Novel CD4+CD8+ Umbilical Cord Blood Regulatory T Cells

Background: Regulatory T cells (Treg), a well defined suppressor cell population with the phenotype of CD4+CD25+CD127- FoxP3+ are emerging as a promising adoptive therapy for a diverse subset of autoimmune diseases and inflammatory disorders. Specificlaly, cord blood (CB) Tregs have been shown to be superior to peripheral blood (PB) Tregs in their function and stability. Here, we define a novel subset of CB Tregs CD4+CD8+CD25+CD127- FoxP3+ that is not very well defined in the PB population and explore its' differentiation characteristics. Method: Tregs were isolated from CB or PB as described previously and cultured in the presence of CD3/28 beads and IL-2. On the day of harvest, cells were further sorted for CD8+ cell population and labelled as CD8+CD4+ (double positive CB Tregs: DP) and the leftover fraction as CD8-CD4+ (single positive CB Tregs: SP). Cells fractions were analyzed using flow cytometry and for cytokine assay, cells were activated (5 hours) with 1X Cell Stimulation Cocktail with Golgi inhibitors and stained for IFN-γ, IL-4, IL-17, IL-10 or TGFβ1 as per manufacturer's instructions. Additional cytokine assay was performed on the culture supernatants. DNA methylation assay was performed for the TSDR region as per manufacturer's instructions. Single cell RNAseq was performed at Albert Einstein Hospital and analyzed at Methodist Hospital. A threshold for original counts were applied to all four conditions, i.e. only transcripts with original counts>10 in all four. This step reduced the number of genes from 34626 to 16628; subsequently stringent cutoffs pairs showed two sets of genes- 1) Fold change>1.5, p-value<0.1, and 2) Fold change<2/3, p-value<0.1, this led to 73 rows, all with defined transcript names. Enrichment analysis using ConsensusPathDB for the above two gene sets were run to identify all pathways with at least one member-gene overlapping with either list, and the enrichment p-value for the pathway<0.1. Result: We cultured the Tregs isolated from CB or PB for 14 days and no evidence of a DP Tregs (0) was seen in PB as compared to a clearly defined DP Treg (8-26%) population in CB cultures. No differences were seen between the SP and DP CB Tregs in terms of the markers for mechanisms of suppression (FoxP3,Helios,CD39 ), homing (PSGL1, CLA, CD62L, CD49d), TGF-beta signaling (LAP, TGF-beta, GARP) and T cell signaling (LAG23, PD1, Icos, Tim3, CTLA-4, GITR). DP CBTregs consisted of a significantly higher expression of CCR4hiCXCR3hiCD95hipopulation. DP CB Tregs demonstrated a significantly higher IL-10 secretion compared to SP CB Tregs (Fig 1A) which correlated with the level of IL-10 secreted in the cell cuture supernatant (data not shown). While the degree of suppression of the proliferating conventional T cells was similar by the DP and SP CB Tregs (data not shown), a significantly higher demethylation of FoxP3 promoter and first intron region was observed in the DP vs SP CB Treg population (n=3) (Fig 1B). A total of 337 pathways were identified from Up-406 gene set, 80 of these pathways had enrichment p-value<0.05 and q-value<0.25 (Fig 1C). The Top 10 pathways enriched with members of Up-406 genes include (Fig 1D): TGF-β Receptor Signaling; Interleukin-10 signaling; Inflammatory bowel disease; IL12 signaling mediated by STAT4. A total of 158 pathways were identified from Down-314 gene set, 19 of these pathways had enrichment p-value<0.05 and q-value<0.25. The Top 10 pathways enriched with Down-314 genes included Immuno-regulatory interactions between a Lymphoid and a non-Lymphoid cell; ESC Pluripotency Pathways; Downstream signaling in naive CD8+ T cells; PDGF receptor signaling network; Nef Mediated CD8 Down-regulation (Fig E, F). Conclusion: We have identified a novel double positive CD4+CD8+ CB Treg population. Additional experiments for evaluating suppressor mechanism in addition to IL-10 secretion are ongoing. Figure 1 Disclosures Verma: Stelexis: Equity Ownership, Honoraria; Acceleron: Honoraria; Celgene: Honoraria; BMS: Research Funding; Janssen: Research Funding. Iyer:Genentech/Roche: Research Funding; Incyte: Research Funding; Seattle Genetics, Inc.: Research Funding; Novartis: Research Funding; Arog: Research Funding; Bristol-Myers Squibb: Research Funding. Parmar:Cellenkos Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Adoptive Therapy with Cord Blood Regulatory T Cells Can Treat Graft Vs Host Disease

Blood ◽

10.1182/blood-2019-129395 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1940-1940

Author(s):

