WT1-Specific T Cells Exhibiting a Non-Exhausted, Functional Phenotype Can Be Generated From The Natural Repertoire Of Healthy Donors For Clinical Use

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4504-4504 ◽  
Author(s):  
Sabine Schmied ◽  
Anne Richter ◽  
Mario Assenmacher ◽  
Juergen Schmitz
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Ex Vivo ◽  
Leukemic Cells ◽  
Target Cells ◽  
Clinical Use ◽  
Healthy Donors ◽  
Effector Cytokines

Background The Wilms tumor antigen 1 (WT1) is a self-antigen expressed at high levels in leukemic cells, but not in healthy tissue. As WT1 expression in leukemic cells drives leukemogenesis, it is a favorable target antigen for immunotherapy, e.g. adoptive transfer of allogeneic T cells, to prevent or treat leukemic relapse after stem cell transplantation (Cheever et al., Clin Cancer Res 2009;15(17)). WT1-specific CD8+ T cells have been detected in healthy individuals at low frequencies (Rezvani et al., Blood 2003;102). However, a comprehensive characterization of CD4+ and CD8+WT1-specific T cells is missing and the efficient expansion of a polyclonal WT1-reactive T cell population for clinical use has remained a major challenge. In this study we aim to directly ex vivo characterize WT1-specific T cells present in the blood of healthy donors at high-resolution and to develop a rapid method for the generation of functionally potent, polyclonal CD4+ and CD8+WT1-specific T cells for clinical use. Methods For direct ex vivo analysis of CD4+ WT1-specific T cells peripheral blood mononuclear cells (PBMC) of healthy blood donors were in vitro stimulated with a pool of overlapping peptides spanning the WT1 protein for 7 hours. Subsequently CD154 (CD40L)-expressing cells were magnetically enriched and flow cytometrically examined for expression of effector cytokines and their differentiation status. Presence and phenotype of CD8+ WT1-specific T cells have been studied after stimulation of presorted naïve and memory T cell populations with WT-1 peptide pool for 30 hours, magnetic enrichment of CD137+ (4-1BB) cells and subsequent staining using pMHCI-Tetramers. For the generation of polyclonal WT1-specific CD4+ and CD8+ T cells PBMC were in vitro activated with WT-1 peptide pool for 30 hours. CD137+cells were magnetically selected and expanded for 9 days in the presence of the cytokines IL-7, IL-15 and IL-21 at low doses. Expanded T cells were analyzed for their phenotype, the expression of co-stimulatory and exhaustion markers and were tested for their functionality and cytotoxicity by restimulation experiments with antigen-loaded target cells. Results Ex vivo frequencies of WT1-specific T cells are low, 1 to 10 WT1-specific CD154+ CD4+ T cells can be detected within 1x106 CD4+ T cells. In about 80% of healthy donors (n=15) a CD4+ memory response, accompanied by production of effector cytokines like IFNγ, TNFα and IL-2, against WT1 peptides is present. Additionally, in all donors naïve WT1-specific CD4+ T cells can be detected. In contrast, detected CD137+CD8+ WT1-reactive T cells exhibit a naïve phenotype (CD45RA+CCR7+) in all donors (n=5), no WT1-reactive CD8+T cells could be enriched from presorted memory T cells. To evaluate the usefulness of our improved short-term expansion protocol to generate potent WT1-specific T cell cultures for clinical use, we characterized CD137 enriched and expanded T cells. Notably, a high frequency of CD4+ and CD8+ T cells show specific reactivity against WT1-presenting autologous cells as detected by production of effector cytokines like IFNγ, TNFα and IL-2 after antigen-specific restimulation. Cytotoxic activity against antigen-loaded target cells could be shown by direct flow-cytometry-based cytotoxicity assays and antigen-specific upregulation of the degranulation marker CD107a. Stainings using multiple WT1-MHCI-tetramers furthermore confirmed antigen-specificity and suggested polyclonality within the CD8+T cell population. In contrast to previous expansion protocols our polyclonally expanded T cells exhibit a favourable, unexhausted memory phenotype, express co-stimulatory markers CD27 and CD28 and the IL7R-a chain (CD127) which has been shown to mark cells with stem T cell like properties. Furthermore exhaustion markers like CD279 (PD-1), CD178 (FasL) and CD57 are scarcely expressed. Conclusions Functional, polyclonal, CD4+ and CD8+ WT1-specific, reactive T cells can be efficiently enriched directly ex vivo from the natural repertoire by magnetic separation of T cells after antigen-specific stimulation. Phenotypic and functional characterization revealed a non-exhausted phenotype of expanded WT1-specific T cells, thereby suggesting good persistence and functionality of the obtained T cell product in vivo. Thus, our approach holds great potential for the GMP-compliant generation of WT1-specific T cells for future clinical use. Disclosures: Schmied: Miltenyi Biotec GmbH: Employment. Richter:Miltenyi Biotec GmbH: Employment. Assenmacher:Miltenyi Biotec GmbH: Employment. Schmitz:Miltenyi Biotec: Employment.

