Glucagon-like Peptide-2 (GLP-2) Treatment Reduces Gvhd-Related Mortality in a Pre-Clinical Transplant Model Via Expansion of Macrophages with Tolerogenic Potential

The gastrointestinal (GI) tract is a major target in GVHD. Conditioning-induced damage and mucosal barrier disruption are important factors in GVHD, however therapies targeting these processes have not been identified. Glucagon-like-peptide 2 (GLP-2) is an enterocyte-specific growth factor produced by L cells that has regenerative potential in models of GI damage. Its impact on the mucosal immune system has not been elucidated. We sought to examine the therapeutic and immunologic effect of GLP-2 in murine GVHD. We employed a major MHC-mismatched GVHD model (C57BL/6J → BALB/cJ). Mice were treated with 800nmol/kg/day of Elsiglutide (a GLP-2 analogue, provided by Helsinn) or vehicle beginning on D+1 for 30 days. Treatment with GLP-2 significantly improved survival and GVHD scores (Fig. 1A), while increasing small intestine mass and villi length (Fig 1B). GLP-2 also reduced T-cell infiltration into the jejunum (Fig. 1C). Analysis of intestinal immune cells by 28-color flow cytometry revealed dramatic differences between treatment groups in both myeloid- and T-cells. On D+14, GLP-2 led to an increased proportion of donor CSF-1R+ macrophages in the lamina propria (LP) (Fig. 2A) - cells that support the maintenance of the intestinal stem cell niche (Sehgal, Nat Commun, 2018). On D+21 the LP donor myeloid compartment was further altered, especially in MHC IIlow F4/80+ CD64+ macrophages (Fig. 2B, C). Here GLP-2 treatment expanded macrophages with lower expression of the co-stimulatory molecules CD80 and CD86 as well as the phagocytic marker CD206, whilst increasing the inhibitory molecule SIRPα, consistent with a tolerogenic phenotype. GLP-2 treatment also increased CX3CR1 expression on MHC IIlow macrophages with reduced Ly6C - a phenotype associated with physiologic macrophage maturation and linked to the resolution of colitis (Zigmond, Immunity, 2012). Vehicle-treated mice, conversely, had predominance of Ly6Chigh MHC IIlow LP macrophages reminiscent of an early infiltrating phenotype and near absence of mature macrophages, suggesting an impaired monocyte-macrophage transition that was restored by GLP-2. In addition, GLP-2 treatment led to significant changes in donor intraepithelial lymphocytes on D+21 (Fig. 2D), where CD8 T cells exhibited decreased CD27, CD103 and CXCR3 expression but higher PD-1, suggesting less activation. To assess potential mechanisms for the differences in macrophage and T-cell phenotype, we examined the impact of GLP-2 on the intestinal microbiota. A syngeneic BALB/cJ model was used to explore the effects of GLP-2 independent of GVHD. Stool samples from D+0, D+14, and D+28 were subjected to 16S rRNA sequencing. Vehicle-treated mice had distinct β-diversity clusters at all time-points, showing a transplant effect on the microbiota (Fig. 3A). GLP-2-treated mice had near-complete cluster overlap between D+0 and D+14, suggesting attenuation of the impact of conditioning. GLP-2 treated mice were significantly enriched for Akkermansia muciniphila and Bacteroidales S24-7 family at D+14 and D+28 (Fig. 3B). These taxa have been associated with anti-inflammatory properties and A. muciniphila abundance is linked to epithelial mucin production, which is increased by GLP-2. We then assessed the role of microbial communities in the protective effect of GLP-2 by conducting an allogeneic transplant with 3 caging conditions; 1) vehicle and GLP-2 treated mice caged together, 2) caged separately, or 3) caged separately plus oral antibiotics. We observed a clear cage effect where co-housing the treatment groups improved the survival of vehicle treated mice (Fig. 3C), suggesting transferal of the therapeutic effect via the microbiome. Antibiotic administration also dampened the beneficial effect of GLP-2. Finally, we conducted a GvL experiment by co-transplanting Luc-A20 and monitoring tumor progression via bioluminescence imaging. Both GLP-2 and vehicle-treated mice eliminated the tumor, whereas mice receiving T-cell depleted bone marrow showed tumor progression (Fig. 3D). In summary, our results demonstrate high therapeutic potential of GLP-2 in GVHD. GLP-2 administration led to reduced mortality, modified the microbiome and altered the intestinal immune response to a more tolerogenic state. This novel mechanism sheds light on the role of the enteroendocrine system in maintaining gut homeostasis and sets the stage for therapeutic clinical trials. Figure 1 Disclosures Uhlemann: Allergan: Research Funding; GSK: Research Funding; Merck: Research Funding. Reshef:Gilead: Consultancy; Magenta: Consultancy; Novartis: Consultancy; Monsato: Consultancy; Atara: Consultancy; BMS: Consultancy.

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The Impact of Exercise Serum on Selected Parameters of CD4+ T Cell Metabolism

Immuno ◽

10.3390/immuno1030008 ◽

2021 ◽

Vol 1 (3) ◽

pp. 119-131

Author(s):

Jana Palmowski ◽

Kristina Gebhardt ◽

Thomas Reichel ◽

Torsten Frech ◽

Robert Ringseis ◽

...

Keyword(s):

T Cells ◽

Oxygen Consumption ◽

T Cell ◽

Real Time ◽

Cd4 T Cells ◽

Cell Metabolism ◽

Cd4 T Cell ◽

Autologous Serum ◽

Cell Phenotype ◽

The Impact

CD4+ T cells are sensitive to peripheral changes of cytokine levels and metabolic substrates such as glucose and lactate. This study aimed to analyze whether factors released after exercise alter parameters of human T cell metabolism, specifically glycolysis and oxidative phosphorylation. We used primary human CD4+ T cells activated in the presence of autologous serum, which was collected before (CO) and after a 30-min exercise intervention (EX). In the course of activation, cells and supernatants were analyzed for cell viability and diameter, real-time oxygen consumption by using PreSens Technology, mRNA expression of glycolytic enzymes and complexes of the electron transport chain by real-time PCR, glucose, and lactate levels in supernatants, and in vitro differentiation by flow cytometry. EX did not alter T cell phenotype, viability, or on-blast formation. Similarly, no difference between CO and EX were found for CD4+ T cell activation and cellular oxygen consumption. In contrast, higher levels of glucose were found after 48 h activation in EX conditions. T cells activated in autologous exercise serum expressed lower HK1 mRNA and higher IFN-γ receptor 1. We suggest that the exercise protocol used was not sufficient to destabilize the immune metabolism of T cells. Therefore, more intense and prolonged exercise should be used in future studies.

