cytokine administration. Grafts collected with GM-CSF+ G-CSF contained significantly fewer DC2 cells and T-cells (median DC2 dose of 2.1 x 10E6/kg and CD3 dose of 197x 10E6/kg) compared with grafts from donors who received G-CSF alone (median DC2 dose of 3.8 x 10E6/kg (p=.01) and CD3 dose of 320 x 10E6/kg (p=0.001)). There was no difference in the content of CD34+ or DC1 in the grafts, nor in the ratio of CD4:CD8 T-cells between grafts collected with the 2 cytokine combinations. T-cell proliferation and cytokine secretion in response to mitogens was not different between grafts collected from the two groups. To date, there is no difference in the frequency of GvHD or relapse between the patients transplanted with the grafts collected from the 2 cytokine cohorts. Conclusions: The addition of GM-CSF to the mobilization regimen results in significantly fewer DC2 cells and T-cells in the blood HPC graft which could impact immune function and GvL following allogeneic HPC transplant. Clinical outcomes and further analysis of TH1/TH2 polarization of T-cells in grafts collected with either G-CSF or G-CSF+GM-CSF are in progress..

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.

scholarly journals B cell depletion impairs vaccination-induced CD8+ T cell responses in a type I interferon-dependent manner

2021 ◽  
pp. annrheumdis-2021-220435
Author(s):  
Theresa Graalmann ◽  
Katharina Borst ◽  
Himanshu Manchanda ◽  
Lea Vaas ◽  
Matthias Bruhn ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cell Expansion ◽  
De Novo ◽  
T Cell Responses ◽  
Type I ◽  
Cell Responses ◽  
T Cell Expansion

ObjectivesThe monoclonal anti-CD20 antibody rituximab is frequently applied in the treatment of lymphoma as well as autoimmune diseases and confers efficient depletion of recirculating B cells. Correspondingly, B cell-depleted patients barely mount de novo antibody responses during infections or vaccinations. Therefore, efficient immune responses of B cell-depleted patients largely depend on protective T cell responses.MethodsCD8+ T cell expansion was studied in rituximab-treated rheumatoid arthritis (RA) patients and B cell-deficient mice on vaccination/infection with different vaccines/pathogens.ResultsRituximab-treated RA patients vaccinated with Influvac showed reduced expansion of influenza-specific CD8+ T cells when compared with healthy controls. Moreover, B cell-deficient JHT mice infected with mouse-adapted Influenza or modified vaccinia virus Ankara showed less vigorous expansion of virus-specific CD8+ T cells than wild type mice. Of note, JHT mice do not have an intrinsic impairment of CD8+ T cell expansion, since infection with vaccinia virus induced similar T cell expansion in JHT and wild type mice. Direct type I interferon receptor signalling of B cells was necessary to induce several chemokines in B cells and to support T cell help by enhancing the expression of MHC-I.ConclusionsDepending on the stimulus, B cells can modulate CD8+ T cell responses. Thus, B cell depletion causes a deficiency of de novo antibody responses and affects the efficacy of cellular response including cytotoxic T cells. The choice of the appropriate vaccine to vaccinate B cell-depleted patients has to be re-evaluated in order to efficiently induce protective CD8+ T cell responses.

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