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

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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Single Injection of Cord Blood Regulatory T Cells Can Delay the Manifestations of Systemic Lupus Erythematosus

Blood ◽

10.1182/blood-2019-131436 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1938-1938 ◽

Cited By ~ 2

Author(s):

Mi-Ae Lyu ◽

Joseph D. Khoury ◽

Mitsutaka Nishimoto ◽

Ke Zeng ◽

Meixian Huang ◽

...

Keyword(s):

Systemic Lupus Erythematosus ◽

Flow Cytometry ◽

T Cells ◽

Single Dose ◽

Regulatory T Cells ◽

Lupus Erythematosus ◽

Research Funding ◽

Single Injection ◽

Systemic Lupus ◽

Adoptive Therapy

Background: Systemic Lupus Erythematosus (SLE) is a chronic inflammatory autoimmune disorder with multi-organ involvement, including skin rash, joint pain, neurological dysfunction, pulmonary fibrosis, vasculitis, and renal failure. Previously it has been reported that SLE patients have a lower percentage of regulatory T cells (Tregs) and when compared to healthy population, Tregs derived from SLE patients show defect in their suppressor function. Our group at MD Anderson Cancer Center has already 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 graft vs host disease (GVHD). Furthermore, adoptive therapy with CB Tregs is being explored as a therapy for bone marrow failure including aplastic anemia as a single agent in the non-transplant setting. Therefore, we hypothesized that adoptive therapy with CB Tregs may be utilized for the treatment of SLE. Method: For examining the efficacy of CB Tregs in vivo, we developed a humanized SLE model, where female Rag2-/-γc-/- mice were transplanted with 3 ~ 4 x 106 human SLE-PBMCs by intravenous injection on day 0. The mice were allowed to develop disease (control) and at 1 weeks post-transplant a single dose of 1x107 CB Tregs was injected through the tail vein (treatment). Mice peripheral blood (PB) was assessed weekly for their cell compartment composition by using flow cytometry; double stranded DNA (dsDNA IgG) and plasma cytokines. Mice were monitored twice per week for weight loss, GVHD score and survival. Weekly urine collection was performed to analyze for albumin and creatinine. At the time of euthanasia, harvested organs were analyzed by flow cytometry, and immunohistochemistry. Result: Single injection of CB Tregs was sufficient to slow down the phenotype of SLE as shown in figure 1A, where the physical appearance of CB Treg treated mice was significantly better than the control and it correlated with a significantly lesser CD3+ infiltrates in the spleen of the treatment vs. control mice (Figure 1B) and a similar finding was observed in the CD20 infiltrate in the renal tissue (data not shown). While a widespread parakeratosis in the skin of the control mice, almost complete resolution was observed in the treatment arm (figure 1C). In addition, a lack of lymphoid infiltrate in the kidney and resolution of splenic lymphoid hyperplasia was observed in the CB Treg recipients (data not shown). In another experiment, we studied the role of weekly injection of 1x107 CB Tregs with the first injection administered at week 4 after the lupus inoculation. Significant improvement was observed in the urine albumin (p<0.02) (Fig 1D) as well as urine creatinine (p<0.0006) levels; which correlated with the significant improvement in the dsDNA IgG levels in the treatment arm compared to control (Fig 1E), respectively. We also measure the durability of single dose CB Treg injection, where a decrease in the 5-week plasma inflammatory cytokines including sCD40L (Fig 1F); TNFα (Fig 1G) and IFNγ (Fig 1H) was observed irrespective of CB Treg injection on day+7 or day+28 after the lupus PBMC injection. Conclusion: We conclude that adoptive therapy with CB Tregs is a viable option for SLE and additional studies are planned to optimize the dose and schedule details. Figure 1 Disclosures Khoury: Angle: Research Funding; Stemline Therapeutics: Research Funding; Kiromic: Research Funding. Iyer:Seattle Genetics, Inc.: Research Funding; Novartis: Research Funding; Bristol-Myers Squibb: Research Funding; Genentech/Roche: Research Funding; Incyte: 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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The Significance of mTOR Inhibitor, Everolimus in TGF-β-Induced Regulatory T cells from Cord Blood

Blood ◽

10.1182/blood.v118.21.2180.2180 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2180-2180

Author(s):

Tokiko Nagamura-Inoue ◽

Seiichiro Kobayashi ◽

Kazuo Ogami ◽

Yuki Yamamoto ◽

Kiyoko Izawa ◽

...