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Generation and Characterization Of a CD30 Positive T-Cell Lymphoma Mouse Model Resembling Human Anaplastic Large Cell Lymphoma

Blood ◽

10.1182/blood.v122.21.2450.2450 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2450-2450

Author(s):

Cathrin Klingeberg ◽

Anna Lena Illert ◽

Nicolas Schneider ◽

Christian Peschel ◽

Cornelius Miething ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Large Cell ◽

Cre Recombinase ◽

Double Negative ◽

Ki 67 ◽

The Impact

Abstract Anaplastic large cell lymphomas (ALCL) are a subgroup of aggressive Non-Hodgkin-Lymphomas mainly affecting children and young adults. In 60 % of systemic ALCLs, a translocation t(2;5) (p23;q35) resulting in NPM-ALK fusion gene expression is found. The constitutively activation of ALK tyrosine kinase expressed from the NPM-promoter causes increased proliferation and inhibition of apoptosis thereby promoting cell survival and tumorigenesis. Immunphenotypic characterization of human ALCLs revealed highly CD30-positive cells of T- or Null-Cell-origin and resulted in promising clinical trials with CD30-coupled antibodies. However, the impact of CD30 on diseases development as well as NPM-ALK signal transduction in course of disease remains unclear and appropriate mouse models to answer these questions are missing. In this regard, we established a retroviral murine bone marrow (BM) transplantation model resembling a human ALCL-like T-cell neoplasia. Therefore we use an inducible Cre/loxP system where NPM-ALK expression is controlled and expressed in a special type of early T-cells. For generation of this vector, we inserted a floxed translational ‘stop-cassette’ between the retroviral promoter MSCV-LTR and the NPM-ALK cDNA, which guaranties specific expression of NPM-ALK only in cells, where the enzyme Cre-recombinase is expressed. Recognition of the loxP-sites by Cre-recombinase leads in our system to deletion of the stop-cassette and consequently NPM-ALK expression. Using different Cre-expressing cell types allowed us to study pathogenesis of ALCL in more detail. In our recent study, we infected bone marrow of transgenic mice expressing Cre-recombinase under the control of the Lck-promotor with our MSCV-Stop-NPM-ALK-IRES-EGFP (MSNAIE) vector and transplanted it into lethally irradiated C57Bl6 recipient mice. With a latency of 4-5 months, these mice developed Thy1.2-positive lymphomas and died from neoplastic infiltration of bone marrow and lymphatic organs with T-cells. Immunphenotypic analyses confirmed T-Cell origin of the lymphomas and showed importantly highly CD30-expression. Staining of the different T-cell-subpopulations demonstrated highest NPM-ALK expression in immature CD4/CD8 double negative T-cells and not fully differentiated CD4/CD8 double positive T-cells. Interestingly, FACS-staining of the proliferation marker Ki-67 revealed highest expression in CD4/CD8 double negative T-cells, in contrast to the other subpopulations where Ki-67 is less detected. Therefore we hypothesized, that the lymphoma initiating cell (LIC) must be within this early T-cell population. Most interestingly we found highest CD30-expression just in the same CD4/CD8 negative T-cell population, pointing to a crucial role of CD30 in lymphoma initiation. To further substantiate our hypothesis we performed secondary and tertiary transplantations with different sorted T-Cell subpopulation and indeed, the immature CD4/CD8 double negative population was able to initiate lymphoma growth in recipient mice. Further transplantations by limited dilution will help to identify the leukemia initiating cell in this model. Taken together, our murine LckCre-NPM-ALK bone marrow transplantation model represents a precise and versatile tool to study disease initiation and development resembling human ALCL. Moreover, the impact of specific proteins (e.g. CD30) in the course of disease can be addressed by combining Knockout (e.g. CD30)/LckCre transgenic mice with our model. To this end we crossed CD30/Lck-Cre mice, and preliminary analysis indicate that CD30 expression seems not to be required for the initial onset of disease. Further characterization of the role of CD30 in ALCL is ongoing. Disclosures: No relevant conflicts of interest to declare.

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Impact of Lenalidomide Maintenance Therapy on Immune Polarization and Activation in Multiple Myeloma

Blood ◽

10.1182/blood.v126.23.2966.2966 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2966-2966 ◽

Cited By ~ 1

Author(s):

Manisha Bhutani ◽

David Foureau ◽

Tammy Cogdill ◽

Kyle Madden ◽

Qing Zhang ◽

...

Keyword(s):