Ex Vivo Fludarabine Selectively Depletes Human Naive T-Cell Subsets: A Step Toward Modifying Donor Lymphocyte Infusion.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1071-1071
Author(s):  
Melody M. Smith ◽  
Cynthia R. Giver ◽  
Edmund K. Waller ◽  
Christopher R. Flowers
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Memory T Cells ◽  
Ex Vivo ◽  
T Cell Subsets ◽  
Effector Memory ◽  
Naïve T Cell ◽  
Naive T Cell

Abstract Ex vivo modification of donor lymphocytes with purine analogs (mDL) may help to minimize graft versus host disease (GvHD) while providing beneficial graft versus leukemia (GvL) effects. In a murine model system, we have shown that allogeneic donor splenocytes, treated with fludarabine ex vivo have significantly reduced GvHD activity when transferred to irradiated recipient mice, and retain anti-viral and GvL activities (Giver, 2003). This effect appears to be mediated by relative depletion of donor CD4 CD44low, “naive” T-cells. As a first step toward developing mDL for use in patients, we sought to evaluate the effects of ex vivo fludarabine exposure on human T-cell subsets, and to determine the minimum dose of fludarabine required to achieve this effect. Methods: Peripheral blood mononuclear cell samples from 6 healthy volunteers were evaluated at 0, 24, 48, and 72 hour time points after ex vivo incubation in varying dosages of fludarabine: 2, 5, and 10(n=3) mcg/ml. Fludarabine incubated samples were compared to samples that received no fludarabine (untreated). The total viable cell number was determined and the fractions and absolute numbers of viable CD4 and CD8 naïve and memory T-cells were determined using flow cytometry after incubation with 7-AAD (dead cell stain), CD4, CD8, CD45RA, CD62L, and CCR7 antibodies, and measuring the total viable cells/ml. Results: The numbers of viable CD4 and CD8 T-cells remained relatively stable in control cultures. Without fludarabine, the average viability at 72 hr of naive and memory T-cells were 92% and 77% for CD4 and 86% and 63% for CD 8 (Fig. 1A). Naive CD4 T-cells were more sensitive to fludarabine-induced death than memory CD4 cells. At 72 hr, the average viability of fludarabine-treated naive CD4 T-cells was 33% at 2 mcg/ml (8.2X the reduction observed in untreated cells) and 30% at 5 mcg/ml, while memory CD4 T-cells averaged 47% viability at 2 mcg/ml (2.3X the reduction observed in untreated cells) (Fig. 1B) and 38% at 5 mcg/ml. The average viability of naive CD8 T-cells at 72 hr was 27% at 2 mcg/ml and 20% at 5 mcg/ml, while memory CD8 T-cell viability was 22% at 2 mcg/ml and 17% at 5 mcg/ml. Analyses on central memory, effector memory, and Temra T-cells, and B-cell and dendritic cell subsets are ongoing. The 5 and 10 mcg/ml doses also yielded similar results in 3 initial subjects, suggesting that 2 mcg/ml or a lower dose of fludarabine is sufficient to achieve relative depletion of the naive T-cell subset. Conclusions: Future work will determine the minimal dose of fludarabine to achieve this effect, test the feasibility of using ex vivo nucleoside analog incubation to reduce alloreactivity in samples from patient/donor pairs, and determine the maximum tolerated dose of mDL in a phase 1 clinical trial with patients at high risk for relapse and infectious complications following allogeneic transplantation. Figure Figure

Latent-Membrane 2a Specific CTL’s but Not Latent-Mebrane 1 Specific CTL’s Lysis Efficiently Hodgkin Lymphoma Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4991-4991
Author(s):  
Max S. Topp ◽  
Jan Diekamm ◽  
Olaf Beck ◽  
Georg Rauser ◽  
Kai Hauschulz ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Adoptive Immunotherapy ◽  
Cytokine Production ◽  
Cd8 T Cell ◽  
Rapid Expansion ◽  
Target Cells ◽  
Specific Lysis ◽  
Healthy Donors

Abstract Relapse is the leading cause of treatment failure after allogeneic SCT of Hodgkin Disease (HD). As Ebstein-Barr infection (EBV) is associated with 60% of all HD cases, adoptive immunotherapy with donor derived EBV-specific T-cells lines has resulted in disease control of allogeneic SCT. Potential targets for the adoptively transferred T-cells are the type II latency protein LMP-1 and LMP-2a, which are both homogenously expressed by HD cells. In healthy individuals, both LMP-1 and LMP-2a elicits subdominant CD8+ T-cell responses with frequencies of less than 1:10000. LMP-1 and LMP-2a specific T-cells from 1x108 PBMC derived from HLA A*0201+healthy donors were stimulated with the HLA A*0201 LMP1-epitopes YLLEMLWRL and YLGQNWWTL and the HLA A*0201 LMP-2a epitope CLGGLLTM. Activated T-cells were selected by the cytokine secretion assay and expanded for 10 days. In 85% of donors 1.7 x106 (range 0.7 –4.5 x106; n=13) LMP-1 or LMP-2a specific CD8+ T-cell could be generated with an average purity of 83% as determined by tetramer staining. LMP1- and LMP2a-specific CD8+ T-cells were then expanded 3000 x in 14 d by the rapid expansion protocol and evaluated functionally for cytokine production and specific lysis. Both LMP-1 and LMP-2a specific CD8+ T-cells retained specific cytokine production if stimulated with peptide pulsed targets, efficiently lysed peptid pulsed targets. Surprisingly, if LMP-1 was presented endogenously by EBV positive targets or by targets cells transduced with LMP-1, no cytokine production or specific lysis was detected despite protein expression of LMP-1 in all targets. In contrast, IFN-γ production could be readily detected in LMP-2a-specific CD8+ T-cells after stimulation with target cells processing endogenously the LMP-2a antigen as well as specific lysis of EBV positive target cells. Furthermore, LMP2a specific CD8+ demostrated also specific lyse of Hodgkin-cells expressing the LMP2a (30:1 E/T ratio; 29,3%) where as LMP-1-specific CD8+ T-cells could not lyse HD-cells. In summary, LMP-1 and LMP-2a specific T-cells, although present at undectable levels in healthy donors, can be readily selected and expanded to up to 6x109 antigen-specific T-cells in less than 4 weeks starting from 1x108 PBMC. Based on this data, adoptive immunotherapy of relapsed EBV positive HD after allogeneic SCT should be preferentially performed with LMP-2a specific CD8+ T-cells rather than with LMP-1 specific CD8+ T-cells.

