Alloantigen-Activated Human T-Cells Increase Extracellular Fatty Acid Uptake and Intracellular Lipid Metabolism during Xenogeneic Gvhd

Abstract Background Following activation by recognition of foreign antigens, human T-cells alter their metabolic pathways to meet the increasing energetic demands for efficient immune response. Like cancer cells, alloreactive T-cells show a preference for aerobic glycolysis rather than oxidative phosphorylation, which is referred to as "Warburg effect". Until recently, it has been thought that extracellular fatty acid (FA) uptake and β-oxidation are severely reduced in alloreactive T-cells; however, some studies have indicated that lipid metabolism is rather increased in alloreactive mouse T-cells, and that metabolic pathway of FA can be a promising target for GVHD. To determine the role of lipid metabolism in human alloreactive T-cells after hematopoietic stem cell transplantation, we investigated the metabolic changes in human T-cells in vivo using human-into-mouse xenogeneic GVHD models. Methods NOG mice received 250cGy of total body irradiation (TBI) and were subsequently injected intravenously with human pan T-cells. All mice developed severe GVHD and died within 2 weeks, while mice that received TBI only survived without any symptoms of GVHD. Cells were harvested from GVHD target organs of mice at day 9 after transplantation. For the measurement of glucose and fatty acid (FA) uptake by flow cytometry, cells were stained with fluorescent-labeled deoxyglucose analogue (2-NBDG) and long-chain fatty acid analogue (BODIPY 500/510 C12), respectively. PCR array and extracellular flux analysis were performed according to manufacturer's instructions. Results Glucose uptake, determined by flow cytometry, was significantly increased in human T-cells obtained from GVHD mice. Extracellular FA uptake was also increased in human T-cells in GVHD mice, and was associated with cell proliferation rate. Effector memory T-cells followed by central memory T-cells showed a higher FA uptake than did naive T-cells. These findings were similarly observed in both human CD4+ and CD8+ T-cells. Robust T-cell proliferation was observed even in MHC class I/II deficient (MHC−/−) NOG mice after transplantation, although to a lesser extent than MHC+/+ NOG mice, in a process known as homeostatic proliferation. Extracellular uptake of FA as well as glucose in T-cells was significantly decreased in MHC−/− NOG mice. Of note, even when compared among only fully proliferated T-cells between MHC+/+ and MHC−/− NOG mice, FA uptake was still significantly decreased in MHC−/− NOG mice, suggesting that the recognition of host MHC molecules by allogeneic T-cells accelerate this process. To compare the ability of human naive and memory T-cells to incorporate extracellular FA, we isolated human naive (CD45RA high) and memory (CD45RA low) T-cells and separately injected into NOG mice. Although it has been shown that memory T-cells exhibit different effector functions, the FA uptake in memory T-cells was comparable to that in naive T-cells. This suggests that memory T-cells can also alter their lipid metabolism following encounter with alloantigens. Finally, we assessed the expression of genes associated with lipid metabolism in human T-cells obtained from GVHD mice. Quantitative real-time PCR analysis detected up-regulation of mRNAs encoding the enzymes involved in FA transport including carnitine palmitoyltransferase (CPT1B), fatty acid binding protein (FABP1-4, FABP6, and FABP7), and β-oxidation pathway including acyl-CoA synthase (ACSBG2) and acyl-CoA dehydrogenase (ACAD9-11, ACADS, and ACADL) when compared with T-cells in MHC−/− NOG mice. Similarly, the expression of genes encoding the enzymes in triacylglycerol metabolism such as glycerol kinase (GK, GK2) and lipoprotein lipase (LPL) was up-regulated in GVHD mice. Furthermore, the expression of genes associated with mevalonate pathways such as HMG-CoA synthase (HMGCS1, HMGCS2), was also upregulated. These observations suggest that T-cells activated by alloantigens in vivo promote lipid hydrolysis, mitochondrial FA transport, and β-oxidation, resulting in greater utilization of free FA. Conclusion Human alloreactive T-cells increased extracellular uptake of FA as well as glucose, and intracellular lipid metabolism in response to alloantigens (summarized in the graphical abstract). Therapeutic effects of specific inhibition of lipid metabolic pathways by pharmacological inhibitors including etomoxir are now being investigated in this model. Figure. Figure. Disclosures Fujiwara: Shire: Consultancy; Pfizer: Consultancy; Chugai: Consultancy; Kirin: Consultancy; Kyowa-Hakko: Consultancy; Astellas: Consultancy. Ohmine:Kyowa Hakko Kirin: Speakers Bureau; Takara Bio: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda Pharmaceutical: Speakers Bureau; Celgene Corporation: Speakers Bureau; Chugai Pharmaceutical: Speakers Bureau; Alexion Pharmaceuticals: Speakers Bureau; Ono Pharmaceutical: Consultancy. Muroi:Japanese Red Cross Society: Speakers Bureau; Dickinson and Company: Speakers Bureau; Becton: Speakers Bureau; JCR: Speakers Bureau. Kanda:Astellas: Consultancy, Honoraria, Research Funding; Eisai: Consultancy, Honoraria, Research Funding; Taiho: Research Funding; Nippon-Shinyaku: Research Funding; Chugai: Consultancy, Honoraria, Research Funding; Dainippon-Sumitomo: Consultancy, Honoraria, Research Funding; Pfizer: Research Funding; Otsuka: Research Funding; Shionogi: Consultancy, Honoraria, Research Funding; Kyowa-Hakko Kirin: Consultancy, Honoraria, Research Funding; MSD: Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Asahi-Kasei: Research Funding; Ono: Consultancy, Honoraria, Research Funding; Sanofi: Research Funding; Novartis: Research Funding; Taisho-Toyama: Research Funding; CSL Behring: Research Funding; Tanabe-Mitsubishi: Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Mochida: Consultancy, Honoraria; Alexion: Consultancy, Honoraria; Takara-bio: Consultancy, Honoraria.