Multiple Myeloma ◽

T Cells ◽

T Cell ◽

Maintenance Therapy ◽

T Cell Activation ◽

Research Funding ◽

Cell Activation ◽

Lymphoid Cells ◽

Relative Distribution ◽

The Impact

Abstract BACKGROUND: Lenalidomide is an immunomodulatory drug (IMiD) with co-stimulatory effects on immune effector cells in vitro and is an approved treatment for multiple myeloma (MM), although its mode of action in patients is not well defined. We studied the impact of lenalidomide maintenance therapy, following autologous stem cell transplant (ASCT), on NK and NK-T polarization (i.e. activating or inhibitory molecules) and, T cell activation (early vs. late activation) in patients with multiple myeloma. PATIENTS AND METHODS: In this ongoing prospective study with a targeted enrollment of 28 newly diagnosed multiple myeloma patients, blood samples are being collected at 2 to 3 months post ASCT, before starting lenalidomide maintenance therapy (baseline), and serially after 1, 3 and 6 months of treatment (T+1mo, T+3mo, T+6mo). Using a 9 color flow cytometry panel, peripheral blood samples were analyzed for expression of CD3 and CD56 to define NK (CD56+ CD3-), NKT (CD56+ CD3+), and T cell (CD56- CD3+) subsets. Killer 'inhibitory' Ig-like receptors, (KiR2DS4, KiR3DL1) natural killer group 2 proteins (NKG2a, NKG2D) and natural killer p46 protein (NKp46) expression were quantified to assess polarization of NK, and NK-T cells. Programmed death receptor 1 (PD-1) and T-cell Ig and mucin receptor 3 (Tim3) expression was quantified to assess T cell activation state. Flow cytometry data were acquired on a BD FACSAria II, and analyzed using FlowJo version X software. RESULTS: Samples from 11 patients have been collected and analyzed so far (11 baseline, 6 T+1mo, 4 T+3mo). At baseline lymphoid cells represent 12-46% of white blood cells (WBC), this heterogeneity being mainly driven by a wide range of T cell relative distribution among patients (30-74 % lymphoid cells). Phenotypically, NK cells at baseline mainly express natural cytotoxicity receptors (NKp46 and NKG2D), whereas NK-T cell also express NKG2D but approximately 1/3 also express PD-1 indicating they may be functionally defective. T cells at baseline express early T cell activation markers NKG2D and approximately 1/3 also stained positive for late T cell activation marker PD-1. Lymphoid cells relative distribution among WBC tends to normalize at T+1mo of treatment (15 to 35 % of WBC) before expanding at T+3mo (35 to 43 % of WBC). Phenotypically, across the 27 immune variables analyzed, each multiple myeloma patient displayed high level of immune homeostasis after 1 or 3 months of lenalidomide treatment. Noticeably, Nkp46 expression by NK cell and PD-1 expression by NK-T cells decreased in 4/6 patients and, NKG2D expression by T cell decreased in all but one patient during lenalidomide therapy. CONCLUSION: To our knowledge, this is the first study examining the influence of lenalidomide maintenance on the comprehensive immune repertoire in the post-ASCT setting in MM patients. The wide heterogeneity of NK, NK-T and T cell distribution observed at baseline among lymphoid cells indicates the potential effect of post-ASCT immune reconstitution and immunomodulatory the impact of lenalidomide. The capacity of lenalidomide to mediate effects on several immune cells raises the question as to which, if any, of these changes correlate with clinical responses. In our study, serially collected data from each patient, when completed would determine the impact of lenalidomide immunomodulatory effect of therapeutic efficacy and PFS duration in relation to immune reconstitution stage. Disclosures Cogdill: Millennium: Speakers Bureau; Onyx: Speakers Bureau; Celgene: Speakers Bureau; Novartis: Speakers Bureau. Ghosh:Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Usmani:Sanofi: Honoraria, Research Funding; Millennium: Honoraria, Speakers Bureau; Onyx: Honoraria, Research Funding, Speakers Bureau; Pharmacyclics: Research Funding; Celgene: Honoraria, Speakers Bureau; Janssen Oncology: Honoraria, Research Funding; Array BioPharma: Honoraria, Research Funding.

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CD33 BiTE ® Construct Mediated Immunological Synapse Formation and Downstream Signaling in T Cells Is Dependent on Expression of Costimulatory Molecules on Target Cells

Blood ◽

10.1182/blood-2021-146488 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2237-2237

Author(s):

Anetta Marcinek ◽

Bettina Brauchle ◽

Gerulf Hänel ◽

Sonja M Lacher ◽

Nora Zieger ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Research Funding ◽