CD4+ Regulatory T Cells Specific for the WT1 Antigen Are Present in Acute Myeloid Leukemia Patients: Implication for Immunotherapy.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1933-1933
Author(s):  
Said Dermime ◽  
Cynthia Lehe ◽  
Hazem Ghebeh ◽  
Abdullah Al-Sulaiman ◽  
Ghofran Al Qudaihi ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Cytotoxic Activity ◽  
Cd8 T Cells ◽  
Ex Vivo ◽  
Granzyme B ◽  
Critical Role ◽  
Leukemic Cells ◽  
Cell Contact

Abstract Compelling evidences indicate a key role for regulatory T cells (Tregs) on the host response to cancer and recent studies indicated that the generation of effective WT1-specific cytotoxic T cells can be largely affected by the presence of Tregs. This is the first study to describe human Tregs generated specifically against the WT1 antigen which is overexpressed in several human leukemias and provide the mechanism by which these anti-WT1 Tregs inhibit the immune response in leukemia patients. We have generated T cell lines and clones that specifically recognized a WT1-84 peptide in an HLA DRB1*0402/TCR-Vb8-restricted manner. Importantly, they recognized HLADRB1* 04-matched fresh leukemic cells expressing the WT1 antigen. These clones exerted a Th2 cytokine profile, had a CD4+CD25+Foxp3+GITR+CD127− Tregs phenotype, and significantly inhibited the proliferative activity of allogeneic T cells independently of cell-contact. Priming of allo-reactive T cells in the presence of Tregs strongly inhibited the expansion of NK; NK-T and CD8+ T cells, had an inhibitory effect on NK/NK-T cytotoxic activity but not on CD8+ T cells. Furthermore, priming of T cells with the WT1- 126 HLA-A0201-restricted peptide in the presence of Tregs strongly inhibited the induction of anti-WT1-126 CD8+ CTL responses as evidenced by both very low cytotoxic activity and IFN-g production. Moreover, these Tregs clones specifically produced Granzyme-B and selectively induced apoptosis in WT1-84 pulsed-autologous APCs but not in apoptoticresistant DR4-matched leukemic cells. Importantly, we have also detected anti-WT1-84 IL-5+/Granzyme-B+/Foxp3+ CD4+ Tregs in 5 out of 8 HLA-DR4+ AML patients. These findings suggest a critical role for anti-WT1 Tregs in the inhibition of T cell responses against leukemia. This study may have important implications for the clinical manipulation of Tregs such as immuno-targeting of TCR-Vb-8 with mAbs to eliminate anti-WT1 Tregs in leukemia patients in order to enhance GVL before vaccination with the WT1 antigen. On the other hand, leukemia patients with GVHD should be clinically-tried for vaccination with the current WT1-84 peptide or adoptively-treated with ex-vivo anti-WT1 Treg cells to specifically enhance their frequency, which is known to be very low in these patients.

scholarly journals AMV564 Depletes Myeloid-Derived Suppressor Cells and Expands T Cells: Potential to Address Key Limitations of Cellular Therapies

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1702-1702
Author(s):  
Sterling Eckard ◽  
Bianca Rojo ◽  
Victoria Smith ◽  
Patrick Chun
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Cell Activation ◽  
Ex Vivo ◽  
Suppressor Cells ◽  
Target Cells ◽  
Myeloid Derived Suppressor Cells ◽  
Car T

Abstract Background Myeloid-derived suppressor cells (MDSC) contribute to an immunosuppressive tumor environment and are a barrier to immune therapeutic approaches, including cell-based therapies such as chimeric antigen receptor T cells (CAR T). Despite good overall response rates with certain subsets of B cell leukemias and lymphomas, a significant percentage of patients treated with CAR T therapy do not respond or subsequently relapse. Poor CAR T expansion, poor persistence of infused cells, and clinical treatment failure are associated with tumor and systemic immune dysregulation including high blood levels of peripheral blood monocytic MDSC (M-MDSCs) and interleukin-6, both of which are associated with lack of durable responses 1. In addition, CAR T therapy has been limited by the occurrence of severe cytokine release syndrome (CRS), which is associated with high IL-6 production 2 by myeloid cells such as MDSC. AMV564 is a potent T cell engager that selectively depletes MDSC while promoting T cell activation and proliferation without significant IL-6 induction 3. In phase 1 studies in acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and solid tumors, AMV564 has been demonstrated to be clinically safe and active with some patients achieving complete remissions. Methods Cell lines, primary human cells, and patient samples were analyzed using flow cytometry with appropriate marker panels. T cell activation and cytotoxicity assays were conducted using primary human T cells from healthy donors and target cells (3:1 ratio) for 72 hours. T cell activation using ImmunoCult Human CD3/CD28 served as an assay reference. Results Analysis of patients treated with AMV564 demonstrated statistically significant selective depletion of M-MDSC by cycle 2 (Fig. 1A). While on AMV564 therapy, median IL-6 levels remained below 100 pg/mL despite robust T cell activation and expansion. Granzyme B production by CD8 T cells increased significantly between Cycle 1 and Cycle 2 in patients on therapy, and effector CD8 T cells expand over the course of treatment (Fig. 1B-C). These data collectively support the finding that AMV564 both removes a key source of immune suppression and is a potent agonist of T cell function and differentiation in patients. AMV564 potently activates and expands primary T cells ex vivo. Across donors, peak proliferation was significantly higher with AMV564 than with the CD3/CD28 reference (Fig. 2A). Importantly, T cell viability remained significantly higher with AMV564 when compared to reference control (CD3/CD28), and there was no evidence of activation-induced cell death (AICD) in AMV564-treated samples (Fig. 2B). Conclusions AMV564 depletes MDSC and stimulates expansion and longevity of T cells without significant IL-6 induction, suggesting a possible strategy for improvement in efficacy of cell-based therapy such as CAR T approaches. As circulating M-MDSC both at baseline and after CAR T infusion correlate with poor clinical efficacy 4, AMV564 may have beneficial effects during the conditioning phase of cell therapy, after re-infusion of CAR T products into patients, or both. Ex vivo studies using donor T cells and ongoing in vitro studies using CAR T molecules suggest that AMV564 may provide dual benefit with respect to both depletion of MDSC and T cell agonism. References 1. Jain, et al; Blood 2021; 137 (19): 2621-2633. doi: https://doi.org/10.1182/blood.2020007445 2. Li et al., Sci. Transl. Med. 11, eaax8861 (2019) 3. Eckard et al; Cancer Res 2021; (81) (13 Supplement) 528; DOI: 10.1158/1538-7445.AM2021-528 4. Jain, et al; Blood 2019; 134 (Supplement_1): 2885. doi: https://doi.org/10.1182/blood-2019-131041 Figure 1 Figure 1. Disclosures Eckard: Amphivena Therapeutics: Current Employment. Rojo: Amphivena Therapeutics: Current Employment. Smith: Amphivena Therapeutics: Current Employment. Chun: Amphivena Therapeutics: Current Employment.