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Mobilization of CD8+ Central Memory T-Cells with Enhanced Reconstitution Potential in Mice By a Combination of G-CSF and GMI-1271-Mediated E-Selectin Blockade

Blood ◽

10.1182/blood.v126.23.512.512 ◽

2015 ◽

Vol 126 (23) ◽

pp. 512-512 ◽

Cited By ~ 1

Author(s):

Ingrid G Winkler ◽

Valerie Barbier ◽

Kristen J Radford ◽

Julie M Davies ◽

Jean-Pierre Levesque ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Research Funding ◽

Memory T Cells ◽

Central Memory ◽

Donor Blood ◽

Central Memory T Cells

Abstract T-cells are critical mediators of immune defense against pathogens and cancer. Adoptive T cell immunotherapy and T-cell engineering have promising clinical applications but T cell survival and exhaustion are current limitations. Central memory cells (TCM CD62L+ CCR7+) and their precursors, stem central memory T-cells (TSCM) possess the stem-like properties needed to reconstitute and prolong an effective immune response long-term. These cells have been shown to significantly improve therapeutic efficacy of adoptive T-cell therapy. The challenge remains to harvest good quality TCM-cells for these immunotherapy approaches. The bone marrow (BM) is the major reservoir of CD8+ TCM and their precursors. We have previously shown that E-selectin is expressed in the BM vasculature and drives activation and differentiation of hematopoietic stem cells during G-CSF induced mobilization to the blood. We find therapeutic blockade of E-selectin promotes HSC self-renewal and reconstitution in vivo. We now examine the impact of E-selectin blockade on CD8+ T cell mobilization from the bone marrow to the blood and hypothesize that E-selectin blockade may also dampen the activation/differentiation of this subset. First we administered a standard G-CSF regime (filgastim 250ug/kg/day for 3 days) to mice and then dosed some cohorts with GMI-1271 (40mg/kg BID) from 12 to 72 hours within this 3 day period. Administration of G-CSF alone results in a near complete disappearance of bone marrow resident CD8+ TCM cells, and their apparent migration (increase in numbers) to the blood, while CD8+ subsets in the lymph nodes and spleen were barely affected by G-CSF. Furthermore among T-cell subsets, CD8+ but not CD4+ TCM were specifically mobilized into the blood when GMI-1271 was co-administered for the last 12 to 24 hours of G-CSF. These findings are consistent with reports demonstrating the bone marrow to be a major reservoir for CD8+ but not CD4+ central memory T-cells. Administration of GMI-1271 caused a marked enhancement in mobilization into the blood of CD8+ TCM/SCM (CD62Lhi, CCR7+) cells over treatment with G-CSF alone (p<0.05). To determine the functional consequences of this skewed mobilization following GMI-1271 co-administration, 25 uL of mobilized blood was transplanted into irradiated congenic B6.SJL recipients together with 2x105 congenic BM cells to analyze long-term donor T-cell engraftment in the recipient mice. We found G-CSF mobilized donor blood did not contribute CD8+ TCM cells that can persist post-transplant (<0.5% at 20 weeks post-transplant). In contrast when donor mice were mobilized with G-CSF together with E-selectin blockade (GMI-1271), we found elevated levels of donor blood derived CD8+ T-cells demonstrating robust long-term CD8+ T-cell persistence / regeneration (5.3 ±3.2% of total recipient T-cells, p=0.04). This dramatic boost in donor CD8+ T-cell reconstitution in mobilized blood following GMI-1271 co-administration is likely to be due to the long-term persistence and in vivo amplification of CD8+ TCM cells from donor mobilized blood. Similar in vivo enhancing effects of GMI-1271 were also observed with other mobilizing agents such as combined CXCR4 and VLA-4 blockade and GM-CSF resulting in a significant 4.9-fold boost in donor CD8+ reconstitution with GMI-1271. Importantly, only 12 hours of E-selectin blockade was sufficient to achieve this boost in CD8+ TCM numbers in the blood following G-CSF. In a previous report we have shown that therapeutic blockade of E-selectin promotes HSC self-renewal in vivo. Thus, it is possible that E-selectin blockade boosts mobilization of CD8+ TCM/SCM with stem-like properties into the blood by loosening factors retaining CD8+ TCM/SCM in the bone marrow and/or blocking the E-selectin-mediated activation and differentiation of this T-cell subset. In summary, our studies identify E-selectin blockade as a novel target to improve harvesting of CD8+ TCM/SCM cells with stem-like properties. Blockade of this target with GMI-1271 significantly improves the in vivo reconstitution potential and regenerative properties of CD8+ T-cells from donor blood allowing a valuable source of desired T-cells for use in adoptive immunotherapy and T-cell engineering. Disclosures Winkler: GlycoMimetics Inc: Research Funding. Barbier:GlycoMimetics Inc: Research Funding. Davies:GlycoMimetics Inc: Research Funding. Smith:GlycoMimetics, Inc.: Employment. Fogler:GlycoMimetics, Inc.: Employment. Magnani:GlycoMimetics Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