Synapse Formation ◽

Costimulatory Molecules ◽

Target Cells ◽

Immune Synapse ◽

Downstream Signaling ◽

Signaling Receptors

Abstract BiTE ® (Bispecific T-cell Engager) constructs represent a novel immunotherapeutic strategy that recruits T cells against cancer cells independent of their TCR specificity. Currently, two CD33xCD3 BiTE ® antibody constructs (AMG 330 & AMG 673) are being investigated in phase I dose escalation trials in patients with relapsed/refractory Acute Myeloid Leukemia (AML) with early evidence of acceptable safety and anti-leukemic activity (Ravandi et al., ASH 2020; Subklewe et al., EHA 2020). So far, details of BiTE ® mediated T-cell engagement and information on parameters contributing to their efficacy need more investigation. Therefore, we aimed to characterize the interplay between target and effector cells to deepen our mechanistic understanding of BiTE ® construct mediated T-cell engagement. Previously, we have created a novel in vitro model system with murine Ba/F3 cells expressing human (hu) CD33 ± huCD80 ± huCD86 ± huPD-L1 to study T-cell proliferation and cytotoxicity induced by AMG 330. Using that system, we showed that expression of T-cell co-signaling receptors on target cells modulate AMG 330 induced T-cell activity (Marcinek et al., ASH 2018, EHA 2019). Here, we hypothesize that expression of costimulatory molecules impacts BiTE ® mediated immune synapse formation and consecutive downstream signaling in BiTE ® construct activated T cells. To study whether AMG 330 can induce synapse formation and TCR triggering we used a previously described reconstituted T-cell system, which consists of non-immune (HEK) cells introduced with genes encoding the TCR and other proteins (e.g. CD45) required for the regulation of TCR phosphorylation (James et al., Nature 2012). HEK-T cells were incubated with huCD33 transduced RajiB cells in presence of fluorescently labeled AMG 330 or a control BiTE® (cBiTE) construct to allow cell conjugation. A spinning disc confocal microscope system was used to image cells. To pinpoint the role of T-cell co-signaling receptors in immune synapse formation we incubated differentBa/F3 cell constructs or primary AML (pAML) cells with healthy donor T cells in the presence of AMG 330 and analyzed intensity of LFA-1 expression within the synapse using an Imaging Flow Cytometer. Furthermore, we determined phosphorylation of ZAP70, AKT and ERK in conjugated T cells after various time points by phosphoflow cytometry. We observed that AMG 330, in contrast to cBiTE®, induced TCR triggering reflected by exclusion of CD45 from the RajiB-T-cell-interface. Simultaneously clustering of CD33 occurred in AMG 330 induced cell-cell-interfaces (Fig. 1A/B). The percentage of conjugates formed with huCD33 + Ba/F3 cells was significantly higher in constructs expressing huCD86, compared to those expressing no costimulatory antigens or additional huPD-L1 (Mean % in huCD33 + Ba/F3: 2.8 vs. huCD33 + CD86 +.Ba/F3: 4.2 [p=0.0031] vs. huCD33 + huCD86 + PD-L1 + Ba/F3: 2.8 [p=0.0018]). This was accompanied by LFA-1 accumulation within the T-cell-Ba/F3 cell synapse (Mean of MFI in huCD33 + CD86 +.Ba/F3: 10,933 > huCD33 + huCD86 + PD-L1 + Ba/F3: 7,749 > huCD33 + Ba/F3: 7,028). For downstream signaling in T cells after engagement with Ba/F3 cell constructs in the presence of AMG 330, we observed that kinase phosphorylation was highest after 10 minutes in CD86 co-expressing Ba/F3 cells (Mean % of phosphorylation in T-cell conjugates with huCD33 + vs huCD33 + huCD86 + vs huCD33 + CD86 +.PD-L1 + Ba/F3: pERK 40.9 vs 54.3 [p=0.0064] vs 51.2 %; pAKT: 69.1 vs 81.5 [p=0.0642] vs 75.1 %; pZAP70: 6.9 vs 12.2 [p<0.0001] vs 7.7 % [p<0.0001]) (Fig. 1C). Finally, we evaluated if these finding could also be observed in pAML samples. For that, we determined LFA-1 expression intensity within AMG 330-induced pAML-T-cell synapses. We used CD33 + pAML samples with either high CD86 and no PD-L1 expression or vice versa. Comparing synapse formation between these samples, LFA-1 intensity was 4.6-fold higher in the CD86 + PD-L1 - sample compared to the CD86 - PD-L1 + pAML. Taken together, our data unravel molecular mechanisms of BiTE® construct induced immune synapse formation, highlighting the role of costimulatory molecules in this process. They support the notion that T cell co-signaling receptors like CD86 and PD-L1 modulate T-cell response in an early event manner. Prospective analyses in clinical trials are needed to validate the relevance of checkpoint molecule expression on target cells as a potential predictive biomarker for response. Figure 1 Figure 1. Disclosures Brauchle: Adivo: Current Employment. Lacher: Roche: Research Funding. Kischel: Amgen GmbH Munich: Current Employment. von Bergwelt: Roche: Honoraria, Research Funding, Speakers Bureau; Miltenyi: Honoraria, Research Funding, Speakers Bureau; Mologen: Honoraria, Research Funding, Speakers Bureau; Kite/Gilead: Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau; Astellas: Honoraria, Research Funding, Speakers Bureau; MSD Sharpe & Dohme: Honoraria, Research Funding, Speakers Bureau; BMS: Honoraria, Research Funding, Speakers Bureau. Theurich: Amgen: Consultancy, Honoraria; BMS/Celgene: Consultancy, Honoraria; GSK: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Takeda: Consultancy, Honoraria. Buecklein: Novartis: Consultancy, Other: congress and travel support, Research Funding, Speakers Bureau; Pfizer: Consultancy, Honoraria, Speakers Bureau; Miltenyi: Research Funding; Kite/Gilead: Consultancy, Honoraria, Other: Congress and travel support, Research Funding; BMS/Celgene: Consultancy, Research Funding; Amgen: Consultancy, Honoraria. Subklewe: Janssen: Consultancy; Seattle Genetics: Consultancy, Research Funding; Roche: Research Funding; Novartis: Consultancy, Research Funding, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Klinikum der Universität München: Current Employment; Takeda: Speakers Bureau; MorphoSys: Research Funding; Miltenyi: Research Funding; Gilead: Consultancy, Research Funding, Speakers Bureau; Amgen: Consultancy, Research Funding, Speakers Bureau; BMS/Celgene: Consultancy, Research Funding, Speakers Bureau.

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Abstract P167: Title: Prediction of Arterial Stiffness from Early T-cell Activation among HIV and HCV Co-infected Women in the Women's Interagency HIV Study (WIHS)

Circulation ◽

10.1161/circ.125.suppl_10.ap167 ◽

2012 ◽

Vol 125 (suppl_10) ◽

Author(s):

Roksana Karim ◽

Naoko Kono ◽

Robert Kaplan ◽

Wendy J Mack ◽

Howard N Hodis ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Arterial Stiffness ◽