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Cord Blood ◽

Cell Population ◽

Cd4 T Cells ◽

Mtor Inhibitor ◽

Ex Vivo ◽

Mtor Inhibitors ◽

Ex Vivo Expansion ◽

The Impact

Abstract Abstract 2180 Background: Regulatory T cells (Tregs) play an important role in immune-tolerance to allograft. Cord blood (CB) is rich in naïve T cells and is a promising source of inducible Tregs (iTregs), since it was reported that stable iTregs may be derived exclusively from naïve T cells. However, the standard method for iTregs has not yet been established. Here we studied the impact of mTOR inhibitors, rapamycin (Rap) and everolimus (Eve), on ex vivo expansion of iTregs from CB-CD4+ T cells. Methods: CB-CD4+ T cell were isolated using anti-CD4 monoclonal antibody (MAb)-conjugated magnetic beads, and cultured in a flask coated with anti-CD3/CD28 MAbs and supplemented with IL-2 and TGF-β in the presence or absence of Rap or Eve. After two weeks of culture, the total number of CD4+ T cells was calculated, and the incidence of CD25+Foxp3+ cell population among those was estimated by FACS. Results and Discussions: Both Rap and Eve significantly increased the incidence of CD25+Foxp3+ cell population in CD4+ T cells. However, Rap apparently inhibited their growth and did not increase the absolute number of CD25+Foxp3+ cells in comparison to the control. On the other hand, Eve contributed to efficient expansion of iTregs at the concentration between 1 and 50ng/ml without no significant inhibition of their growth. Expansion of CD4+ T cells with TGF-β and Eve yielded 71.5 ±23.5% purity of CD25+Foxp3+ cells which also expressed CTLA-4 as well as the memory phenotype, while the purity obtained with TGF-β only was 47.4±30.0% and that without TGF-β/Eve was 7.3±4.5%. Thus, an average of 2.95±2.8 x107 iTregs were obtained from the initial input of 5×104 CD4+ T cells. The resulting iTregs with TGF-β, TGF-β/Rap and TGF-β/Eve inhibited the proliferation of CFSE-labeled T cells stimulated with allogeneic dendritic cells. The precise mechanism for Foxp3 induction by mTOR inhibitors still remains to be elucidated. Furthermore, we found that expression of CD26 (DPP-IV) was significantly down-regulated in CD4+ T cells expanded with TGF-β and profoundly with TGF-β/Eve, while CD127 was negative after culture in all the conditions. Mean fluorescence intensity of CD26 indicated 67.5 in CD4+ T cells without TGF-β, 1.58 with TGF-β, 0.18 with TGF-β/Rap and 0.12 with TGF-β/Eve, respectively. Accordingly, CD26 negativity may be an indicator of iTregs together with Foxp3. Conclusion: mTOR inhibitor, Eve, is an efficient co-inducer of iTregs and applicable to ex vivo expansion of iTregs in a clinical setting. Disclosures: No relevant conflicts of interest to declare.

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Third-party umbilical cord blood–derived regulatory T cells prevent xenogenic graft-versus-host disease

Cytotherapy ◽

10.1016/j.jcyt.2013.07.009 ◽

2014 ◽

Vol 16 (1) ◽

pp. 90-100 ◽

Cited By ~ 31

Author(s):

Simrit Parmar ◽

Xiaoying Liu ◽

Shawndeep S. Tung ◽

Simon N. Robinson ◽

Gabriel Rodriguez ◽

...