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Phase I Study of Adoptive Immunotherapy with HA-1-Specific CD8+ and CD4+ Memory T Cells for Children and Adults with Relapsed Acute Leukemia after Allogeneic Hematopoietic Stem Cell Transplantation (HCT): Trial in Progress

Blood ◽

10.1182/blood-2020-137726 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 45-46 ◽

Cited By ~ 1

Author(s):

Elizabeth F Krakow ◽

Corinne Summers ◽

Ann Dahlberg ◽

Merav Bar ◽

Melinda Ann Biernacki ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Phase I ◽

Research Funding ◽

Phase I Trial ◽

Memory T Cells ◽

Cell Immunotherapy ◽

T Cell Immunotherapy

Background: Donor T cells specific for minor histocompatibility (H) antigens can deliver potent, selective anti-leukemic effects after allogeneic HCT when the antigen is negligibly or not expressed by non-hematopoietic tissues, not present in the donor, and expressed by the recipient. We reported a new minor H antigen-directed T-cell therapy that can be deployed after HCT to manage persistent or recurrent measurable residual hematologic malignancies or overt relapse (Blood 2018;131(1):108). We developed a transgene with 4 components: 1) a high-affinity T-cell receptor (TCR) specific for the hematopoietic-restricted minor H antigen, HA-1 that is presented on HLA-A*02:01; 2) a CD8 co-receptor to enhance function of the class I-restricted TCR in CD4+ T cells so they promote cytotoxic CD8+ T cell function and survival; 3) an inducible caspase-9 safety switch, which can be triggered by the drug rimiducid in case of in vivo toxicity; and 4) a CD34-CD20 epitope to facilitate selection of the engineered product during manufacturing and track HA-1 TCR T cells in the recipient. The 21-day manufacturing process entails CD45RA+ naïve T cell depletion (minimizes the risk of GvHD), and subsequent CD4+ and CD8+ separation (provides a consistent 1:1 CD4:CD8 ratio). The separate cultures are transduced with the lentivirus construct iCasp9-HA1-TCR2-RQR-CD8, expanded, and selected using the CD34 marker to ensure removal of untransduced T cells. Study Design and Methods: The single-center phase I trial (NCT03326921) evaluates the feasibility and safety of infusion of HA-1 TCR T-cell immunotherapy. Primary end points are 1) Feasibility of manufacturing and administering HA-1 TCR CD8+ and CD4+ memory T cells and 2) Dose-limiting toxicity of HA-1 TCR T cells. Major inclusion criteria are: HLA-A*02:01-positive, HA-1-positive patients who underwent HCT for acute leukemia, myelodysplastic syndrome, BPDCN, CML, CMML or JMML from a HLA-A*-02:01+/HA-1-negative donor or HLA-A*02:01-negative haploidentical or mismatched donor (excluding umbilical cord). HA-1 genotype screening is performed on patient and donor blood, hair follicle or cheek swab samples shipped to Fred Hutchinson Cancer Research Center. To be eligible for treatment, patients must develop measurable residual disease or overt relapse after HCT but may receive other standard or investigational therapies prior to treatment with HA-1 TCR T-cell immunotherapy if clinically indicated. Some systemic immunosuppression may be continued, but prior grade IV acute GVHD and prior severe chronic GVHD are key exclusions. Two groups, <16 and ≥16 years, will be treated at dose levels ranging from 3 x 106 to30 x 106 cells/kg, in cohorts of 3-6 subjects, up to approximately 24 subjects in total. Fludarabine lymphodepletion will be used in most subjects, followed by a single T-cell infusion, with an option for a subsequent infusion(s) if the subject demonstrates an initial response without severe toxicity. Bone marrow aspirations are performed prior to T-cell infusion and at several time points following infusion. Recruitment and Patient Characteristics: To date, 3 subjects have been treated on the phase I clinical trial and received a total of 5 infusions (Table 1). HA-1 TCR T cell persistence in blood and bone marrow has been documented from >3 months to >13 months. Clear in vivo anti-leukemic activity was observed at the first dose level, including in a subject with aggressive, highly refractory T-ALL and early post-HCT relapse. Outlook: Minor H antigen-specific T-cell immunotherapy may offer effective management of post-HCT relapse while avoiding GvHD and other off-target effects. Due to population genetics of HA-1 and HLA-A*02:01, HA-1 TCR T-cell immunotherapy is applicable to 10-15% of HCT recipients with various hematological malignancies. The ongoing phase I trial is actively recruiting patients. Development of T-cell immunotherapy targeting other minor H antigen/HLA combinations is also underway to increase the broad applicability of minor H antigen-targeted T-cell immunotherapy. Disclosures Krakow: HighPass Bio: Research Funding. Cunningham:HighPass Bio: Research Funding. Vartanian:HighPass Bio: Research Funding. Bleakley:HighPass Bio: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