scholarly journals Anti-BCMA BiTE® AMG 701 Potently Induces Specific T Cell Lysis of Human Multiple Myeloma (MM) Cells and Immunomodulation in the Bone Marrow Microenvironment

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 592-592 ◽  
Author(s):  
Shih-Feng Cho ◽  
Liang Lin ◽  
Lijie Xing ◽  
Jiye Liu ◽  
Tengteng Yu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Half Life ◽  
Cd4 T Cells ◽  
Cell Lysis ◽  
Cd4 T Cell ◽  
Target Cells ◽  
Immunomodulatory Effects ◽  
Positive Target

Abstract The first Bispecific T-cell Engager (BiTE®) targeting B-cell maturation antigen (BCMA) on multiple myeloma (MM) cells and CD3 on T cells is currently in clinical development (NCT02514239). This first-generation BiTE® has a short serum half-life and is delivered by continuous IV infusion. We here investigated the T cell-redirected cytotoxicity and immunomodulatory effects of AMG 701, a BCMA-targeting BiTE® with extended half-life, alone and in combination with lenalidomide (len), in MM cell lines and patient-derived samples. AMG 701 has a plasma half-life of 112 hr. in non-human primates (Cancer Res 2018;78(13 Suppl):Abstract nr LB-299) and is currently being evaluated clinically (NCT03287908). We here show that AMG 701 significantly induced T cell-mediated lysis of BCMA-positive MM cells resistant or sensitive to current anti-MM agents including bortezomib and lenalidomide (len). EC50 values ranged from 0.64-2.54 ng/ml following overnight treatment with AMG 701 at effector to target (E/T) ratio of 10 to 1. Moreover, following overnight treatment AMG 701-induced MM cell lysis remained robust even at low concentrations (< 2 ng/ml) and low E/T ratios (2/1, 1/2). Importantly, the presence of myeloma-supporting osteoclasts or bone marrow stromal cells did not significantly alter the ability of AMG 701 to lyse MM cells. In the presence of BCMA-positive target cells, AMG 701 rapidly induced degranulation of CD4+ and CD8+ T cells in a dose-dependent manner, evidenced by upregulated surface CD107a expression. AMG 701 triggered lysis also induced secretion of IFNγ and TNFα, to a greater extent in CD8+ than CD4+ T cell subsets. Importantly, AMG 701 (1d treatment) induced lysis of autologous patient tumor cells from relapsed and refractory MM. Combined effects of AMG 701 with len were next investigated using effector cells (PBMC or CD3+ T) pretreated with len. Len enhanced AMG 701-mediated T cell lysis of MM cells even in the presence of osteoclasts. In the presence of BCMA-positive target cells, len pretreatment also further enhanced AMG 701-induced secretion of IFNγ and TNFα from T cells, to a greater extent in CD8+ than CD4+ T cell subsets. In the absence of AMG 701, len-specific lysis of MM cells was observed, confirming len-enhanced cytotoxic potential of T cells against MM cells. We next studied the potential immunomodulatory effects of AMG 701 by flow cytometry analysis. AMG 701 was added to co-cultures of MM cells and CellTraceTM violet-labeled effector cells and T cells were analyzed at various time points. After 4d-co-incubation, AMG 701 induced more proliferation of CD8+ than CD4+ T cells (9.94% vs 0.8% at 1 ng/ml; and 47.5% vs 16.7% at 10 ng/ml, respectively). Higher levels of CD25 and CD69 were also observed in CD8+ than CD4+ T cells. Transient but not persistent upregulation of immune checkpoint molecules PD-1, TIM-3, and LAG-3 were seen on CD4+ and CD8+ T cells after coincubation with AMG 701 and BCMA-positive target cells. In parallel, the CD8+/CD4+ T cell ratios were increased (1.21 to 3.48-fold at d1 to d5, p<0.001); and further increased by 7d (1.97 to 8.64 folds at d1 to d8, p<0.001). AMG 701 induced differentiation of CD4+ and CD8+ T cells from naïve T cells to central memory (CM) and effector memory (EM) T cells, as demonstrated by changes in expression of CD62L and CD45RA. Moreover, CM and EM T cells were increased consistently after AMG 701 treatment (median % of CM+EM on CD4+ T cells: 74.8% (d1), 82.5% (d5), and 91.5% (d8), p<0.01; median % of CM+EM on CD8+ T cells: 54.4% (d1), 83.6% (d5), and 91.1% (d8), p<0.001). Furthermore, the proliferating T cells derived from AMG 701-treated co-cultures rapidly and potently lysed MM cells, even those with low levels of BCMA expression. Taken together, these results demonstrate that AMG 701 potently induces T cell-directed lysis of BCMA-positive MM cells in and triggers robust immunomodulatory effects to overcome the immunocompromised BM microenvironment. Moreover, these results provide the rationale for clinical trials based of AMG 701, alone and in combination with lenalidomide, to improve patient outcome in MM. Disclosures Munshi: OncoPep: Other: Board of director. Anderson:Bristol Myers Squibb: Consultancy; C4 Therapeutics: Equity Ownership, Other: Scientific founder; Gilead: Membership on an entity's Board of Directors or advisory committees; OncoPep: Equity Ownership, Other: Scientific founder; Millennium Takeda: Consultancy; Celgene: Consultancy.