Hongbing Ma ◽

Ke Zeng ◽

Mitsutaka Nishimoto ◽

Mi-Ae Lyu ◽

Meixian Huang ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

Regulatory T Cells ◽

Cord Blood ◽

Peripheral Blood ◽

Research Funding ◽

Adoptive Therapy ◽

Host Disease ◽

Graft Vs Host Disease ◽

The Impact

Background Adoptive therapy with regulatory T cells (Tregs) has already been established as a promising strategy for prevention of graft vs. host disease (GVHD) in clinical trials. Our group at MD Anderson Cancer Center has previously shown that a significantly lower dose of cord blood (CB) Tregs as compared to conventional T cells (Tcon) in the donor graft is able to prevent GVHD while preserving the graft vs. leukemia (GVL) effect. Therefore, we now examined the efficacy of using CB Tregs in the treatment of GVHD. Method: Xenogenic GVHD mouse model was established using NOD/SCID/IL2Rgnull (NSG) mice were sublethally irradiated at 300 cGy followed by injection of 1x107 peripheral blood (PB) mononuclear cells on day 0, as previously described. Ex vivo expanded CB Tregs were injected on day -1 (for prophylaxis) or at different days post PBMC injection for treatment. Mice were serially examined for appearance, weight, posture, GVHD score and survival. Serial peripheral blood sampling for flow cytometry and serum cytokine analysis. CB Tregs were also analyzed by flow cytometry. In order to understand the impact of the routine immunosuppressive agents on the function of CB Tregs, we incubated the CB Tregs in culture with cyclosporine (200ng/ml) or sirolimus (20 ng/ml) from day 8 to day 14. Cells were harvested on day 14 and analyzed by flow cytometry and CellTrace Violet suppression assay. Result: A single dose of 1x107 CB Tregs injected at day +7 did not result in a survival difference compared to the control arm (data not shown). Therefore, we froze multiple aliquots of expanded CB Tregs to be injected at different intervals post-transplant. Thawed CB Tregs showed stable phenotype of CD4+25+127lo: 94.7%; intracellular Helios+: 98.5% and intracellular FOXP3+: 99.4% and were able to suppress 87% of the proliferating conventional T-cells (Tcons). In order to compare the efficacy of the CB Tregs for GVHD treatment, we set up 3 arms: i) Control: PBMC alone; ii) Prophylaxis: 1x107 CB Tregs injected on day -1 and iii) Treatment: 1x107 CB Tregs injected on day +4, +7, +18 and +25. The mice in the prophylaxis and treatment arm retained their weight as compared to the control arm (p<0.003) (Fig 1A) and showed significantly better overall survival (P=0.01) (Fig 1B), which correlated with the decrease in circulating inflammatory cytokines including TNFa (Fig 1C). Since the standard of care for acute GVHD still remains high dose steroids, we evaluated the effect of continued exposure to steroids (prednisone-100ug/ml) for a period of 96 hours on the viability of CB Tregs. When compared to CB Tcons, 90.3% CB Tregs remained alive and viable compared to 64.7% of Tcons (Fig 1D). No differences were observed in the intracellular expression of FOXP3 or Helios in the control vs. cyclosporine or sirolimus exposed cells (Fig 1E). Similarly, no significant impact was observed on their suppressor function (Fig 1F). Conclusions: Multiple injections with CB Tregs can effectively treat GVHD. Combination therapy of CB Tregs with the commonly used GVHD treatments can be explored. Figure 1 Disclosures Iyer: Genentech/Roche: Research Funding; Incyte: Research Funding; Seattle Genetics, Inc.: Research Funding; Novartis: Research Funding; Bristol-Myers Squibb: Research Funding; Arog: Research Funding. Parmar:Cellenkos Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Integrated Analysis of Global DNA Methylation and Transcription Reveals a Leukemia Subtype with Extreme Hypermethylation Associated with Silencing of Regulators of Differentiation

Blood ◽

10.1182/blood-2018-99-118521 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2608-2608

Author(s):

Claudia Gebhard ◽

Roger Mulet-Lazaro ◽

Lucia Schwarzfischer ◽

Dagmar Glatz ◽

Margit Nuetzel ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Promoter Methylation ◽