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Engraftment Fitness of Human Central Memory-Derived Effector CD8+ T Cells In Immunodeficient Mice

Blood ◽

10.1182/blood.v116.21.1019.1019 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1019-1019

Author(s):

Xiuli Wang ◽

Berger Carolina ◽

Stanley R. Riddell ◽

ChingLam W Wong ◽

Stephen Forman ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Adoptive Transfer ◽

Tumor Response ◽

Memory T Cells ◽

Effector T Cells ◽

Caspase Activity ◽

Effector Cells ◽

Nog Mice

Abstract Abstract 1019 Development of T cell products that have engineered specificity for CD19 has broad application to adoptive transfer therapy for B-lineage lymphoma and leukemia. Clinical studies have demonstrated the safety and feasibility of T cell transfer as a therapy for patients. But the potency of this strategy has proven challenging, primarily due to issues relating to a lack of persistence of the adoptively transferred cells in patients. The repertoire of memory T cells is heterogeneous with respect to phenotypic, functional, and epigenetic attributes. Memory T cells are divided into sub-populations of 1) effector memory (TEM) cells that distribute to tissue beds and exhibit immediate cytolytic effector functioning, and 2) central memory (TCM) cells that home to lymph nodes based on CD62L/CCR7 expression and are capable of extensive proliferative activity upon re-encountering antigen. Thus the cell-intrinsic programming of distinct memory T cell subtypes, such as TEM and TCM, likely dictate divergent fates of their derived effector cells. To address this important issue, a clear functional dichotomy between TCM- and TEM-derived CD8+ CTLs was recently delineated in a nonhuman primate model, where it was found that virus-specific CD8+ CTL clones derived from TCM, but not TEM precursors, establish persistent and functional memory following adoptive transfer. Here, we extended these studies to human effector T cells using CMV as antigen model system to investigate the engraftment of human CMVpp65-specific CD8+ effector T cells derived in vitro from either sort purified CD45RO+CD62L+ TCM or CD45RO+CD62L- TEM precursors in NOD/Scid IL-2RγCnull (NOG) mice. TCM-derived effector cells (TE(CM)) and TEM-derived effector cells (TE(EM)) were adoptively transferred (i.v) into NOG mice reconstituted with human IL-15 and T cell levels in circulation were evaluated at different time points by FACS. 20% CD8+ TE(CM) and 3% CD8+ TE(EM) were detected on day 14. Then after, engraftment of the CD8+ TE(CM) remained at a steady state of approx 2% of circulating mononuclear cells for 100 days while TE(EM) remained at or below the level of detection, indicating that TE(CM) were superior in their ability to engraft in response to IL-15 as compared to TE(EM) after adoptive transfer (P<0.05). The long-term (100 days) persisting CD8+ TE(CM), harvested from primary recipient mice were found to be capable of engrafting secondary recipients. TcR Vβ analysis of persisting cells demonstrated that CD8+ TE(CM) engraftment was polyclonal, suggesting that homeostatic engraftment fitness is a general feature of these cells. To delineate the mechanism(s) by which TE(CM) exhibit superior in vivo engraftment, TE(CM) and TE(EM) were first labeled with CFSE before in vivo administration. CFSE profiles appear that the TE(EM) proliferated more extensively than TE(CM) early after adoptive transfer as indicated by the percent of cells which diluted CFSE on day 9 (i.e., 80% vs. only 25%, respectively). However, using D2R cleavage as a measure of caspase activity as a surrogate for apoptosis, 5.8% of engrafting TE(CM) were positive for activated caspase activity compared to 31.6% of TE(EM), suggesting that in NOG mice both CD8+ TE(CM) and TE(EM) proliferate in response to IL-15 whereas TE(CM) are intrinsically resistant to caspase activation and apoptosis. We also evaluated the antigen specific responsiveness of engrafted cells. Weekly infusions of irradiated pp65+/A2+ LCL as antigen significantly augmented the levels of circulating CD8+ TE(CM) as compared to no antigen stimulation (P<0.05), whereas CD8+ TE(EM) did not respond to antigen challenge. Moreover, when CMVpp65 specific CD8+ TE(CM) or TE(EM) were infused into CMVpp65+ tumor bearing mice, tumor cells progressed in mice receiving TE(EM) at a rate similar to untreated control mice over a ten day observation period, whereas TE(CM) significantly controlled tumor progression (P<0.05), indicating that CD8+ TE(CM) but not TE(EM) are able to mediate an anti-tumor response. Together these studies confirm that human CD8+ effector T cells derived from TCM precursors are capable of persistence after infusion, can proliferate in in vivo in response to antigen, can mediate an anti-viral or anti tumor response, and are likely the preferred T cells for antigen specific anti-tumor adoptive T cell therapy . Disclosures: No relevant conflicts of interest to declare.