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Cord Blood ◽

Umbilical Cord ◽

Graft Versus Host Disease ◽

Umbilical Cord Blood ◽

Third Party ◽

Host Disease ◽

Graft Versus Host

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Persistence of naive CD45RA+ regulatory T cells in adult life

Blood ◽

10.1182/blood-2005-06-2403 ◽

2006 ◽

Vol 107 (7) ◽

pp. 2830-2838 ◽

Cited By ~ 196

Author(s):

Nabila Seddiki ◽

Brigitte Santner-Nanan ◽

Stuart G. Tangye ◽

Stephen I. Alexander ◽

Michael Solomon ◽

...

Keyword(s):

T Cells ◽

Lymph Node ◽

T Cell ◽

Regulatory T Cells ◽

Cord Blood ◽

Peripheral Blood ◽

Cell Subset ◽

Human Infant ◽

Wide Range ◽

Adult Peripheral Blood

AbstractRegulatory T cells (TREGs) constitutively expressing CD4, CD25, and the transcription factor Foxp3 can prevent a wide range of experimental and spontaneous autoimmune diseases in mice. In humans, CD4+CD25bright T cells, predominantly within the CD45RO+ activated/memory subset in adults and the CD45RA+ naive T-cell subset in infants, are considered to be the equivalent subset. Using novel combinations of monoclonal antibodies (mAbs), we examined expression of CD25 in human infant thymus, cord blood, adult peripheral blood, lymph node, and spleen. In addition to the CD4+CD25bright T cells, subfractionation on the basis of CD45 splice variants indicated that all samples contained a second distinct population of cells expressing a slightly lower level of CD25. In adult peripheral blood, this population expressed a naive CD45RA+ phenotype. The corresponding population in lymph node, spleen, and cord blood showed some evidence of activation, and expressed markers characteristic of TREGs, such as cytotoxic T lymphocyte-associated antigen 4 (CTLA-4). Sorted CD4+CD25+CD45RA+ T cells from both cord and adult blood expressed very high levels of mRNA for Foxp3 and manifested equivalent suppressive activity in vitro, indicating that they are bone fide members of the regulatory T-cell lineage. Targeting naive TREGs in adults may offer new means of preventing and treating autoimmune disease.

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Adiponectin/AdipoR1 Axis Promotes IL-10 Release by Human Regulatory T Cells

Frontiers in Immunology ◽

10.3389/fimmu.2021.677550 ◽

2021 ◽

Vol 12 ◽

Author(s):

Patricia Ramos-Ramírez ◽

Carina Malmhäll ◽

Omar Tliba ◽

Madeleine Rådinger ◽

Apostolos Bossios

Keyword(s):

Flow Cytometry ◽

T Cells ◽

Regulatory T Cells ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Foxp3 Expression ◽

P38 Map Kinase ◽

Adiponectin Receptor ◽

Peripheral Blood Mononuclear ◽

Cytokine Milieu

BackgroundAdiponectin is an important immunomodulatory mediator in inflammatory conditions. While we previously showed that adiponectin receptor 1 (AdipoR1) is expressed in murine regulatory T cells (Tregs), its expression in human Tregs remain unknown. Here, we examined the expression of AdipoR1 in human Tregs and whether its ligand, globular adiponectin (gAd) affects the Treg ability to secrete IL-10 and the role of Type 2 (T2) inflammation in such process.MethodsHuman Tregs from peripheral blood were analyzed by flow cytometry for AdipoR1, Helios and IL-10 expression. CD4+ T cells enriched from peripheral blood mononuclear cells (PBMCs) were cultured in the presence or the absence of gAd or the chemical adiponectin receptor agonist, AdipoRon, or in a T2 cytokine milieu. Flow cytometry was then used to assess intracellular IL-10, IL-10 secreting cells, FOXP3 and Helios expression, and phosphorylated p38 MAP kinase (MAPK). IL-10 levels in CD4+ T cell supernatants were quantified by ELISA.ResultsWe found that a subset of human Tregs expressed AdipoR1. Importantly, more Helios- cells expressed AdipoR1 than Helios+ cells. Likewise, there was a higher frequency of IL-10+ cells within Helios- AdipoR1+ Tregs compared to Helios+ AdipoR1+ Tregs. In contrast, the IL-10 mean fluorescence intensity (MFI) was higher in Helios+ AdipoR1+ Tregs compared to Helios-AdipoR1+ Tregs. When human CD4+ T cells were treated with gAd or AdipoRon, a significant increase in IL-10 secretion, FOXP3 expression, and p38 MAPK phosphorylation was observed in Helios- AdipoR1+ Tregs. Interestingly, gAd under T2 cytokine milieu significantly increased the intracellular levels of IL-10, mainly in Helios+ AdipoR1+ Tregs, and IL-10 levels in supernatants of CD4+ T cells.ConclusionsCollectively, our findings suggest that adiponectin/AdipoR1 axis promotes IL-10 release by Tregs, mainly in Helios- Tregs, and the effect was amplified by T2 inflammation in Helios+ Tregs.