Research Funding ◽

Promoter Hypermethylation ◽

Ctcf Binding ◽

P Value ◽

Global Dna Methylation ◽

Employment Equity ◽

Equity Ownership

Abstract Acute myeloid leukemia (AML) represents a highly heterogeneous myeloid stem cell disorder classified based on various genetic defects. Besides genetic alterations, epigenetic changes are recognized as an additional mechanism contributing to leukemogenesis, but insight into the latter process remains minor. Using a combination of Methyl-CpG-Immunoprecipitation (MCIp-chip) and MALDI-TOF analysis of bisulfite-treated DNA in a cohort of 196 AML patients we previously demonstrated that (cyto)genetically defined AML subtypes, including CBFB-MYH11, AML-ETO, NPM1-mut, CEBPA-mut or IDH1/2-mut subtypes, express specific DNA-methylation profiles (Gebhard et al, Leukemia, 2018). A fraction of AML patients (5/196) displayed a unique abnormal hypermethylation profile that was completely distinct from any other AML subtype. These patients present immature leukemia (FAB M0, M1) with various chromosomal aberrations but very few mutations (e.g. no IDH1/2, KRAS, DNMT3A) that might explain the CpG island methylator phenotype (CIMP) phenotype. The CIMP patients showed high resemblance with a recently reported CEBPA methylated subgroup (Wouters et al, 2007 and Figueroa et al, 2009), which we confirmed by MCIp-chip and MALDI-TOF analysis. To explore the whole range of epigenetic alterations in the CIMP-AML patients we performed in-depth global DNA methylation and gene expression analyses (MCIp-seq and RNA-seq) in 45 AML and 12 CIMP patients from both studies. Principle component analysis and t-distributed stochastic neighbor embedding (t-SNE) revealed that CIMP patients express a unique DNA-methylation and gene-expression signature that separated them from all other AMLs. We could discriminate promoter methylation from non-promoter methylation by selecting MCIp-seq peaks within 3kb around TSS. Promoter hypermethylation was highly associated with repression of genes (PCC = -0.053, p-value = 0.00075). Hypermethylation of non-promoter regions was more strongly associated with upregulation of genes (PCC = 0.046, p-value = 4.613e-06). Interestingly, differentially methylated regions also showed a positive association with myeloid lineage CTCF binding sites (27% vs 18% expected, p-value < 2.2e-16 in a chi-square test of independence). Methylation of CTCF sites causes loss of CTCF binding, which has been reported to disrupt boundaries between so-called topologically associated domains (TADs), allowing enhancers located in a particular TAD to become accessible to genes in adjacent TADs and affect their transcription. Whether this is the case is under investigation. In this study we particularly focused on the role of hypermethylation of promoters in CIMP-AMLs. Promoters of many transcriptional regulators that are involved in the differentiation of myeloid lineages of which several are frequently mutated in AML were hypermethylated and repressed, including CEBPA, CEBPD, IRF8, GATA2, KLF4, MITF or MAFB. Notably, HMGA2, a critical regulator of myeloid progenitor expansion, exhibited the largest degree of CIMP promoter hypermethylation compared to the other AMLs, accompanied by a reduction in gene expression. Moreover, multiple members of the HOXB family and KLF1 (erythroid differentiation) were methylated and repressed as well. In addition, these patients frequently showed hypermethylation of many chromatin factors (e.g. LMNA, CHD7 or TET2). Hypermethylation of the TET2 promoter could result in a loss of maintenance DNA demethylation and therefore successive hypermethylation at CpG islands. We carried out regulome-capture-bisulfite sequencing on CIMP-AMLs compared to other AML samples and normal blood cell controls and confirmed methylation of the same transcription and chromatin factor promoters. We conclude that these leukemias represent very primitive HSCPs which are blocked in differentiation into multiple hematopoietic lineages, due to the absence of regulators of these lineages. Although the underlying cause for the extreme hypermethylation signature is still subject to ongoing studies, the consequence of promoter hypermethylation is silencing of key lineage regulators causing the differentiation arrest in these cells. We argue that these patients may particularly benefit from therapies that revert DNA methylation. Disclosures Ehninger: Cellex Gesellschaft fuer Zellgewinnung mbH: Employment, Equity Ownership; GEMoaB Monoclonals GmbH: Employment, Equity Ownership; Bayer: Research Funding. Thiede:AgenDix: Other: Ownership; Novartis: Honoraria, Research Funding.

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The JAK1/2 Inhibitor Ruxolitinib Downregulates the Immune Checkpoint Protein B7-H3 in Multiple Myeloma

Blood ◽

10.1182/blood-2019-131047 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1824-1824

Author(s):

Ning Xu ◽

Nicole Ng ◽

Mingjie Li ◽

Erin Yu ◽

Eric Sanchez ◽

...

Keyword(s):