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Activation of T and NK Cells Following Lenalidomide Therapy in Patients with Follicular Lymphoma.

Blood ◽

10.1182/blood.v120.21.2766.2766 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2766-2766

Author(s):

Seema Rawal ◽

Nathan Fowler ◽

Min Zhang ◽

Zhiqiang Wang ◽

Tariq Muzzafar ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cell ◽

Nk Cells ◽

Research Funding ◽

Memory T Cells ◽

T Cell Subsets ◽

Central Memory T Cells

Abstract Abstract 2766 Background: Lenalidomide plus rituximab therapy is a highly effective and well-tolerated therapy in patients (pts) with follicular lymphoma (FL). In a Phase II trial, this combination induced a complete remission rate of 87% in pts with advanced stage untreated FL (Fowler et al, Ann Oncol, 2011; 22; suppl 4:137). A randomized Phase III trial was recently initiated to compare this combination with current standard of care therapies in pts with FL. Although lenalidomide is known to be an immunomodulatory drug with effects on a variety of immune cells in vitro, its effects have not been well studied in vivo in humans. Understanding the in vivo effects of lenalidomide could lead to novel combination strategies to enhance the efficacy and improve clinical outcome in FL and other malignancies. Methods: Pts received lenalidomide 20 mg/day on days 1–21 of each 28-day cycle and rituximab was given at 375 mg/m2on day 1 of each cycle. Peripheral blood mononuclear cells (PBMC) were phenotyped by multiparametric flow cytometry at baseline, on cycle 2 day 15 (C2D15), and at the end of cycle 6. In addition, peripheral blood (PB) samples were collected in PAXgene Blood RNA tubes at baseline and on C2D15 for whole genome gene expression profiling (GEP). Results: Immunophenotyping of baseline and end of cycle 6 PBMC (n=17) showed that the percentages and absolute numbers of CD3+, CD4+, CD8+, TCRgd, and Foxp3+ regulatory T cells; and NK, NKT, and myeloid dendritic cells were not significantly different between the two time points. However, a significant increase in CD4+CD45RO+ (p<0.01) and CD8+CD45RO+ (p=0.04) memory T cells was observed post-therapy. Further characterization of CD4+ T cells showed a significant increase in central memory T cells (p<0.001) and a decrease in naïve (p<0.01) and terminally differentiated (p<0.01) T cells, but no change in effector memory T cells. The increase in CD8+ central memory T cells was marginally significant (p=0.06). Plasmacytoid dendritic cells (PDC) were also significantly increased (p=0.02). In contrast, no such changes in T cell subsets or PDC were observed in FL pts (n=9) treated with 6 cycles of R-CHOP chemotherapy that received equal number of rituximab doses and analyzed at similar time points (baseline and end of cycle 6). To understand lenalidomide-induced changes on a molecular level, we compared GEP data at C2D15 vs. baseline for 7 pairs of PB samples. The paired significance analysis of microarrays method, based on Student's t test, identified 1,748 differentially expressed genes (DEG; 713 up, 1035 down), without a fold-change threshold, in C2D15 samples vs. baseline. Results were influenced by rituximab-induced depletion of B cells in C2D15 samples, but there were many changes that suggested altered PBMC physiology. Noteworthy up-regulated genes (>1.5 fold) included genes associated with T and NK cell activation including BATF, CCR2, CD1B, CD2, CD160, CTLA4, CXCR3, ICOS, and LAG3; and CD163 and CD209, phagocytic receptors expressed on monocytes/macrophages. Down-regulated genes (>1.5 fold) included CXCR5, which mediates B cell migration into follicles; and IL1B and TNFSF13B (BAFF), which are produced by activated macrophages and induce B cell proliferation. Gene set enrichment analysis of all GEP results, and Ingenuity Pathway Analysis of DEGs, indicated up regulation of multiple pathways and processes including ribosomal and mitochondrial components involved in translation and oxidative phosphorylation, CTLA4 signaling in cytotoxic T cells, and differentiation and signaling by ICOS and CD28 in T helper cells. We confirmed up regulation of CTLA4, ICOS, and LAG3 at the protein level in C2D15 PBMC by flow cytometry. Furthermore, treatment of PBMC derived from untreated FL pts with lenalidomide in vitro resulted in up regulation of these molecules in T and/or NK cells consistent with our in vivo results. Conclusions: In FL pts, lenalidomide induced multiple changes in the immune system including increases in PDC and memory T cell subsets, activation of T and NK cells, and down-regulation of certain genes mediating B cell migration and proliferation. These results provide insights into the mechanism of action of lenalidomide and suggest that it can be combined with other immunostimulatory agents such as therapeutic vaccines, adoptive T cell therapy strategies, and immune checkpoint inhibitors to further enhance its efficacy in FL and other malignancies. Disclosures: Fowler: Celgene: Research Funding. Heise:Celgene Corporation: Employment, Equity Ownership. Lacerte:Celgene: Honoraria. Samaniego:Celgene: Research Funding. Neelapu:Celgene Corporation: Research Funding.