Functional Defects of T Cells of NHL Patients after Different Chemotherapy Regimens Activated By CD19/CD3 Tetravalent Bispecific TandAb® AFM11

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4130-4130
Author(s):  
Johannes Duell ◽  
Dragana Slavkovic ◽  
Margarete Karg ◽  
Uwe Reusch ◽  
Florian Eisele ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Healthy Volunteers ◽  
Cd4 T Cells ◽  
Target Cells ◽  
Cytotoxicity Assays ◽  
Healthy Donors ◽  
Significant Difference ◽  
Pretreated Patients ◽  
Functional Defects

Abstract Introduction: T-cell engaging immunotherapies such as bispecific T-cell recruiting antibodies such as the recently approved blinatumomab, or chimeric antigen receptor T-cells (CAR-T) have emerged as highly active therapeutics in patients with refractory or relapsed hematological malignancies such as ALL or NHL. However, a relevant number of heavily pretreated patients progress or do not respond to these novel therapies. The objective of this study was to determine whether patients´ treatment history impacts the T-cell engagement of novel immunotherapies by analyzing activity of AFM11 - a CD19-directed tandem diabody (TandAb®) construct with T-cells obtained from patients after different chemotherapeutic regimens (R-Bendamustine, R-CHOP, HD-BEAM). Methods: T-cells were isolated and enriched from NHL patients 4-6 weeks after different therapeutic regimens and characterized side by side with T-cells enriched from healthy volunteers by flow cytometry. The responsiveness of T-cells from NHL patients to AFM11 was compared with T-cells from healthy volunteers in proliferation and cytokine release assays. In addition, enriched T-cells were used as effector cells at limiting effector-to-target (E:T) ratios in heterologous cytotoxicity assays with NALM-6 target cells in the presence of AFM11 or an appropriate control TandAb. Results: We found less CD3+ cells (median 148.1 cells/µL in patient group vs. 1232.2 cells/µL in healthy donors), less NK-cells (median 35.9 in patient group vs. 217.8 cells/µL in healthy donors), and no B-cells in the selected patients. Among memory T-cells, the percentages of both central (CD45RO+/CD62L+) and effector memory (CD45RO+/CD62L-) CD4+ and CD8+ cells were significantly increased in patient PBMC. Finally, we examined the percentages of regulatory T-cells (Treg) among the CD4+ T-cells of patients and healthy donors. Overall, patients contained higher proportions of Treg. Patients who received R-Bendamustine or HD-BEAM treatment had more Treg, while the percentage of Treg in patients after R-CHOP was comparable to healthy donors. Whereas CD8+ T-cells from patients and healthy volunteers showed similar proliferative response upon AFM11 stimulation in the presence of target cells, CD4+ T-cells from patients proliferated significantly more than CD4+ T-cells from healthy volunteers. No difference was observed among the patient groups with different prior chemotherapeutic regimens. T-cells from patients produced significantly higher amounts of IFN-γ than T-cells of healthy donors upon stimulation with AFM11 and target cells. In contrast, the production of IL-10 was significantly lower by T-cells from patients when compared to healthy donors. Surprisingly, most of the T-cells from healthy donors and patients did not produce IL-6. In heterologous cytotoxicity assays T-cells from healthy donors induced statistically significant higher specific lysis of NALM-6 in the presence of AFM11. There was no statistically significant difference between T-cells isolated from patients after different chemotherapies at an E:T ratio of 1:2. However, at lower E:T ratios there was statistically significant difference among T-cells from patients after different chemotherapy regimens. T-cells isolated from patients who received R-CHOP treatment had the strongest lysis capacity compared to the two other groups. The lowest efficacy of AFM11-mediated target cell lysis was observed with T-cells from patients after treatment with HD-BEAM. Conclusions: In conclusion, T-cells of NHL patients after different chemotherapy regimens are reduced in number and have functional defects. However, AFM11 is able to activate patient T-cells for potent target cell lysis with similar efficacy as T-cells from healthy volunteers at higher E:T ratios. Only at limiting effector cell counts a lower efficacy was observed for T-cells from patients. T-cells from R-CHOP treated patients are the most active among the tested treatment groups and display similar responsiveness as T-cells from healthy donors. Lower activity was measured with T-cells from R-Bendamustine pretreated patients and substantial lower cytotoxic activity for T-cells from patients after HD-BEAM treatment. The correlation of these findings with in vivo response will be evaluated in an ongoing Phase I trial with AFM11. Disclosures Reusch: Affimed: Employment, Patents & Royalties: Patents. Einsele:Celgene: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Speakers Bureau. Treder:Affimed: Employment.