Gene Expression ◽

T Cells ◽

Immune Checkpoint ◽

Research Funding ◽

Flow Cytometric Analysis ◽

Cell Activity ◽

Cytokine Signaling ◽

Checkpoint Protein ◽

Equity Ownership

Introduction: The JAKSTAT pathway plays a critical role in the regulation of hematopoietic pathways and immunological cytokine signaling. The JAK pathway is also involved in tumor cell proliferation and drug resistance in multiple myeloma (MM). Thus, inhibition of the JAK pathway should be a potentially effective strategy for treating MM patients. B7-H3 is an immune checkpoint protein in the B7 superfamily and has been shown overexpressed in several tumors. Immune checkpoint blockade may suppress tumor progression or enhance anti-tumor immune responses. In this study, we investigated the effects of the JAK1/2 inhibitor ruxolitinib (Rux) on B7-H3 in MM. Materials and Methods: Bone marrow mononuclear cells (BMMCs) were collected from MM patients after obtaining IRB approval. Single-cell suspensions were prepared from human MM LAGλ-1A xenografts which had been grown in severe combined immunodeficient mice. HS-5 stromal and SUP-T1 T cells were purchased from ATCC. The cells were cultured and treated with or without RUX and then subjected to qRT-PCR, flow cytometric analysis, and western blot analysis. For qRT-PCR, total RNA was extracted and applied to cDNA synthesis, followed by qPCR. Gene expression was analyzed in MM BMMCs alone or co-cultured with stromal cells or T cells with or without Rux treatment (1μM) in vitro. Results: We identified increased B7-H3 expression in MMBMMCs from patients with progressive disease (PD) patients compared to those in complete remission (CR). Rux significantly reduced B7-H3 expression in MMBMMCs in patients with PD, MM cells (U266), and BM from patients in PD when co-cultured with stromal cells (HS-5) after 48-72 hours. Rux decreased B7H3 expression in the human MM xenograft model LAGλ-1A when cultured ex vivo. In addition, Rux suppressed B7-H3 at protein levels as shown with flow cytometric analysis and western blotting, consistent with the gene expression results. Next, we tested whether B7-H3 blockade by Rux could potentially restore exhausted T cell activity against myeloma cells in MMBM. We found that Rux can increase IL-2 and CD8 gene expression in MMBM with lower plasma percentages (< 30%) but not among those with higher plasma cell percentages (>70%). Rux also elevated IL-2 and CD8 gene expression in BM when it was cocultured with T cells (SUP-T1), suggesting Rux may mediate immunological cytokine signaling. B7-H3-neutralizing antibody increased CD8 gene expression in MMBM in vitro, suggesting that one of the mechanisms through which Rux upregulates CD8 T cells in MMBM may be via downregulation of B7-H3. Conclusion: The immune checkpoint protein B7-H3 is overexpressed in MMBM in PD compared to CR patients. The JAK1/2 inhibitor Rux can decrease B7-H3 expression and increase IL-2 and CD8 expression in BM in vitro. Our results provide evidence for Rux inhibiting the immune checkpoint protein B7-H3 which may potentially restore exhausted T-cell activity in the MMBM tumoral microenvironment. Disclosures Chen: Oncotraker Inc: Equity Ownership. Berenson:Amgen: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Sanofi: Consultancy; Sanofi: Consultancy; Amag: Consultancy, Speakers Bureau; Amag: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; OncoTracker: Equity Ownership, Other: Officer; OncoTracker: Equity Ownership, Other: Officer; Bristol-Myers Squibb: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Incyte Corporation.: Consultancy, Research Funding; Incyte Corporation.: Consultancy, Research Funding; Takeda: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau.

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Cooperation between Cancer Cells and Regulatory T Cells to Promote Immune-escape through Integrin αvβ8-Mediated TGF-β Activation

10.21203/rs.3.rs-208701/v1 ◽

2021 ◽

Author(s):

Alexandra Laine ◽

Ossama Labiad ◽

Hector Hernandez-Vargas ◽

Sebastien This ◽

Amélien Sanlaville ◽

...

Keyword(s):

T Cells ◽

T Lymphocytes ◽

Regulatory T Cells ◽

Cancer Cells ◽

Immune Escape ◽

Antibody Treatment ◽

Inactive Form ◽

Treg Population ◽

Cytotoxic Function ◽

Made In

Abstract Among the strategies allowing cancer cells to escape the immune system, the presence of TGF-b in the tumor micro-environment is one of the most potent. However, TGF-b is secreted in an inactive form and mechanisms responsible for its activation within the tumor remain unknown. Here, we demonstrate that regulatory T cells (Tregs) compose the main cells expressing the b8 chain of avb8 integrin (Itgb8) in the tumors and that the Itgb8pos Treg population activates TGF-b produced by the cancer cells and stored in the tumor micro-environment. Itgb8 ablation in Tregs impaired TGF-b signaling in T lymphocytes present in the tumor but not in the tumor draining lymph nodes. The cytotoxic function of CD8pos T lymphocytes infiltrating the tumors was subsequently exacerbated leading to an efficient control of the tumor growth. Similar observations were made in patient tumors after anti-Itgb8 antibody treatment. Thus, this study reveals that Tregs work in concert with cancer cells to produce bioactive-TGF-b and create a powerful-immunosuppressive micro-environment.

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The MEK Inhibitor Trametinib Enhances Diverse T Cell Reconstitution with Suppressing Xenogeneic Graft-Versus-Host-Disease

Blood ◽

10.1182/blood-2019-128913 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1929-1929

Author(s):

Hidekazu Itamura ◽

Hiroyuki Muranushi ◽

Takero Shindo ◽

Kazutaka Kitaura ◽

Seiji Okada ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Research Funding ◽