Hiv Infection ◽

T Cell Activation ◽

Cell Activation ◽

Infection Status ◽

Activation Markers ◽

Color Flow ◽

The Impact

Introduction: Activation of T-lymphocytes, a hallmark of HIV infection, reaches a set point early in HIV infection and persists even after viral suppression with highly active antiretroviral therapy (HAART). Early T-cell activation predicts subsequent CD4 depletion, progression to AIDS and survival. HIV-infected subjects are at high risk for premature atherosclerosis. Little is known regarding the impact of early T cell activation on arterial stiffness. While Kaplan et al. (2011) were the first and only group to show a cross-sectional association, we investigate here if early T cell activation can predict future arterial stiffness. Hypothesis: High early T cell activation will predict increased arterial stiffness, measured 5.5 (IQR=2.5-7.5) years later, in HIV and HCV co-infected women. Methods: A longitudinal study nested within the WIHS, an ongoing prospective cohort study. Percentages of CD4 and CD8 T cell activation, assessed by CD38 and HLA-DR co-expression using 3-color flow cytometry, were measured on average 5.5 years before arterial stiffness assessments (carotid artery distensibility, and Young’s elastic modulus for elasticity) using B-mode carotid ultrasound. Multiple linear regression models evaluated the association between log-transformed T cell activation markers (independent variables) and arterial stiffness (dependent variable). Analyses were stratified by HCV co-infection status and by pre- and post-HAART assessment of T cell activation. Results: A total of 376 HIV+ women (185 HCV+) were included in the analysis. Participants were on average 46(SD=9) years old, 59% Black, and 49% were current smokers. Activation of both CD4 and CD8 T cells significantly univariately predicted reduced distensibility and elasticity among HIV-infected women. CD4 activation continued to significantly predict distensibility (β(SEM)= −3.51(1.30) 10 −6* N −1* m 2 , p=0.01), and elasticity (0.11(0.04)10 5* N * m 2 , p=0.004) with adjustment for age, race, BMI, smoking, ART, CD4 count, and HIV RNA. CD8 activation was no longer associated after adjustment. When stratified by HCV co-infection status, the prediction of arterial stiffness parameters from early CD4 activation was somewhat stronger among the HIV+/HCV+ women compared to HIV+/HCV- women (β(SEM)= −4.44(1.93), p=0.02 vs. −3.04(1.84), p=0.10 for distensibility, and 0.17(0.06), p=0.003 vs. 0.09(0.05), p=0.09 for elasticity); however the test for interaction was not statistically significant. In a subset of 188 women, CD4 activation measured both pre- and post-HAART significantly predicted later arterial stiffness. Conclusions: CD4 activation level predicts future arterial stiffness in HIV-infected women, perhaps more markedly among HCV co-infected women. These data confirm the proinflammatory impact of activated T cells that can cause vascular dysfunction and shed light on the early onset of atherogenesis.

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Identification of PD1 and TIM3 As Checkpoints That Limit Chimeric Antigen Receptor T Cell Efficacy in Leukemia

Blood ◽

10.1182/blood.v126.23.852.852 ◽

2015 ◽

Vol 126 (23) ◽

pp. 852-852 ◽

Cited By ~ 7

Author(s):

Saad S Kenderian ◽

Marco Ruella ◽

Olga Shestova ◽

Michael Klichinsky ◽

Miriam Y Kim ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Therapy ◽

Cell Line ◽

Research Funding ◽

Hematological Malignancies ◽

Complete Response ◽

Checkpoint Blockade ◽

Blocking Antibodies

Abstract Chimeric antigen receptor T cell (CART) therapy has developed as a powerful and potentially curative therapy in hematological malignancies over the last few years. CD19 directed CART cells have resulted in impressive complete response rates of ~90% in acute lymphoblastic leukemia that are durable for the majority of patients. However, the overall response rates in other malignancies such as chronic lymphocytic leukemia are around 50%. This could be partially related to CART exhaustion and dysfunction induced by leukemia cells. In this study, we aim to evaluate the role of inhibitory receptors/pathways in inducing CART cell dysfunction and exhaustion in hematological malignancies. As a tumor model, we used an acute myeloid leukemia (AML) cell line (MOLM14) and primary AML samples and treated them with CD33 or CD123 directed CART cells (second generation CARs using 4-1BB and CD3z signaling domains and a lentiviral vector). Incubation of primary AML samples or MOLM14 cell line with CD33 or CD123 directed CARTs resulted in a significant up-regulation of PD-L1 on tumor cells after 24 hours of incubation (0% on day 0 vs 80% on day 1, P <0.001), and up-regulation of PD-1 and TIM-3 on T cells 3-7 days post co-culture (8% of T cells expressed PD-1 on day 0 vs 43% on day 3, P=0.03 and 13% of T cells expressed TIM-3 on day 0 vs 71% on day 3, p=0.001, Figure 1). For in vivo experiments, we used NSG (NOD-SCID-g-/-) mice and engrafted them with the MOLM14 cell line. Treatment of these AML xenografts with suboptimal doses of CD33 or CD123 CARTs resulted in initial anti-tumor responses, followed by disease relapses in 40-60% of the mice (Figure 2A). T cells were isolated from the bone marrow of these mice and analyzed for differential expression of inhibitory receptors. There was a significantly increased up-regulation of TIM-3 receptors on T cells isolated from mice with relapsed disease compared with T cells isolated from mice in remission after CART cell therapy (Figure 2B). Next, we investigated the role of adding checkpoint blockade to improve T cell function ex vivo after CART cell therapy. Marrows of mice that relapsed after CART cell therapy contained both residual CART cells and leukemia and were used to model the administration of checkpoint blockade in the setting of CART cell exhaustion. Cells were cultured with PD-1 or TIM-3 blocking antibodies or the combination of both (10 ug/ml for 72 hours). CART cell effector functions such as cytokine production and Ki-67 proliferation marker improved in the presence of checkpoint antagonists especially when both PD-1 and TIM-3 blocking antibodies were combined (figure 2C). Finally, we tested the combination of checkpoint blockade with CARTs in AML xenografts. In this approach, we treated MOLM14 xenografts with suboptimal doses of CD33 or CD123 directed CARTs or with control untransduced T cells (UTD), with or without checkpoint blocking antibodies. NSG mice bearing MOLM14 AML xenografts were established. Engraftment was confirmed by bioluminescent imaging. The tumor bearing mice were then treated with suboptimal doses (0.25-0.5 x106 total T cells I.V) of CD33 or CD123 directed CARTs or with control untransduced T cells (UTD). Mice also received PD-1 blockade, TIM-3 blockade or the combination of both on days 3, 6, 9 and 12 post T cell therapy, with the rationale for early checkpoint blockade being based on our in vitro observations of early upregulation of inhibitory ligands on AML after exposure to CART cells. Mice were then followed with serial imaging to assess disease burden. The addition of checkpoint antagonists to untransduced T cells, in particular anti-TIM3, did not lead to an anti-leukemic effect. However, the addition of PD-1 or TIM-3 blockade to CART cell therapy resulted in a synergistic anti-tumor activity as shown in Figure 3. The durable complete response rate was: 45% for treatment with CART123 alone, 80% for treatment with CART123+PD-1 blockade, 100% for treatment with CART123+TIM-3 blockade, and 80% for treatment with CART123+ both PD-1 and TIM-3 blockade). Our preclinical results indicate that PD-1 and TIM-3 pathways are involved in CART exhaustion and dysfunction in AML. Combination of checkpoint inhibitors with CART cells may lead to enhanced efficacy in AML and other hematological malignancies. Current studies are investigating mechanisms of synergy and the role of these combinations in other hematological malignancies. Figure 1. Figure 1. Figure 2. Figure 2. Figure 3. Figure 3. Disclosures Kenderian: Novartis: Patents & Royalties, Research Funding. Ruella:Novartis: Patents & Royalties, Research Funding. Porter:Novartis: Patents & Royalties, Research Funding. June:University of Pennsylvania: Patents & Royalties: financial interests due to intellectual property and patents in the field of cell and gene therapy. Conflicts of interest are managed in accordance with University of Pennsylvania policy and oversight; Novartis: Research Funding. Gill:Novartis: Patents & Royalties, Research Funding.