scholarly journals Evolving Exhaustion of T Cells during the Course of the Disease in AML Can be Abrogated By CD33 BiTE ® Construct Mediated Cytotoxicity

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1172-1172
Author(s):  
Maryam Kazerani Pasikhani ◽  
Anetta Marcinek ◽  
Bettina Brauchle ◽  
Jonathan Jonas Taylor ◽  
Helena Domínguez Moreno ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Research Funding ◽  
Cell Function ◽  
Target Cells ◽  
Cell Cytotoxicity ◽  
T Cell Cytotoxicity ◽  
Support Research

Abstract Novel immunotherapeutic strategies like BiTE ® (bispecific T cell engager) constructs aim to eradicate neoplastic cells by TCR-independent T-cell activation, and therefore rely on the function of autologous T cells. Currently, their efficacy is also evaluated in heavily pre-treated patients with relapsed/refractory acute myeloid leukemia (AML). Previous data demonstrated dysfunction in CD8 + T cells of AML patients (Knaus et al 2018). Thus, we aimed to characterize the progressive modulation of T-cell activity over the course of AML progression to improve the optimal application of T-cell based immunotherapeutic approaches. Bone marrow mononuclear cells (BMMCs) from AML patients at time of initial diagnosis (ID), complete remission (CR), relapse (RL), as well as of age-matched healthy donors (HD) were analyzed for T-cell subset distribution and expression of exhaustion markers by flow cytometry. Additionally, T-cell function was assessed after stimulation with 1) CD3/CD28 beads; 2) AMG 330, a CD33/CD3 specific BiTE ® construct, after incubation with OCI-AML3 target cells; or 3) AMG 330 in an autologous ex vivo long-term culture system after incubation with primary AML cells (pAML). After 6 days, T cell proliferation, expression of effector molecules and cytokines, and AMG 330-mediated T-cell cytotoxicity were assessed by flow cytometry. Lastly, we performed longitudinal bulk RNA-sequencing on 5000 sorted T cells from 7 matched ID-RL primary AML samples. Immunophenotypic analysis of BM T-cell subsets revealed a shift from T NAIVE toward central/effector memory subsets during AML progression. We observed lower percentages of T NAIVE in RL (n=3) compared to CR (n=3) CD8 + T cells(11.8 vs. 45.2%, p=0.07; RL vs. CR). Conversely, RL patients showed increased percentages of CD8 + memory T cells (T CM: 23.4 vs. 6.7%; T EM: 29.4 vs. 20.2%; T EMRA: 35.3 vs. 27.8%; RL vs. CR). Further characterization of exhaustion markers exhibited a significantly higher percentage of both CD4 + and CD8 + T cells expressing 2B4 (CD244) in ID (n=19) and RL (n=13) compared to HD (n=10, both p &lt; 0.001). A higher percentage of PD-1 + CD8 + and TIM-3 + CD4 + T cells was detected in both ID and RL relative to HD (all p &lt; 0.05). However, a significantly increased percentage of CD8 + T cells expressing TIM-3 and CD160 was detected in ID relative to HD (p &lt; 0.05). Intriguingly, RL CD4 + T cells demonstrated a significantly higher level of LAG3 compared to ID (p &lt; 0.01). In line with phenotypic exhaustion features, ID (n=4) and RL (n=5) CD8 + T cells showed reduced proliferation compared to HD (n=4) CD8 + T cells after CD3/CD28 bead stimulation (both p &lt; 0.01). Correspondingly, we observed a marked reduction in the expression of Granzyme B (GZMB) by CD8 + T cells (both p &lt; 0.05). Interestingly, when compared to ID, RL CD4 + T cells showed decreased TNF-α secretion (p &lt; 0.05). In contrast to these findings, AMG 330-mediated T cell cytotoxicity against OCI-AML3 target cells was superior with RL T cells compared to ID T cells (p &lt; 0.001). The percentage of GZMB + CD8 + T cells strikingly enhanced in RL relative to ID (p &lt; 0.01). In an autologous setting with pAML samples, T cells from RL patients (n=6) showed higher AMG 330-mediated cytotoxicity compared to ID (n=9) T cells (67.7 vs. 35.2; RL vs. ID). In our longitudinal RNA-sequencing, differentially expressed genes analysis detected 61 up- and 30 downregulated genes (log2 FC &gt; 1 or &lt; -1; p &lt; 0.01) in RL T cells compared to their matched ID counterparts. Among the significantly upregulated genes in RL, we identified genes associated with memory T cell function (TP53INP2, DUSP4) and exhaustion (NR4A1, TOX2). Moreover, Gene set enrichment analysis showed significant enrichment of gene signatures associated to memory and exhausted T cells (normalized enrichment score (NES)=1.2 and 1.3; p-value= 0.026 and 0.008, respectively), depletion of oxidative phosphorylation (NES=-2.05; p adj &lt; 0.0001) and protein secretion (NES=-1.49; p adj &lt; 0.05) gene signatures in RL vs. ID T cells. Taken together, our data show that patient T cells acquire an activated/exhausted phenotype upon AML progression. However, this is not reflected in the T-cell effector functions upon AMG 330 stimulation, in contrast to bead stimulation. These observations may highlight the significant role of the AML target cells in shaping a T-cell response. To this end, we will further analyze the longitudinal communication between T cells and their corresponding AML blasts. Disclosures Brauchle: Adivo: Current Employment. Kischel: Amgen GmbH Munich: Current Employment. Buecklein: BMS/Celgene: Consultancy, Research Funding; Amgen: Consultancy, Honoraria; Kite/Gilead: Consultancy, Honoraria, Other: Congress and travel support, Research Funding; Miltenyi: Research Funding; Novartis: Consultancy, Other: congress and travel support, Research Funding, Speakers Bureau; Pfizer: Consultancy, Honoraria, Speakers Bureau. Subklewe: Novartis: Consultancy, Research Funding, Speakers Bureau; MorphoSys: Research Funding; Roche: Research Funding; Miltenyi: Research Funding; Seattle Genetics: Consultancy, Research Funding; Gilead: Consultancy, Research Funding, Speakers Bureau; BMS/Celgene: Consultancy, Research Funding, Speakers Bureau; Amgen: Consultancy, Research Funding, Speakers Bureau; Janssen: Consultancy; Pfizer: Consultancy, Speakers Bureau; Takeda: Speakers Bureau; Klinikum der Universität München: Current Employment.