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Novel CD4+CD8+ Umbilical Cord Blood Regulatory T Cells

Blood ◽

10.1182/blood-2019-131948 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4446-4446

Author(s):

Meixian Huang ◽

Amit Verma ◽

Ke Zeng ◽

Mi-Ae Lyu ◽

Mitsutaka Nishimoto ◽

...

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Cord Blood ◽

Research Funding ◽

P Value ◽

Q Value ◽

Double Positive ◽

Treg Population ◽

Equity Ownership ◽

Cytokine Assay

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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Flow Cytometric Detection of the Expression of CXCR4 on CD34+ Cells and the Distribution of Lymphocyte Subsets in Allogeneic Hematological Grafts.

Blood ◽

10.1182/blood.v106.11.5195.5195 ◽

2005 ◽

Vol 106 (11) ◽

pp. 5195-5195

Author(s):

Lulu Lu ◽

Yongping Song ◽

Baogen Ma ◽

Xiongpeng Zhu ◽

Xudong Wei ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

T Cells ◽

Cord Blood ◽

Peripheral Blood ◽

Lymphocyte Subsets ◽

Color Flow ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Mobilized Peripheral Blood

Abstract Background and objectives: Normal human bone marrow (BM), cord blood (CB) and mobilized peripheral blood (MPB) are the most commonly used sources for allogeneic hematopoietic stem cell transplantation (HSCT). The aim of this study was to detect the expression of CXCR4 on CD34+ cells and to assess the distribution of lymphocyte subsets in each type allograft. Methods: CD34+ cells were separated from BM (n=30), CB (n=30) and MPB (n=30) by the CD34 MultiSort Kit immunomagnetic bead system. The expression of CXCR4 on CD34+cells was assayed by double color flow cytometry. The lymphocyte subsets in each type of allograft were detected by three-color flow cytometry. The groups of monoclonal antibodies were used as the following: CXCR4-PE/CD34−Pecy5, CD8−FITC/CD4−R-PE/CD3−TC, CD45RA-FITC/CD45RO-PE/CD4−Pecy5, CD45RA-FITC/CD45RO-PE/CD8−Pecy5, and CD3−FITC/CD16+56-PE. Isotype-specific antibodies were used as controls. Results: The expression of CXCR4 of cord blood and mobilized peripheral blood CD34+ cells was lower than that of bone marrow cells (BM 40.21%±6.72%, CB 20.93%±3.96%, MPB 20.93%±3.96%, P <0.05). The difference between cord blood and mobilized peripheral blood was not significant (P>0.05). The CD3+CD8low and CD3+CD4−CD8low subsets were higher in BM than that of CB and MPB (BM 8.61%±1.40%, CB 3.31%±0.88%, MPB 5.11%±0.76%,P<0.01). The relative frequencies of the naïve CD45RA+ CD45RO− phenotype among CD4+ and CD8high T cells were highest in CB, and it was higher in MPB than in BM grafts (BM 28.09%±4.52%, 41.86 %±3.31%; CB83.83%±12.24%, 86.69%±6.12%; MPB 43.58%±4.54%, 57.64%±4.77%, P<0.01). Naïve T cells (CD45RA+ CD45RO−) were mobilized preferentially compared to memory T cells (CD45RA− CD45RO+)(P <0.01); The relative frequencies of NKT (CD3+CD16+56+) among lymphocytes were lower in CB than that in BM and MPB (CB 0.77±0.19, BM4.15±1.10, MPB 4.13±0.84, P<0.01). Conclusion: BM, CB and MPB allografts differ widely in cellular makeup of CD34+ cells and lymphocyte subsets, which are associated with the distinct characteristics after allogeneic HSCT from different allogeneic hematological sources.