Mek Inhibitor ◽

Effector Memory ◽

Graft Versus Host ◽

T Cell Reconstitution ◽

Tcr Repertoire ◽

Repertoire Diversity ◽

Equity Ownership

Introduction: Early immune reconstitution without severe graft-versus-host disease (GVHD) is required for the success of allogeneic hematopoietic stem cell transplantation (allo-HSCT). We showed that MEK inhibitors suppress GVHD but retain antiviral immunity and graft-versus-tumor (GVT) effects (Shindo, Blood2013; Itamura, Shindo, JCI Insight2016). Furthermore, we have shown that they attenuate graft rejection but spare thymic function following rat lung transplantation (Takahagi, Shindo, Am J Respir Cell Mol Biol2019). Here we analyzed their effects on human polyclonal T cell reconstitution in xenogeneic transplant by evaluating T-cell receptor (TCR) repertoire diversity. Methods: As a xenogeneic GVHD model, human PBMCs were infused to NOD/Scid/JAK3null mice, immunodeficient mice lacking T/B/NK cells, after total body irradiation. Vehicle, tacrolimus, or the MEK inhibitor trametinib was administered from day 0 through 28 or day 15 through 28. Human TCR repertoire diversity was evaluated by an adapter ligation PCR method with next generation sequencing (Shindo, Oncoimmunol2018) in the liver, lung, and spleen. The assignment and frequencies of TCRαV/J clones were determined at the single-cell level. Their diversity and clonality were evaluated by Inv. Simpson's index 1/λ. Results: Trametinib prolonged their survival compared with vehicle (median survival: 88 vs 46 days, p<0.05). It enhanced engraftment of human leukocytes in peripheral blood (human CD45+cells: 11.0 vs 2.5%), but prevented their infiltration into the lung (human CD45+cells on day 60: 1.5 vs 6.5%). Treatment with vehicle resulted in skewed TCR repertoire with limited clones in the spleen, liver and lung. Interestingly, expansion of one specific clone (TRAV20/J10) was commonly observed, which might reflect the GVHD-inducing pathological clone (Fig. 1: 3D graphs show the frequencies of TCRαV/J clones). However, trametinib enabled diverse and polyclonal T cell engraftment without the TRAV20/J10 clone. While CD4+and CD8+T cells within injected human PBMCs mainly consisted of naïve (CD45RA+CD27+) and central memory (CD45RA-CD27+) T cells, infiltrating T cells in each organ showed effector memory (CD45RA-CD27-) T cell phenotype. Of note, CD8+T cells in the bone marrow, spleen, and lung of trametinib-treated recipients showed reduced effector memory T cells (CD45RA-CD27-) compared with vehicle-treated mice at day 28 (bone marrow 21.7 vs 74.7%, p<0.01; spleen 66.3 vs 88.7%, p<0.05; lung 33.0 vs 72.5%, p<0.05), which indicating that MEK inhibition suppresses functional differentiation of human T cells in vivo. Furthermore, trametinib treatment from day 14 to 28 still ameliorated clinical GVHD score, and maintained polyclonal T cell repertoire. Conclusions:GVHD can be characterized with skewed TCR repertoire diversity and expansion of pathological T cell clones in the target tissues. Trametinib suppresses GVHD but maintains polyclonal T cell reconstitution, even in established GVHD. These results explain the facts that MEK inhibitors separate GVHD from GVT effects/antimicrobial immunity. Furthermore, MEK inhibition enhances immune reconstitution after allo-HSCT, which would avoid post-transplant complications. Disclosures Shindo: Novartis: Research Funding. Kitaura:Repertoire Genesis Inc.: Employment. Okada:Bristol-Myers Squibb: Research Funding; Japan Agency for Medical Research and Development: Research Funding. Shin-I:BITS Co., Ltd: Equity Ownership. Suzuki:Repertoire Genesis Inc.: Equity Ownership. Takaori-Kondo:Celgene: Honoraria, Research Funding; Novartis: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; Ono: Research Funding; Takeda: Research Funding; Kyowa Kirin: Research Funding; Chugai: Research Funding; Janssen: Honoraria; Pfizer: Honoraria. Kimura:Ohara Pharmaceutical Co.: Research Funding; Novartis: Honoraria, Research Funding.

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Combination of NKTR-255, a Polymer Conjugated Human IL-15, with CD19 CAR T Cell Immunotherapy in a Preclinical Lymphoma Model

Blood ◽

10.1182/blood-2019-125960 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2866-2866 ◽

Cited By ~ 2

Author(s):

Cassie Chou ◽

Simon Fraessle ◽

Rachel Steinmetz ◽

Reed M. Hawkins ◽

Tinh-Doan Phi ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Research Funding ◽