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Diffuse Large B Cell Pdtx in Humanized Mice Are Valuable Models to Study Host-Lymphoma Interactions and Immune-Modulating Agents

Blood ◽

10.1182/blood-2021-151598 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2406-2406

Author(s):

Giorgia Zanetti ◽

Giuseppina Astone ◽

Luca Cappelli ◽

William Chiu ◽

Maria Teresa Cacciapuoti ◽

...

Keyword(s):

T Cells ◽

Immune System ◽

T Cell ◽

B Cell ◽

Research Funding ◽

Ex Vivo ◽

Human Immune System ◽

Human T Cells ◽

Human Immune

Abstract Introduction: Immunotherapy is a promising therapeutic intervention for cancer treatment. Activation of the immune system via checkpoint blockade has been shown to produce antitumor responses in patients with both solid and hematological tumors. However, many patients do not respond to checkpoint inhibitors, and additional therapies are needed to treat these patients. Testing immunotherapies requires a functional human immune system; thus, it is difficult to evaluate their effectiveness using conventional experimental models. For this reason, establishing in vivo models that closely reproduce not only human tumors, but also their interactions with the human immune system, has become mandatory. Methods: We developed a humanized mouse model and combined it with a patient-derived tumor xenograft (PDTX). Humanized mice (HuMice) were generated by transplantation of cord blood or mobilized peripheral blood CD34+ hematopoietic stem and progenitor cells into preconditioned immunodeficient mice. We compared human engraftment in 3 different mouse strains: NSG (NOD.Cg-Prkdc scidIl2rg tm1Wjl/SzJ), NSGS (NOD.Cg-Prkdc scidIl2rg tm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ) and NBSGW (NOD.Cg-Kit W-41J Tyr + Prkdc scid Il2rg tm1Wjl/ThomJ). Immune cell profiling and distribution was performed using flow cytometry and immunohistochemistry. The B cell receptor (BCR) repertoire was evaluated using an RNA-based NGS assay. To evaluate the maturation and functionality of T cells developing in HuMice we performed proliferation, degranulation and intracellular cytokine staining. Results: Two months after CD34+ cell transplantation, we observed high levels of human hematopoietic chimerism in all the 3 strains. NSGS mice supported high-level chimerism as early as 1 month after transplantation, with more than 25% of human CD45+ cells in the blood. In all mice the majority of human circulating leukocytes were CD19+ B cells. An early appearance of CD3+ human T cells was detected in NSGS mice as compared to the other strains. Notably, the T cell expansion correlated with a decrease in relative B cell abundance while the myeloid cell contribution to the graft remained steady. We documented the differentiation of CD4+ and CD8+ human T cells at a 2:1 ratio. The characterization of the T cell subsets revealed that the majority was represented by CD45RA-CCR7- effector memory cells in both the spleen and the blood of HuMice. Nevertheless, recipient mice did not exhibit overt signs of graft-versus-host disease. We also evaluated the cytotoxic potential of T cells isolated from the spleen of HuMice: ex vivo peptide antigen (i.e. EBV) presentation let to generation of effective and specific cytotoxic T-cells. After assessing a functional human immune system reconstitution in HuMice, we challenged them in vivo with low-passage tumor fragments from a diffuse large B cell lymphoma (DLBCL) PDTX. All tumor implants were successfully engrafted in both HuMice and non-humanized controls. Remarkably, all the 3 HuMice strains showed a significant reduction in the tumor volume and/or eradication compared to matched non-humanized controls. Flow cytometry analysis of the peripheral blood of humanized PDTX revealed that the tumor engraftment elicited a significant expansion of CD3+ T cells and cytotoxic CD8+ lymphocytes. Moreover, tumors developing in HuMice exhibited intermediate to high levels of tumor infiltrating T lymphocytes commingling with the neoplastic B cells, as determined by immunohistochemistry. Large areas of necrosis were often observed in PDTX of HuMice. Infiltrating CD3+ cells were TIGIT, PD-1 and Lag-3 positive, and did not efficiently proliferate ex vivo: all features consistent with an exhaustion phenotype. PDTX of HuMice often displayed larger areas of necrosis. Conclusions: Collectively, our data demonstrate that a robust reconstitution can be achieved in different strains of immunocompromised mice and that HuMice elicit effective anti-lymphoma responses. PDTX HuMice represent a powerful platform to study host-tumor interactions, and to test novel immune-based strategies (CAR-T, bifunctional Abs) and new pharmacological approaches to counteract T-cell exhaustion. Figure 1 Figure 1. Disclosures Scandura: Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Constellation: Research Funding; MPN-RF (Foundation): Research Funding; CR&T (Foudation): Research Funding; European Leukemia net: Honoraria, Other: travel fees . Roth: Janssen: Consultancy; Merck: Consultancy.