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Massive, Clinical Grade Expansion Of Polyclonal T Cells Using Blinatumomab For Adoptive Autologous Cellular Therapy Of CLL Patients

Blood ◽

10.1182/blood.v122.21.3272.3272 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3272-3272 ◽

Cited By ~ 1

Author(s):

Josée Golay ◽

Anna D’amico ◽

Gianmaria Borleri ◽

Maria Chiara Finazzi ◽

Giulia Quaresmini ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

B Cells ◽

Peripheral Blood ◽

T Cell Subsets ◽

Th2 Cells ◽

Adoptive Therapy ◽

Specific Patient

Abstract Background The combined use of chemotherapy and monoclonal antibodies has proved highly effective for the treatment of CLL but often results in severe life threatening immunosuppression. The development of adoptive therapy with autologous T cells could be clinically relevant to overcome these problems. Methods We have devised a novel, simple and efficient method for ex vivo expansion of normal autologous T cells from the peripheral blood of CLL patients for adoptive therapy, using blinatumomab (CD3xCD19) and rhIL-2 in serum-free medium. The complete phenotype of in vitro expanded T cells was analyzed by flow cytometry and their cytotoxic activity by calcein release assays. Results We performed 18 expansions of T cells, starting from a very small volume of peripheral blood from untreated CLL patients (mean 10.3 ml, range 2-30 ml) that contained a mean of 9.2x106 T cells (range 0.4 to 51x106)(Fig.1). This method allowed reproducible expansion in about 21 days of a mean 410x106 CD3+ T cells (range 71 to 2184x106). The mean fold expansion of T cells in about 3 weeks of in vitro culture was 224 (range 4.4-1326). The only significant contaminant in final Blinatumomab Expanded T cell cultures (BET) were NK cells (mean 18.5%). Indeed addition of blinatumomab and rhIL-2 to the cultures led to a rapid decrease in CLL B cells, which took place from days 7 to 14 onwards and resulted in their complete depletion within 3 weeks (mean 0.2% CLL B cells at days 18-25). Only in one case, a significant percentage of CLL B cells could be observed at the end of culture, but this was due to the particularly high percentage neoplastic cells in the starting population in this patient (98%), resulting in relatively late depletion of these cells, which took place between days 14 and 21, and therefore remained detectable in this case at day 24 (3.8% CLL B cells at day 24). Despite the very low percentage of starting T cells in this specific patient (1.2%), 152x106 T cells could be obtained, equivalent to a 42 fold expansion. In the 18 expansions performed, the resulting BET cells contained both CD4+ and CD8+ cells in varying proportions (median 46.2% and 44.4% respectively). Only in two cases the final product was composed predominantly of CD4+ cells (95%). Expanded T cells were polyclonal, as shown by TCR Vβ expression which was within the normal range by flow cytometry. Indeed CMV specific clones, detected by CMV peptide (pp65495-503)-loaded HLA-A*0201 tetramer, were expanded using this method and detected in equivalent proportion before and after expansion. Final T cells were composed predominantly of the effector and central memory subsets. Th1 were slightly prevalent over Th2 cells (means 20% and 10%, respectively), whereas Th17 and Treg were less than 1%. Since CLL derived T cells have been shown previously to have enhanced expression of the synapse regulators CD272 and CD279 compared to normal T cells, leading to impaired immunological synapse formation, we have analyzed these markers in both starting and BET cells from 4 patients. We observed that CD272 and CD279 diminished in BET compared to the starting CLL T populations (from 73% to 19% and 61% to 18%, respectively). These data suggest that stimulation and expansion with blinatumomab and rhIL-2 has normalized expression of these regulators on CLL T cells. Indeed BET were highly cytotoxic against CD19+ targets cell lines or primary CLL cells, with 70-90% lysis at a 3:1 effector target ratio in presence of blinatumomab. Finally BET were compared to Xcellerated cells expanded using anti-CD3/CD28 Dynabeads and rhIL-2. The expansion protocols using either blinatumomab or anti-CD3/CD28 Dynabeads showed equivalent efficiency and comparable cell composition at the end of culture. Further comparison of the T cell subsets present in BET or CD3/CD28 cultures is in progress. Conclusions These data altogether suggest that the use of blinatumomab and rhIL-2 provides a reproducible, simple and GMP-compliant protocol, allowing expansion of large numbers of autologous polyclonal T cells depleted of CLL cells, from relatively small volumes of peripheral blood from CLL patients. This approach is an attractive option for adoptive therapy in these patients after immunosuppressive treatments. Disclosures: No relevant conflicts of interest to declare.