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Striking Clinical, Molecular and Immunological Outcomes in Non-Human Primate Hematopoietic Stem Cell Transplantation Using a Novel OX40L Blockade Agent, KY1005, Combined with Rapamycin

Blood ◽

10.1182/blood.v128.22.3341.3341 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3341-3341

Author(s):

Victor Tkachev ◽

Scott N. Furlan ◽

Ben Watkins ◽

Betty Zheng ◽

Daniel Hunt ◽

...

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

Research Funding ◽

Cd8 T Cell ◽

Effector Memory ◽

T Cell Proliferation ◽

Hematopoietic Stem ◽

Free Survival ◽

The Impact

Abstract While calcineurin inhibition (CNI)-based strategies remain the mainstay for GVHD prevention, CNI are notoriously antagonistic to immune tolerance induction. Rapamycin (Rapa) has been shown to be more pro-tolerogenic; however, the best agents to combine with Rapa are still undetermined, and it remains a second-line GVHD prevention strategy without clear superiority over CNI. Finding tolerogenic partners for Rapa, therefore, represents a critical unmet need in the field. Of the possible partners for Rapa, the OX40/OX40L pathway represents an important target. OX40 is a costimulatory receptor expressed on activated human T cells, which, upon interaction with OX40L delivers activation signals to conventional T cells (Tconv) promoting their proliferation, survival and clonal expansion. Notably, these same OX40/OX40L signals may either inhibit or promote Treg functions, depending on context, suggesting that blockade of this pathway may simultaneously control Tconv activation while permitting Treg homeostasis. During GVHD in non-human primates (NHP), we found OX40L upregulation on myeloid dendritic cells and OX40 upregulation on activated T cells in recipients treated with multiple immunosuppressive agents, including Rapa (Fig 1). These data provided strong rationale for testing KY1005, a novel human monoclonal antibody that binds to OX40L and blocks its interaction with OX40, as a potential partner with Rapa. We tested the outcomes of prophylactic blockade of this pathway on NHP GVHD, using KY1005 alone and in combination with Rapa. These experiments utilized our previously published NHP GVHD model, in which GVHD is studied after T cell-replete haplo-identical HCT. KY1005 was dosed at 10mg/kg weekly from days -2ˆ+54 and Rapa was continued through Day +100. Prophylaxis with KY1005 alone provided initial evidence for its in vivo activity, with control of CD4>CD8 T cell proliferation and mitigation of the expansion of CD4>CD8 T effector/memory cells. Consistent with the partial control of T cell activation, these recipients demonstrated improved GVHD-free survival versus unprophylaxed controls, but disease ultimately broke through (Median Survival Time (MST) = 19.5 days with KY1005 (n=4) compared to 8 days in unprophylaxed recipients (n= 10, Fig 2)). We next investigated the impact of OX40L blockade + Rapa. We have published that Rapa as a monotherapy minimally controlled both immunologic and clinical disease, with an MST = 14 days (n=6). Combined prophylaxis was striking: recipients given KY1005+Rapa (n=5) maintained robust health throughout the entire experiment (MST >100d), and demonstrated high levels of donor T cell chimerism (86 +/- 3% at Day 100), rapid hematopoietic reconstitution, and had a terminal GVHD Grade of 0, compared to a Grade of III-IV in both KY1005- and Rapa-monotherapy cohorts. Immunologic analysis demonstrated synergistic control of both CD4 and CD8 T cell proliferation, restoring it to the level observed during autologous immune reconstitution, and resulting in a concomitant abrogation of CD4 and CD8 memory/effector expansion while preserving T cells with a na•ve phenotype. In striking contrast to the inhibition of Tconv activation by KY1005+Rapa, recipients of dual therapy demonstrated intact Treg reconstitution post-HCT, which resulted in a favorable Treg:Tconv ratio of 5.4 vs 1.4:100 in KY1005+Rapa treated compared to untreated recipients (p < 0.05). Transcriptomic analysis confirmed the unique immunologic state conferred by KY1005+Rapa on purified T cells, with gene arrays from these recipients demonstrating separation from all other transplant cohorts in Principal Component space (Figure 3A) and Class Neighbor Analysis identifying unique expression modules that tracked with KY1005 + Rapa prophylaxis (Figure 3B red and blue boxes). These results underscore the critical role of OX40/OX40L signaling in the development of GVHD and demonstrate the striking control of GVHD in KY1005+Rapa recipients. They represent the first demonstration of uniform, long-term GVHD-free survival in the primate model of high-risk haplo-identical HCT, and the first therapeutic strategy that simultaneously controls Tconv activation while supporting Treg homeostasis in this model. They suggest that OX40L blockade + Rapa is a novel, evidence-based combinatorial strategy to control GVHD that is an exceptional candidate regimen for clinical translation. Disclosures Tkachev: Kymab Ltd: Patents & Royalties: US Patent 9,382,325, Research Funding. Casson:Kymab Ltd: Employment. Kirby:Kymab Ltd: Employment, Patents & Royalties: US Patent 9,382,325. Bland-Ward:Kymab Ltd: Employment, Patents & Royalties: US Patent 9,382,325. Kean:Juno Therapeutics, Inc: Research Funding.