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The neutralizing function of the anti-HTLV-1 antibody is essential in preventing in vivo transmission of HTLV-1 to human T cells in NOD-SCID/¿cnull (NOG) mice

Retrovirology ◽

10.1186/preaccept-6892873451261068 ◽

2014 ◽

Vol 11 (1) ◽

pp. 74

Author(s):

Mineki Saito ◽

Reiko Tanaka ◽

Hideki Fujii ◽

Akira Kodama ◽

Yoshiaki Takahashi ◽

...

Keyword(s):

T Cells ◽

Human T Cells ◽

Nog Mice

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The neutralizing function of the anti-HTLV-1 antibody is essential in preventing in vivo transmission of HTLV-1 to human T cells in NOD-SCID/γcnull (NOG) mice

Retrovirology ◽

10.1186/s12977-014-0074-z ◽

2014 ◽

Vol 11 (1) ◽

Cited By ~ 6

Author(s):

Mineki Saito ◽

Reiko Tanaka ◽

Hideki Fujii ◽

Akira Kodama ◽

Yoshiaki Takahashi ◽

...

Keyword(s):

T Cells ◽

Human T Cells ◽

Nog Mice

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Nanoparticle-Mediated Lipid Metabolic Reprogramming of T Cells in Tumor Microenvironments for Immunometabolic Therapy

Nano-Micro Letters ◽

10.1007/s40820-020-00555-6 ◽

2021 ◽

Vol 13 (1) ◽

Author(s):

Dongyoon Kim ◽

Yina Wu ◽

Qiaoyun Li ◽

Yu-Kyoung Oh

Keyword(s):

T Cells ◽

Fatty Acid ◽

Lipid Metabolism ◽

Tumor Microenvironment ◽

Fatty Acid Metabolism ◽

Acid Metabolism ◽

Metabolic Reprogramming ◽

Killing Effect ◽

Tumor Tissues

Highlights aCD3/F/AN, anti-CD3e f(ab′)2 fragment-modified and fenofibrate-encapsulated amphiphilic nanoparticle, reprogrammed mitochondrial lipid metabolism of T cells. aCD3/F/AN specifically activated T cells in glucose-deficient conditions mimicking tumor microenvironment, and exerted an effector killing effect against tumor cells. In vivo treatment with aCD3/F/AN increased T cell infiltration, cytokine production, and prevented tumor growth. Abstract We report the activation of anticancer effector functions of T cells through nanoparticle-induced lipid metabolic reprogramming. Fenofibrate was encapsulated in amphiphilic polygamma glutamic acid-based nanoparticles (F/ANs), and the surfaces of F/ANs were modified with an anti-CD3e f(ab′)2 fragment, yielding aCD3/F/ANs. An in vitro study reveals enhanced delivery of aCD3/F/ANs to T cells compared with plain F/ANs. aCD3/F/AN-treated T cells exhibited clear mitochondrial cristae, a higher membrane potential, and a greater mitochondrial oxygen consumption rate under glucose-deficient conditions compared with T cells treated with other nanoparticle preparations. Peroxisome proliferator-activated receptor-α and downstream fatty acid metabolism-related genes are expressed to a greater extent in aCD3/F/AN-treated T cells. Activation of fatty acid metabolism by aCD3/F/ANs supports the proliferation of T cells in a glucose-deficient environment mimicking the tumor microenvironment. Real-time video recordings show that aCD3/F/AN-treated T cells exerted an effector killing effect against B16F10 melanoma cells. In vivo administration of aCD3/F/ANs can increase infiltration of T cells into tumor tissues. The treatment of tumor-bearing mice with aCD3/F/ANs enhances production of various cytokines in tumor tissues and prevented tumor growth. Our findings suggest the potential of nanotechnology-enabled reprogramming of lipid metabolism in T cells as a new modality of immunometabolic therapy.