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Ex vivo Fucosylation Enhances Regulatory T Cell Anti-Graft Versus Host Disease Potency in Mice

Blood ◽

10.1182/blood.v124.21.660.660 ◽

2014 ◽

Vol 124 (21) ◽

pp. 660-660

Author(s):

Simrit Parmar ◽

Xiaoying Liu ◽

Yvon Eric ◽

Patrick zweidler-mcCay ◽

Nina Shah ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Mouse Model ◽

Graft Versus Host Disease ◽

Peripheral Blood ◽

Conflicts Of Interest ◽

Preventive Strategy ◽

Adoptive Therapy ◽

Host Disease ◽

Graft Versus Host

Abstract Emerging preclinical data indicate a potential therapeutic use of regulatory T cells (Tregs) to suppress or eliminate graft versus host disease (GVHD). Adoptive therapy with Tregs has been examined as a prophylactic strategy for GVHD. Thus far, 1:1 ratio of Tregs to conventional T cells (Tcons) has been required to prevent GVHD, and such high targeted cell doses are hard to achieve in a clinical setting due to variability in Treg frequency and expansion potential. As a result, novel strategies are needed to generate clinically-effective Treg products. Additionally, since the ability of Tregs to enter inflamed tissues has been shown to be critically dependent on their ability to bind E- and P-selectins, we sought to exploit this pathway to improve Treg homing. Incubation of culture expanded Cord Blood (CB) Tregswith fucosyltransferase-VI (FTVI) increased the degree of fucosylation from 8% to 64%. Importantly, fucosylated Tregs are able to suppress T cell proliferation in an in vitro allogeneic mixed lymphocyte assay and show preferential increase in the E-selectin binding ability when compared to untreated Tregs. We injected 10e6 Tregs vs. 10e6 fucosylated Tregs followed by 10e7 peripheral blood mononuclear cells in the sublethally irradiated NOD/SCID gamma null (NSG) xenogenic GVHD mouse model, and recipients of untreated Tregs showed weight loss as early as day 6 whereas fucosylated Treg recipients retained weight until day 12 (Fig 1). Fucosylated Tregs were detected in the peripheral blood of mice until day 31 as opposed to day 12 for untreated Tregs. As compared to recipients of untreated Tregs, lower numbers of GVH-inducing allogeneic T-cells were detected in the secondary lymphoid organs in the fucosylated Treg recipients (Table 1). The overall survival of fucosylated Treg recipients was significantly superior to that of untreated Treg recipients (Fig 2). We conclude that prophylactic treatment with fucosylated Tregs prevents GVHD and improves survival in a xenogenic mouse model at doses that are i) at less than 1:1 ratio with conventional T-cells (Tcon) and ii) are lower as compared to untreated Tregs. We believe that by overcoming the 1:1 dose requirement of Tregs: Tcons, we will be able to effectively translate Treg based adoptive therapy into the clinic for the prevention of GVHD. Establishing fucosylated Tregs as a preventive strategy for GVHD will result in a major breakthrough in the field of stem cell transplantation eliminating a major life threatening outcome of the therapy. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures No relevant conflicts of interest to declare.

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