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THU0031 ABATACEPT ALTERS THE FREQUENCY OF IMMUNOREGULATORY AND EFFECTOR T CELL SUBPOPULATIONS IN RHEUMATOID ARTHRITIS

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2020-eular.4894 ◽

2020 ◽

Vol 79 (Suppl 1) ◽

pp. 229.2-229

Author(s):

B. Dreo ◽

B. Prietl ◽

S. Kofler ◽

H. Sourij ◽

A. Lackner ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

T Cells ◽

T Cell ◽

Autoimmune Diseases ◽

T Cell Subsets ◽

Cell Phenotype ◽

Effector T Cell ◽

Cell Subpopulations ◽

T Cell Subpopulations ◽

The Impact

Background:Under physiological conditions, T regulatory cells (Tregs) are responsible for the downregulation of the immune response. In autoimmune diseases, such as rheumatoid arthritis (RA), auto-inflammation is driven by an imbalance of activation and downregulation of immunological pathways. Thus, treatment plans for autoimmune diseases often involve the enhancement of immunoregulatory pathways by administering inhibitors of costimulation, i.e. CTLA-4-Ig (abatacept, ABA). ABA binds specifically to CD80 and CD86 on antigen presenting cells (APC). Consequently, T cell activation via the CD28 receptor is blocked. Previous studies have demonstrated surprising effects of abatacept on Tregs, specifically decreased frequency of these cells but enhancement in their function1. Whether these alterations can only be found in patients with ABA treatment, or whether they are also present in patients receiving other anti-inflammatory drugs is currently unknown.Objectives:The aim of our research was to delineate the impact of ABA on the different subsets of effector and regulatory T cells in RA and compare these findings with patients receiving tocilizumab (TCZ) or rituximab (RTX).Methods:Peripheral blood samples from 56 RA patients (median ± SE; age: 60.5 ± 1.3 years, female ratio: 0.7, disease duration: 17.9 ± 2.1 years; respectively) were drawn over a sampling period of 2 years. Freshly isolated PBMCs of RA patients were stained with fluorochrome-labelled antibodies and T cell subsets were identified by flow cytometric means. CD3+CD4+T cells were further classified using different T cell markers (CD25, CD127, CD39, CD95). All cytometric measurements were performed using a standardized BD LSR-Fortessa platform. RA patients were compared according to their treatment with ABA, TCZ or RTX.Results:Eighteen out of 56 RA patients (32%) received ABA, 25 patients (45%) received TCZ and 13 patients (23%) were under CD20+ cell depletion therapy with RTX. RA patients receiving ABA displayed a significant decrease in CD3+CD4+CD25+CD127dimTregs (3.7% ± 0.4) compared to patients with TCZ (5.4% ± 0.4, p = 0.041) and patients under RTX treatment (7.52% ± 0.93, p = 0.026). CD39+Tregs were significantly higher in RA patients treated with TCZ (49.5% + 3.2, p = 0.000) or RTX (50.5% ± 5.3, p = 0.026) compared to patients receiving ABA (24.5% ± 3.1). In addition, the frequency of CD95+Tregs was significantly reduced in ABA patients compared to RTX patients (59.6% ± 3.1 vs.76.7% ± 3.6, p = 0.014; respectively). Interestingly, T cells displaying an effector T cell phenotype (CD3+CD4+CD25+/-CD127+) were increased in ABA treated patients compared to RTX treated patients (59.6% ± 3.1 and 76.7% ± 3.6, p = 0.002). Since none of our patients were a non-responder or had high disease activity, we could not analyse whether these changes are associated with treatment outcome.Conclusion:Our data demonstrate that blockage of T cell stimulation via ABA leads to characteristic alterations in different regulatory and effector T cells not seen in patients treated with TCZ or RTX. Further studies must clarify whether the analysis of regulatory and effector T cell subpopulations before treatment initiation can be used as biomarker for treatment response.References:[1]Álvarez-Quiroga C, Abud-Mendoza C, Doníz-Padilla L, et al. CTLA-4-Ig therapy diminishes the frequency but enhances the function of treg cells in patients with rheumatoid arthritis.J Clin Immunol. 2011;31(4):588-595.doi:10.1007/s10875-011-9527-5Acknowledgments:Work done in “CBmed” was funded by the Austrian Federal Government within the COMET K1 Centre Program, Land Steiermark and Land Wien.Disclosure of Interests:None declared

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Mechanisms of Resistance and Determinants of Response of the GPRC5D-Targeting T-Cell Redirecting Bispecific Antibody JNJ-7564 in Multiple Myeloma

Blood ◽

10.1182/blood-2020-141187 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 8-9

Author(s):

Christie P.M. Verkleij ◽

Marloes Broekmans ◽

Amy Wong ◽

Sonja Zweegman ◽

Raluca Verona ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Research Funding ◽