Board Member ◽

Ad Hoc ◽

Advisory Board ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

Equity Ownership

Background CD19 CAR T immunotherapy has been successful in achieving durable remissions in some patients with relapsed/refractory B cell lymphomas, but disease progression and loss of CAR T cell persistence remains problematic. Interleukin 15 (IL-15) is known to support T cell proliferation and survival, and therefore may enhance CAR T cell efficacy, however, utilizing native IL-15 is challenging due to its short half-life and poor tolerability in the clinical setting. NKTR-255 is a polymer-conjugated IL-15 that retains binding affinity to IL15Rα and exhibits reduced clearance, providing sustained pharmacodynamic responses. We investigated the effects of NKTR-255 on human CD19 CAR T cells both in vitro and in an in vivo xenogeneic B cell lymphoma model and found improved survival of lymphoma bearing mice receiving NKTR-255 and CAR T cells compared to CAR T cells alone. Here, we extend upon these findings to further characterize CAR T cells in vivo and examine potential mechanisms underlying improved anti-tumor efficacy. Methods CD19 CAR T cells incorporating 4-1BB co-stimulation were generated from CD8 and CD4 T cells isolated from healthy donors. For in vitro studies, CAR T cells were incubated with NKTR-255 or native IL-15 with and without CD19 antigen. STAT5 phosphorylation, CAR T cell phenotype and CFSE dilution were assessed by flow cytometry and cytokine production by Luminex. For in vivo studies, NSG mice received 5x105 Raji lymphoma cells IV on day (D)-7 and a subtherapeutic dose (0.8x106) of CAR T cells (1:1 CD4:CD8) on D0. To determine optimal start date of NKTR-255, mice were treated weekly starting on D-1, 7, or 14 post CAR T cell infusion. Tumors were assessed by bioluminescence imaging. Tumor-free mice were re-challenged with Raji cells. For necropsy studies mice received NKTR-255 every 7 days following CAR T cell infusion and were euthanized at various timepoints post CAR T cell infusion. Results Treatment of CD8 and CD4 CAR T cells in vitro with NKTR-255 resulted in dose dependent STAT5 phosphorylation and antigen independent proliferation. Co-culture of CD8 CAR T cells with CD19 positive targets and NKTR-255 led to enhanced proliferation, expansion and TNFα and IFNγ production, particularly at lower effector to target ratios. Further studies showed that treatment of CD8 CAR T cells with NKTR-255 led to decreased expression of activated caspase 3 and increased expression of bcl-2. In Raji lymphoma bearing NSG mice, administration of NKTR-255 in combination with CAR T cells increased peak CAR T cell numbers, Ki-67 expression and persistence in the bone marrow compared to mice receiving CAR T cells alone. There was a higher percentage of EMRA like (CD45RA+CCR7-) CD4 and CD8 CAR T cells in NKTR-255 treated mice compared to mice treated with CAR T cells alone and persistent CAR T cells in mice treated with NKTR-255 were able to reject re-challenge of Raji tumor cells. Additionally, starting NKTR-255 on D7 post T cell infusion resulted in superior tumor control and survival compared to starting NKTR-255 on D-1 or D14. Conclusion Administration of NKTR-255 in combination with CD19 CAR T cells leads to improved anti-tumor efficacy making NKTR-255 an attractive candidate for enhancing CAR T cell therapy in the clinic. Disclosures Chou: Nektar Therapeutics: Other: Travel grant. Fraessle:Technical University of Munich: Patents & Royalties. Busch:Juno Therapeutics/Celgene: Consultancy, Equity Ownership, Research Funding; Kite Pharma: Equity Ownership; Technical University of Munich: Patents & Royalties. Miyazaki:Nektar Therapeutics: Employment, Equity Ownership. Marcondes:Nektar Therapeutics: Employment, Equity Ownership. Riddell:Juno Therapeutics: Equity Ownership, Patents & Royalties, Research Funding; Adaptive Biotechnologies: Consultancy; Lyell Immunopharma: Equity Ownership, Patents & Royalties, Research Funding. Turtle:Allogene: Other: Ad hoc advisory board member; Novartis: Other: Ad hoc advisory board member; Humanigen: Other: Ad hoc advisory board member; Nektar Therapeutics: Other: Ad hoc advisory board member, Research Funding; Caribou Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; T-CURX: Membership on an entity's Board of Directors or advisory committees; Juno Therapeutics: Patents & Royalties: Co-inventor with staff from Juno Therapeutics; pending, Research Funding; Precision Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Eureka Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Kite/Gilead: Other: Ad hoc advisory board member.

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Itolizumab As a Potential Therapeutic for the Prevention and Treatment of Graft Vs Host Disease

Blood ◽

10.1182/blood-2019-122787 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5603-5603 ◽

Cited By ~ 1

Author(s):

Cherie Tracy Ng ◽

Jeanette Ampudia ◽

Robert J. Soiffer ◽

Jerome Ritz ◽

Stephen Connelly

Keyword(s):