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Synthetic mycobacterial diacyl trehaloses reveal differential recognition by human T cell receptors and the C-type lectin Mincle

Scientific Reports ◽

10.1038/s41598-021-81474-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Josephine F. Reijneveld ◽

Mira Holzheimer ◽

David C. Young ◽

Kattya Lopez ◽

Sara Suliman ◽

...

Keyword(s):

T Cells ◽

Fatty Acid ◽

Immune System ◽

T Cell ◽

Cross Reactivity ◽

Lipid Binding ◽

Human Immune System ◽

Human T Cell ◽

Human T Cells ◽

Pure Compounds

AbstractThe cell wall of Mycobacterium tuberculosis is composed of diverse glycolipids which potentially interact with the human immune system. To overcome difficulties in obtaining pure compounds from bacterial extracts, we recently synthesized three forms of mycobacterial diacyltrehalose (DAT) that differ in their fatty acid composition, DAT1, DAT2, and DAT3. To study the potential recognition of DATs by human T cells, we treated the lipid-binding antigen presenting molecule CD1b with synthetic DATs and looked for T cells that bound the complex. DAT1- and DAT2-treated CD1b tetramers were recognized by T cells, but DAT3-treated CD1b tetramers were not. A T cell line derived using CD1b-DAT2 tetramers showed that there is no cross-reactivity between DATs in an IFN-γ release assay, suggesting that the chemical structure of the fatty acid at the 3-position determines recognition by T cells. In contrast with the lack of recognition of DAT3 by human T cells, DAT3, but not DAT1 or DAT2, activates Mincle. Thus, we show that the mycobacterial lipid DAT can be both an antigen for T cells and an agonist for the innate Mincle receptor, and that small chemical differences determine recognition by different parts of the immune system.

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627 The DLL3-targeted half-life extended bispecific T cell engager (HLE BiTE®) immune-oncology therapy AMG 757 has potent antitumor activity in neuroendocrine cancer

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0627 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A663-A663

Author(s):

Keegan Cooke ◽

Juan Estrada ◽

Jinghui Zhan ◽

Jonathan Werner ◽

Fei Lee ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Growth ◽

Mouse Models ◽

T Cell Activation ◽

Phase 1 ◽

Phase 1 Study ◽

Human T Cells

BackgroundNeuroendocrine tumors (NET), including small cell lung cancer (SCLC), have poor prognosis and limited therapeutic options. AMG 757 is an HLE BiTE® immune therapy designed to redirect T cell cytotoxicity to NET cells by binding to Delta-like ligand 3 (DLL3) expressed on the tumor cell surface and CD3 on T cells.MethodsWe evaluated activity of AMG 757 in NET cells in vitro and in mouse models of neuroendocrine cancer in vivo. In vitro, co-cultures of NET cells and human T cells were treated with AMG 757 in a concentration range and T cell activation, cytokine production, and tumor cell killing were assessed. In vivo, AMG 757 antitumor efficacy was evaluated in xenograft NET and in orthotopic models designed to mimic primary and metastatic SCLC lesions. NSG mice bearing established NET were administered human T cells and then treated once weekly with AMG 757 or control HLE BiTE molecule; tumor growth inhibition was assessed. Pharmacodynamic effects of AMG 757 in tumors were also evaluated in SCLC models following a single administration of human T cells and AMG 757 or control HLE BiTE molecule.ResultsAMG 757 induced T cell activation, cytokine production, and potent T cell redirected killing of DLL3-expressing SCLC, neuroendocrine prostate cancer, and other DLL3-expressing NET cell lines in vitro. AMG 757-mediated redirected lysis was specific for DLL3-expressing cells. In patient-derived xenograft and orthotopic models of SCLC, single-dose AMG 757 effectively engaged human T cells administered systemically, leading to a significant increase in the number of human CD4+ and CD8+ T cells in primary and metastatic tumor lesions. Weekly administration of AMG 757 induced significant tumor growth inhibition of SCLC (figure 1) and other NET, including complete regression of established tumors and clearance of metastatic lesions. These findings warranted evaluation of AMG 757 (NCT03319940); the phase 1 study includes dose exploration (monotherapy and in combination with pembrolizumab) and dose expansion (monotherapy) in patients with SCLC (figure 2). A study of AMG 757 in patients with neuroendocrine prostate cancer is under development based on emerging data from the ongoing phase 1 study.Abstract 627 Figure 1AMG 757 Significantly reduced tumor growth in orthotopic SCLC mouse modelsAbstract 627 Figure 2AMG 757 Phase 1 study designConclusionsAMG 757 engages and activates T cells to kill DLL3-expressing SCLC and other NET cells in vitro and induces significant antitumor activity against established xenograft tumors in mouse models. These preclinical data support evaluation of AMG 757 in clinical studies of patients with NET.Ethics ApprovalAll in vivo work was conducted under IACUC-approved protocol #2009-00046.

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