Differential Regulation Of Chemokine Receptor Expressions On T Lymphocyte Subsets In Healthy Donors After Mobilization With Rhg-CSF and Its Correlation With Acute GvHD

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3296-3296 ◽  
Author(s):  
Han-Yun Ren ◽  
Meng Wang ◽  
Xiang-Juan Ma ◽  
Yu-Jun Dong ◽  
Zhi-Xiang Qiu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Chemokine Receptor ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Chemokine Receptors ◽  
T Cell Subsets ◽  
Down Regulation ◽  
Healthy Donors ◽  
Before And After

Abstract Introduction This study is aimed to investigate chemokine receptors (CCR5, CCR6, CCR7, CCR9, CXCR3 and CCR2) expression on T cell subsets in healthy donors after mobilization with recombinant human granulocyte colony-stimulating factor (rhG-CSF) and analyze its correlation with acute graft-versus-host disease (aGVHD) and to understand the possible mechanisms underlying rhG-CSF-induced immune tolerance. Methods Sixty-eight healthy donor and their recipient pairs of family donor allogeneic hematopoietic stem cell transplantation (allo-HSCT) were included in this study. The expressions of chemokine receptors on CD4+ and CD8+ T cells in the peripheral blood (PB) before and after mobilization was detected using flow cytometry (FCM) respectively. Six chemokine receptors (CCR2, CCR5, CCR6, CCR7, CCR9 and CXCR3) were detected on T cell subsets in all the donors, and CCR5 and CCR7 were detected only in eighteen of all the donors. The expressions of chemokine receptor before and after mobilization was compared and its correlation with II-IV aGVHD were analyzed. Results After rhG-CSF mobilization, the expression of CCR9 on CD4+ T cells and CCR7 on CD8+ T cells were significantly upregulated compared with that before mobilization (p<0.05). However, the mean value of CCR5, CCR6 and CXCR3 expression on CD4+ and CD8+ T cell subsets in PB after mobilization didn’t differ significantly compared with that before mobilization(p>0.10). However, different individuals showed apparent inconsistencies. According to the changes of chemokine receptor expression on CD4+ and CD8+ T cell subsets, the evaluable donors and their relevant recipients were divided into the down-regulated group and the non-down-regulated (unchanged or up-regulated ) group. The incidence of grade II to IV aGVHD in the two groups were compared in their corresponding recipients. In the univariate analysis, mismatched HLA (p=0.046), down-regulation of CCR7 expression on donor CD4+ T cell subsets (p=0.010), unchangeableness or up-regulation of CCR5 expression on donor CD4+ T cell subsets (p=0.032) and CCR6 down-regulation on donor CD8+ T cells (p=0.045) were risk factors for recipients to develop II-IV aGVHD. In the multivariate analysis, down-regulation of CCR7 expression on donor CD4+ T cells after rhG-CSF was independent risk factor for II-IV aGVHD [RR=3.5, 95% CI (1.3-9.4), p=0.012], while CCR5 down-regulation on CD4+ T cells could reduce the incidence of II-IV aGVHD [RR=0.3, 95% CI (0.1-0.8), p=0.031]. Conclusions rhG-CSF mobilization could lead to differential regulation of chemokine receptors expression on T cell subsets, which might cause different effects on the migration of T cells in vivo, and decrease T cells trafficking towards GVHD target organs, and thus reduce the incidence of aGVHD after transplantation. Disclosures: No relevant conflicts of interest to declare.

Most antiviral CD8 T cells during chronic viral infection do not express high levels of perforin and are not directly cytotoxic

Blood ◽  
2003 ◽  
Vol 101 (1) ◽  
pp. 226-235 ◽  
Author(s):  
Dong Zhang ◽  
Premlata Shankar ◽  
Zhan Xu ◽  
Brooke Harnisch ◽  
Gang Chen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Chronic Infection ◽  
Ex Vivo ◽  
Barr Virus ◽  
Cell Functions ◽  
Healthy Donors ◽  
Epstein Barr ◽  
Antigenic Exposure