Weight Loss ◽

T Cells ◽

T Cell ◽

Peripheral Blood ◽

Research Funding ◽

Chronic Gvhd ◽

Vehicle Control ◽

Control Group ◽

Employment Equity ◽

Equity Ownership

Background: CD6 is a co-stimulatory receptor, predominantly expressed on T cells, that binds to activated leukocyte cell adhesion molecule (ALCAM), a ligand expressed on antigen presentation cells and various epithelial and endothelial tissues. The CD6-ALCAM pathway plays an integral role in modulating T cell activation, proliferation, differentiation and trafficking and is central to inflammation. While effector T cell (Teff) are CD6hi and upregulate expression upon activation, regulatory T cells (Treg) remain CD6lo/-, making this an attractive target to modulate Teff activity while preserving Treg activity. Early studies by Soiffer and colleagues demonstrated using T12, an anti-CD6 monoclonal antibody (mAb) that ex-vivo depletion of CD6+ donor cells prior to transplantation decreased the incidence of both acute and chronic GVHD, highlighting the importance of CD6+ cells in GVHD pathogenesis and validating it as a therapeutic target. However, it remains to be shown whether modulating the CD6-ALCAM pathway in vivo can attenuate GVHD. We investigated the use of itolizumab, a humanized anti-CD6 mAb that has demonstrated clinical efficacy in other autoimmune diseases, as both a preventive and therapeutic treatment for GVHD, using a humanized xenograft mouse model. Methods: Humanized xenograft mice were generated by intravenous transfer of 2x10^7 human PBMCs into 6-8 weeks old NOD/SCID IL2rγ-null (NSG). To investigate the ability of itolizumab to prevent GVHD, mice were dosed with either 60μg or 300μg of itolizumab, 150μg of abatacept (CTLA4-Ig), or vehicle, starting one day prior to PBMC transplantation. To investigate the therapeutic effect of itolizumab, mice were dosed with either 150μg of itolizumab or vehicle, starting at Day 5 post-PBMC transfer, when transplanted T cells are already activated. All treatments were administered IP every other day. Weight and disease scores were monitored throughout the study. At Days 18 and 35, peripheral blood was evaluated by flow cytometry to examine T cell prevalence, and tissues were collected for histological examination of pathology and T cell infiltration. Results: When administered as prevention (Day -1), treatment with either 60μg or 300μg of itolizumab significantly decreased mortality compared to the vehicle control (100% vs. 10%); this decrease was similar to the positive control group treated with abatacept (Figure 1). At 60μg, itolizumab-treated mice demonstrated significant reductions in the prevalence of human T cells in peripheral blood vs. vehicle-treated mice at Day 18 (<0.2% vs. 74.5%; p < 0.001). The reduction in peripheral T cells was accompanied by reductions in tissue-infiltrating T cells in lung (85-fold) and gut (9.5-fold), as well as reductions in disease scores and weight loss. When administered therapeutically, treatment with itolizumab was associated with a survival rate of 50% compared to 10% in the control group (Figure 2). Similarly, peripheral T cell prevalence (34.3% vs. 65.1%; p < 0.001), weight loss, and disease scores were inhibited by itolizumab compared to vehicle control mice. Conclusions: These data suggest that systemic treatment with itolizumab can modulate pathogenic Teff cell activity, establishing this antibody as a potential therapeutic for patents with GvHD. A phase I/II study using itolizumab as first line treatment in combination with steroids for patients with aGVHD is currently ongoing (NCT03763318). Disclosures Ng: Equillium: Employment, Equity Ownership. Ampudia:Equillium: Employment. Soiffer:Mana therapeutic: Consultancy; Kiadis: Other: supervisory board; Gilead, Mana therapeutic, Cugene, Jazz: Consultancy; Juno, kiadis: Membership on an entity's Board of Directors or advisory committees, Other: DSMB; Cugene: Consultancy; Jazz: Consultancy. Ritz:Equillium: Research Funding; Merck: Research Funding; Avrobio: Consultancy; TScan Therapeutics: Consultancy; Talaris Therapeutics: Consultancy; Draper Labs: Consultancy; LifeVault Bio: Consultancy; Celgene: Consultancy; Aleta Biotherapeutics: Consultancy; Kite Pharma: Research Funding. Connelly:Equillium: Employment, Equity Ownership.

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Abstract T MP18: In Vivo Expansion of Regulatory T cells with Interleukin-2/Interleukin-2-antibody Complex Protects against Transient Ischemic Stroke

Stroke ◽

10.1161/str.46.suppl_1.tmp18 ◽

2015 ◽

Vol 46 (suppl_1) ◽

Author(s):

Haiyue Zhang ◽

Peiying Li ◽

Yanqin Gao ◽

Jun Chen ◽

Xiaoming Hu

Keyword(s):

T Cells ◽

Ischemic Stroke ◽

Brain Injury ◽

Regulatory T Cells ◽

Interleukin 2 ◽

Ischemic Brain Injury ◽

Ischemic Brain ◽

Treg Population ◽

Antibody Complex

Background and Purpose: Our previous work documents the transfer of regulatory T cells (Tregs) in rodent models of ischemic stroke protects acute ischemic brain injury by regulating poststroke inflammatory response and thereby ameliorating BBB disruption. However, the low number of Tregs restricts the clinical feasibility of Treg transfer. Recently, in vivo expansion of Tregs with IL-2/IL-2-antibody complex (IL-2/IL-2Ab) was validated protective in autoimmune diseases model,renal ischemia reperfusion model and atherosclerosis. Here we investigate the beneficial effect of IL-2/IL-2Ab on ischemic stroke and decipher the underlying mechanisms. Methods: IL-2/IL-2Ab or isotype IgG was ip injected into C57/BL6 mice for 3 consecutive days. The mice are then subjected to 60-minute middle cerebral artery occlusion (MCAO) or sham operation. Brain infarction, inflammation and neurological performance was assessed up to 7 days after reperfusion. Results: Flow cytometry analysis reveals a marked increase of CD4+CD25+Foxp3+ Tregs in the blood, lymph nodes and spleens collected from IL-2/IL-2Ab-treated mice as compared to those from isotype-treated controls. Such Treg elevation could be observed since 3 days after IL-2/IL-2Ab injection and lasts until 7 days after MCAO. Immunochemistry staining confirms the increased number of Foxp3+ cells in the spleen at 3 days after MCAO in IL-2/IL-2Ab-treated mice. IL-2/IL-2Ab promotes function recovery up to 7 days after stroke, as revealed by significantly improved performance in corner test (n=6-9, ***p<0.001), rotarod test (n=8, **p<0.01), cylinder test (n=8, **p<0.01) and adhesive removal test (n=3, *p<0.05). Quantification of TTC staining and microtubule-associated protein (MAP2) staining shows reductions in brain infarct volume at 3 days (n=5-9,*p<0.05) and 7 days (n=7-9,*p<0.01), respectively, after MCAO. Meanwhile, we observed reduced infiltration of peripheral immune cells (CD3+ T cells, MPO+ neutrophils and F4/80+ macrophages) into the ischemic brain. Conclusions: Our finding suggests that IL-2/IL-2Ab treatment is a novel and clinical feasible immune therapy to expand Treg population in vivo, reduce post-stroke inflammatory responses and protect against ischemic brain injury.