Bispecific Antibody ◽

Cell Lysis ◽

Advisory Committees ◽

Hla Dr ◽

Tnf Α ◽

The Impact

Introduction: New immunotherapies directed against CD38, SLAMF7 or BCMA have significantly improved the outcome of multiple myeloma (MM) patients. However, most patients eventually relapse, underscoring the need for additional immunotherapeutic targets. We have previously shown that expression levels of GPRC5D, an orphan G protein-coupled receptor, are significantly higher on MM cells, compared to normal plasma cells or other immune cells. We also showed that the novel GPRC5DxCD3 bispecific antibody (BsAb) JNJ-7564, has promising anti-MM activity in patient-derived BM samples (Verkleij et al., EHA 2019). To elucidate which factors contribute to the observed heterogeneity in ex vivo response, we analyzed the impact of tumor and patient characteristics on efficacy of JNJ-7564. We further investigated whether tumor-intrinsic factors may be determinants of response by also testing in these assays JNJ-7957, a BCMA-targeting BsAb that differs from JNJ-7564 only in the tumor-antigen-binding domain. Methods: Bone marrow (BM) samples obtained from 13 newly diagnosed (ND), 17 daratumumab-naive relapsed/refractory (DARA-naive RR; median of 3 prior therapies) and 15 daratumumab-refractory (DARA-R, median of 6 prior therapies) MM patients were analyzed for tumor- and immune cell composition, and subsequently incubated with JNJ-7564 (0.00128-4.0 µg/mL) or JNJ-7957 (0.8 µg/mL). After 48 hours, MM cell lysis was assessed by flow cytometry. Luciferase-transduced MM cell lines were incubated with JNJ-7564 (0.032-4.0 µg/mL) in the presence of healthy peripheral blood mononuclear cells (PBMCs), purified CD4+CD25- T-cells or regulatory T-cells (Tregs). After 48 hours, MM cell lysis was assessed by bioluminescence assay. Results: We found no difference in JNJ-7564 efficacy with respect to disease stage (NDMM vs DARA-naive RRMM vs DARA-R MM, P=0.48). Importantly, the presence of high-risk cytogenetic abnormalities [del(17p), t(4;14) and t(14;16)] did not impair JNJ-7564 efficacy. The level of target expression was an important determinant of response, as evidenced by superior MM cell lysis in samples with higher than median GPRC5D expression, when compared to lower GPRC5D expression (Fig. 1A). Inferior MM cell lysis was observed in older patients (>67 years), in samples with low T-cell counts or low effector:target (E:T) ratios, and in those with a high frequency of PD-1+ T-cells, HLA-DR+ activated T-cells, or Tregs. These determinants of response also affected JNJ-7564-mediated T-cell activation and degranulation. To further analyze the impact of Tregs, we performed additional cell line experiments. Purified Tregs impaired T-cell proliferation, and were significantly less potent to kill MM cells when redirected by JNJ-7564, compared to CD4+CD25- T-cells (Fig. 1B). This was accompanied by reduced secretion of IFN-γ, TNF-α, IL-2 and granzyme B. To evaluate the impact of BM stromal cells (BMSCs) on JNJ-7564 activity, MM cell lines were co-incubated with PBMCs and patient-derived BMSCs. Direct cell-cell contact hampered MM cell lysis, while indirect contact (transwell) did not affect JNJ-7564 activity. Direct contact also decreased secretion of TNF-α and IL-2, and reduced GPRC5D expression on MM cells, contributing to BMSC-mediated resistance to JNJ-7564. Finally, we simultaneously evaluated the single agent activity of both JNJ-7564 and JNJ-7957 (0.8 µg/mL, dose whereby a plateau in MM cell lysis was observed with both BsAbs) in 40 BM samples. MM cell lysis induced by both agents was strongly correlated (Fig. 1C). In 6 samples, both agents exhibited poor activity (<45% lysis), whereas in 9 samples very good activity was observed (>80% lysis). Comparison of characteristics between these groups showed that a low E:T ratio (Fig. 1D) and high frequency of Tregs (Fig. 1E) significantly impaired efficacy of both BsAbs, suggesting patient-specific factors can determine response to T-cell redirectors targeting different antigens. Conclusion: We show that tumor-related factors, such as GPRC5D expression, as well as differences in the composition of the BM microenvironment, including E:T ratio, frequency of PD-1+ or HLA-DR+ T-cells or immune-suppressing Tregs or BMSCs, contribute to the variability in response to JNJ-7564. Our data indicate that strategies aiming at optimizing E:T ratio (e.g. induction therapy) or Treg depletion, may improve response to T-cell redirecting antibodies in MM. Disclosures Wong: Jhonson & Jhonson: Current Employment. Zweegman:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees. Verona:Johnson & Johnson: Current Employment, Current equity holder in publicly-traded company. Adams:Johnson & Johnson: Ended employment in the past 24 months. Mutis:Janssen Pharmaceuticals: Research Funding; Genmab: Research Funding; Takeda: Research Funding; Onkimmune: Research Funding; Gadeta: Research Funding. van de Donk:Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Ferrer: Membership on an entity's Board of Directors or advisory committees.

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Altered T-Lymphocyte Biology Following High-Dose Melphalan and Autologous Stem Cell Transplantation With Implications for Adoptive T-Cell Therapy

Frontiers in Oncology ◽

10.3389/fonc.2020.568056 ◽

2020 ◽

Vol 10 ◽

Author(s):

Thomas Mika ◽

Swetlana Ladigan-Badura ◽

Abdelouahid Maghnouj ◽

Bakr Mustafa ◽

Susanne Klein-Scory ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

T Cell ◽

Stem Cell Transplantation ◽

Cell Transplantation ◽

Autologous Stem Cell Transplantation ◽

Cell Phenotype ◽

High Dose ◽

Car T ◽

The Impact

In relapsed and refractory multiple myeloma (MM), adoptive cell therapies (ACT) including CAR-T-cells are under clinical investigation. However, relapse due to T-cell exhaustion or limited persistence is an obstacle. Before ACT are considered in MM, high-dose (HD) melphalan followed by autologous stem-cell transplantation (autoSCT) has been administered in most clinical situations. Yet, the impact of HD chemotherapy on T-cells in MM with respect to ACT is unclear. In this study, T-lymphocytes’ phenotypes, expansion properties, lentiviral transduction efficacy, and gene expression were examined with special respect to patients following HD melphalan. Significant impairment of T-cells’ expansion and transduction rates could be demonstrated. Expansion was diminished due to inherent disadvantages of the predominant T-cell phenotype but restored over time. The quantitative fraction of CD27−/CD28− T-cells before expansion was predictive of T-cell yield. Following autoSCT, the transduction efficacy was reduced by disturbed lentiviral genome integration. Moreover, an unfavorable T-cell phenotype after expansion was demonstrated. In initial analyses of CD107a degranulation impaired T-cell cytotoxicity was detected in one patient following melphalan and autoSCT. The findings of our study have potential implications regarding the time point of leukapheresis for CAR-T-cell manufacturing. Our results point to a preferred interval of more than 3 months until patients should undergo cell separation for CAR-T therapy in the specific situation post-HD melphalan/autoSCT. Monitoring of CD27−/CD28− T-cells, has the potential to influence clinical decision making before apheresis in MM.

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