Abstract Despite the frequency of HIV-specific CD8 T cells, most HIV-infected patients do not control viral replication without antiviral drugs. Although CD8 T cells are important in containing acute HIV and simian immunodeficiency virus (SIV) infection, CD8 T-cell functions are compromised in chronic infection. To investigate whether functional deficits are specific to HIV, the phenotypic and functional properties of HIV, Epstein-Barr virus (EBV), and cytomegalovirus (CMV)–specific CD8 T cells, labeled with HLA A2.1 or B8 tetramers, were compared in 35 HIV-infected and 9 healthy donors. Cytotoxic T lymphocytes express the cytolytic molecules perforin and granzymes, and are thought to be CD45RA+CD27−. Although most HIV- specific cells are antigen experienced and express granzyme A (median, 85%), few express high levels of perforin (median, 10%) or CD45RA (median, 14%) or have down-modulated CD27 (median, 12%). Perforin expression by HIV-specific cells is not significantly different from that of EBV- or CMV-specific cells in the same donors or in healthy donors. EBV- and CMV-specific cells, like HIV-specific cells, are often not cytotoxic when tested directly ex vivo. HIV-specific T-cell expression of other phenotypic markers is similar to that of EBV- and CMV-specific CD8 T cells in healthy donors. However, CMV-specific cells (and, to a lesser extent, EBV-specific cells) in HIV-infected donors are more likely to be CD27−, CD45RA+, and GzmA+. These results suggest that the chance to eradicate an infection by T-cell–mediated lysis may be undermined once an infection becomes chronic. Impaired antiviral cytotoxicity during chronic infection is not specific to HIV but likely represents the immune response to chronic antigenic exposure.

scholarly journals Eomes and IL-10 Regulate Anti-Tumor Activity of T Cells in Chronic Lymphocytic Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4288-4288
Author(s):  
Laura Llaó Cid ◽  
Philipp M Rößner ◽  
Ekaterina Lupar ◽  
Bola S Hanna ◽  
Jérôme Paggetti ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Research Funding ◽  
Ex Vivo ◽  
Cd8 T Cell ◽  
Lymphocytic Leukemia ◽  
Increased Risk ◽  
Leukemia Development

Introduction: Genome-wide association studies showed that a single-nucleotide polymorphism (SNP) affecting the transcription factor Eomesodermin (Eomes) is associated with a significantly increased risk to develop chronic lymphocytic leukemia (CLL). Eomes and its paralogue T-bet are known master regulators of CD8+ effector T cells and CD4+ T helper cells and critical for T cell-mediated immune responses against pathogens and cancer. While Eomes has been shown to be essential for effector function of CD8 T cells, its role in CD4 T cells is less well understood. Recent work suggested that Eomes drives the development of IL-10 and IFNγ co-producing, FoxP3-negative, regulatory T cells, named Tr1 cells, a population that was found enriched in inflamed tissues in patients with chronic inflammatory disorders. In CLL, the T cell compartment is altered with an enrichment of effector and memory T cells that were shown to control leukemia development in a mouse model, but also to acquire a dysfunctional or exhausted state. Methods: We investigated Eomes-expressing CD4 and CD8 T cells in blood and lymph node (LN) samples of patients with CLL, as well as in the Eµ-TCL1 mouse model of CLL by flow cytometry, CyTOF, mRNA sequencing, and ex vivo functional assays. The role and function of these cells was explored in bone marrow-chimeric mice harbouring an Eomes-deficient hematopoietic microenvironment that were used for adoptive transfer of TCL1 leukemia, as well as by co-transfer experiments of Eomes- or IL-10R-deficent CD4 or CD8 T cells with TCL1 leukemia cells in Rag2-/- mice lacking B and T cells. Results: We detected an accumulation of Eomes-expressing CD4 and CD8 T cells in CLL patients, which was more severe in LN compared to blood samples, and significantly stronger in CLL LN compared to reactive LN samples as non-cancer control (Fig. 1A). This was in line with an observed expansion of Eomes-positive T cells in the spleen of leukemic Eµ-TCL1 mice and upon adoptive transfer of TCL1 leukemia. Eomes expression in CD8 T cells correlated with the expression of CD69, IFNγ and PD-1, suggesting a link between Eomes and CD8 T cell activation and function in CLL. The importance of Eomes in CD8 T cell-mediated control of CLL was demonstrated in mice that were transplanted with TCL1 leukemia, where Eomes-deficient CD8 T cells failed to control leukemia development. As we detected significantly less Eomes-deficient CD8 T cells in these mice compared to respective wildtype controls, and a lower percentage of them was positive for the proliferation marker Ki-67, we conclude that Eomes drives the differentiation and expansion of CD8 T cells in mice with CLL-like disease. We further explored Eomes-expressing CD4 T cells in CLL by transcriptome analysis, flow cytometry and ex vivo functional assays, and observed increased expression of IFNγ and IL-10, as well as inhibitory receptors, like PD-1, BLIMP-1 and LAG3, features that are described for Tr1 cells. Transfer of Eomes-deficient or wildtype CD4 T cells in Rag2-/- mice that were injected with TCL1 leukemia cells, lead to a comparable expansion of CD4 T cells independent of Eomes. But even though wildtype CD4 T cells were able to control leukemia development in this setting, Eomes-deficient CD4 T cells failed to do so (Fig. 1B). As Eomes is a known driver of IL-10 expression, we tested whether IL-10R signalling in CD4 T cells is involved in the anti-tumor activity by performing respective CD4 T cell transfer experiments comparing this time wildtype with IL-10R-deficent CD4 T cells. Interestingly, lack of IL-10R in CD4 T cells lead to a reduction in anti-tumor control (Fig. 1C) and therefore suggests that IL-10 is involved in Eomes-driven regulation of CD4 T cell-mediated immune control. Conclusions: In summary, we conclude that Eomes is required for CD8 T cell-mediated control of CLL, and Eomes+ PD-1+ IL-10-producing CD4 T cells contribute to adaptive immunity in CLL. The increased risk of developing CLL in individuals harbouring a SNP in the Eomes gene might be therefore explained by a negative impact of this alteration on CD4 and/or CD8 T cell-mediated immune control of CLL. Disclosures Stilgenbauer: Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; GSK: Consultancy, Honoraria, Research Funding, Speakers Bureau; Hoffmann La-Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau; Pharmacyclics: Other: Travel support; AbbVie: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; AstraZeneca: Consultancy, Honoraria, Research Funding, Speakers Bureau.

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