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Distribution of CD4+CD25highFoxP3+regulatory T-cells in umbilical cord blood

The Journal of Maternal-Fetal & Neonatal Medicine ◽

10.3109/14767058.2012.666591 ◽

2012 ◽

Vol 25 (10) ◽

pp. 2058-2061 ◽

Cited By ~ 9

Author(s):

Hanah Kim ◽

Hee-Won Moon ◽

Mina Hur ◽

Chul-Min Park ◽

Yeo-Min Yun ◽

...

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Cord Blood ◽

Umbilical Cord ◽

Umbilical Cord Blood

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CD4+CD25+ Regulatory T Cells from Cord Blood Have Functional and Molecular Properties of T Cell Anergy.

Blood ◽

10.1182/blood.v104.11.316.316 ◽

2004 ◽

Vol 104 (11) ◽

pp. 316-316

Author(s):

Lequn Li ◽

Wayne R. Godfrey ◽

Stephen B. Porter ◽

Ying Ge ◽

Carle H. June ◽

...

Keyword(s):

Cell Cycle ◽

T Cells ◽

Regulatory T Cells ◽

Cord Blood ◽

Cell Lines ◽

Cell Cycle Progression ◽

Map Kinases ◽

Cyclin E ◽

Cyclin A ◽

Cycle Progression

Abstract CD4+CD25+ regulatory T cells (Tr) are negative regulators of immune responses. Studies of human Tr are restricted by their small numbers in peripheral blood and their hypoproliferative state. A recently established method achieved in vitro expansion and generation of Tr cell lines (Godfrey et al; Blood 2004,104:453-61). This approach facilitates the evaluation of cultured Tr cells as a novel form of immunosuppressive therapy and provides a system for molecular analysis of Tr. Activation of Ras and MAP kinases is mandatory for IL-2 production, viability and cell cycle progression of T cells. In anergic T cells activation of these signaling events is impaired, whereas activation of Rap1 is retained. Subsequently, anergic cells have defective IL-2 production, impaired cell cycle progression, and increased susceptibility to apoptosis. In the current study, we sought to determine the signaling and biochemical properties of Tr. Human CD4+CD25+ (Tr) and control CD4+CD25− (Tc) cell lines were generated from human cord blood cells. We examined activation of Ras, Rap1 and MAP kinases as well as cell cycle progression and cell viability, in response to TCR/CD3-plus-CD28 mediated stimulation. Stimulation was done for 15 min, 2 and 16 hrs for assessment of signaling events or for 24, 48 and 72 hrs for assessment of cell cycle progression and viability. Although activation of Rap1 was not affected, activation of Ras was reduced in Tr as compared to Tc. Activation of JNK and Erk1/2 MAP kinases was also significantly impaired. Both Tr and Tc entered the cell cycle and expressed cyclin E and cyclin A at 24 and 48 hrs of culture. However, p27 was downregulated only in Tc and not in Tr and hyperphosphorylation of Rb, which is the hallmark of cell cycle progression, was detected only in the Tc and not in the Tr population. At 72 hrs of culture, expression of cyclin E and cyclin A was dramatically diminished in Tr whereas it remained unchanged in Tc. More strikingly, expression of p27 in Tr was increased to levels higher than background. Since Tr do not produce IL-2, we examined whether addition of exogenous IL-2 would downregulate p27 and rescue Tr from defective cell cycle progression, similarly to its effect on anergic cells. Addition of exogenous IL-2 resulted in decrease of p27, sustained increase of cyclin E and cyclin A and cell cycle progression. Besides inhibiting cell cycle progression, p27 also promotes apoptosis. Therefore, we examined whether Tr had a higher susceptibility to apoptosis. As determined by Annexin V staining, Tr had a high degree of apoptosis only at 72 hrs of culture, when p27 expression was highly upregulated. Exogenous IL-2 reversed both p27 upregulation and apoptosis. Addition of IL-2 to Tr, also resulted in sustained IL-2-receptor-mediated activation of Erk1/2 at levels equivalent to those of Tc. Thus Tr cells share many biochemical and molecular characteristics of anergy, including defective TCR/CD3-plus-CD28-mediated activation of Ras and MAP kinases, increased expression of p27, defective cell cycle progression and high susceptibility to apoptosis. Moreover, these results suggest that TCR/CD3-mediated and IL-2 receptor-mediated signals converge at the level of MAP kinases to determine the fate of Tr cells towards expansion or cell cycle arrest and subsequent apoptosis.

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