GSK3β Inhibition Promotes T Cell Reconstitution and Preserves Naive T Cell Phenotype in Bone Marrow Reconstituted Mice

Abstract Abstract 1890 Stem cell transplantation has become a widely used procedure in the treatment of haematological and non-haematological clinical disorders. Unfortunately, cure is often hampered by relapse of the underlying disease, graft-versus-host disease (GVHD), or severe opportunistic infections. Slow T-cell reconstitution is regarded as primarily responsible for infections, GVHD, and relapse, therefore, enhancing immune reconstitution is important. Glycogen synthase kinase-3β (GSK3β) was recently identified as an important regulator of T cell function acting through the Wingless (Wnt) pathway. The effect of in vivo administration of GSK3β inhibitor 6-Bromoindirubin 3'-oxime (BIO) was examined in a humanised mouse model. Mice transplanted with highly purified cord blood CD34+ stem cells demonstrated efficient multilineage reconstitution including myeloid, B and T cell lymphoid compartments. The presence of human CD4 and CD8 single positive human T cells was abundant in peripheral blood (PB). De novo generated T cells exhibited low CD31 expression in the naïve CD4+ T cells suggesting prolonged post-thymic proliferative history of these cells. This is not completely surprising considering that graft recipient mice are characterized by impaired thymopoiesis following irradiation. Human T cells at various stages of differentiation including late effector T cells were recorded by detecting the expression of CD62L, CD45RA and CD45RO. Late memory T cell skewing was observed in PB and spleen of graft recipient mice. Activation of human T cells expressing CD25 was registered in the spleen, however, the recipients of the graft did not exhibit any signs of GVHD suggesting normal positive and negative selection occurring in the thymus during human T cell development in this mouse model. Human T cells isolated from the spleen of transplanted mice exhibited strong proliferative responses to mitogenic and allogeneic stimulation, however, they did not demonstrated any CTL activity tested following vaccination with human leukaemia U937 cells. In vivo administration of GSK3β inhibitor promoted T cell reconstitution in mice transplanted with human CD34+haematopoietic progenitor cells. Per cell output of T cells from CD34+ and CD34+CD38- primitive bone marrow (BM) progenitor cells was higher in BIO-treated mice while CD19+ B cell output was reduced suggesting T-cell developmental skewing in expense of B cell development. In vitro analysis of CD34+ progenitor cells co-cultured with bone marrow stroma MS5 cells has demonstrated inhibited B-cell development following BIO-treatment. CD31 expression in naïve CD4+ T cells was not up-regulated by BIO suggesting that GSK3β inhibition does not act to increase thymic output of T cells. GSK3β inhibition also increased naïve/memory T cell ratio in reconstituted mice. A similar effect was observed in mice transplanted with mature cord blood (CB)-derived T cells. delayed naive to memory T cell transition is likely related to decreased T cell activation and proliferation demonstrated ex vivo. BIO reduced IFNγ and TNFα production in human T cells. BIO increased naïve T cell production in mitogenically stimulated T cells and in mixed lymphocyte cultures. GSK3β inhibition preserved naïve T cell gene expression profile and suppressed the expression of genes activated during effector T cell differentiation. BIO actrivated β-catenin sigbnaling and up-regulated IL7Rα expression. IL7 signalling prevents activated T cell death following effector differentiation suggesting that the mechanism triggered by BIO may act through the inhibition of activated T cell death. In addition, BIO down-regulated negative regulator of IL7Rα SOCS1 as well as CTLA4 and PDCD1 both up-regulated during effector differentiation. Thus clinically GSK3β inhibition acting to prevent late memory T cell skewing and preserving a subset of naïve T cells may increase T cell diversity and improve T cell responses in the recipients of CB transplant particularly in adult patients with impaired thymic function. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Tumor-Targeted Human CD4+ CD25hi Regulatory T Cells Effectively Inhibit T Cell –mediated Rejection of CD19+ Tumors in An in Vivo xenotransplant Tumor Model

Blood ◽

10.1182/blood.v112.11.3901.3901 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3901-3901

Author(s):

James Lee ◽

Michel Sadelain ◽

Renier J. Brentjens

Keyword(s):

T Cells ◽

T Cell ◽

Regulatory T Cells ◽

B Cell ◽

Clinical Setting ◽

Effector T Cells ◽

Effector T Cell ◽

Human T Cells ◽

Anti Tumor Activity

Abstract The genetic targeting of human T cells to selected tumor antigens offers a novel means to investigate human immunobiology and treat cancer. T cells may be genetically modified to target specific antigens through the introduction of genes encoding chimeric antigen receptors (CARs). We have previously demonstrated that human T cells targeted in this manner to the CD19 antigen, expressed on normal B cells as well as most B cell tumors, eradicate systemic human CD19+ B cell malignancies in SCID-Beige mice. However, in the clinical setting, the anti-tumor efficacy of these T cells may be impaired by endogenous suppressive elements of the host immune system, including CD4+ CD25hi Foxp3+ regulatory T cells (Tregs). Significantly, Tregs are often increased in the blood and infiltrate the tumor of cancer patients which has been correlated with poor patient outcome and ineffective anti-tumor immunity. In order to study the in vivo impact of Tregs on adoptive therapy with CD19 targeted effector T cells, we developed a murine model wherein human Tregs, similarly targeted to the tumor, are infused prior to adoptive transfer of targeted cytotoxic T cells. To do so, we initially isolated natural Tregs from healthy donor peripheral blood mononuclear cells. Isolated Tregs were subsequently modified to express CARs through retroviral gene transfer. Subsequently, CAR+ Tregs were rapidly expanded either by activation on NIH-3T3 fibroblasts modified to express CD19 and the CD80 costimulatory ligand (3T3(CD19/CD80)), or non-specifically using CD3/CD28 antibodycoated magnetic beads. Expanded CAR+ Tregs exhibited potent suppressive function in vitro inhibiting both effector T cell proliferation as well as cytotoxicity. In vivo, CAR+ Tregs specifically traffic to established tumor in SCID-Beige mice. Significantly, injection of CD19-targeted Tregs into SCID-Beige mice bearing established human CD19+ tumors at 24 hours prior to infusion with CD19-targeted effector T cells, completely abrogated effector T cell function even at Treg:Teff ratios as low as 1:8. We further found that full suppression was dependant both on Treg localization to the tumor site as well as in vivo activation through the CAR. Finally, we show that a pre-conditioning regimen with low-dose cyclophosphamide, which failed to eradicate tumor, was able to reverse the CAR+ Treg mediated inhibition and restore the anti-tumor activity by the targeted effector T cells. In conclusion, we have developed a robust model ideally suited to the study of in vivo Treg-Teff interactions. Furthermore, the data generated from this model to date have significant implications with respect to the application of adoptive T cell therapies in the clinical setting. Namely, the presence of endogenous Tregs at the site of tumor is likely to significantly compromise the anti-tumor activity of adoptively transferred tumor targeted T cells. This inhibition may be reversed by preconditioning regimens designed to eradicate endogenous Tregs. The findings presented here should be considered in the design of future clinical trials utilizing T cell-based adoptive therapies of cancer.

Download Full-text

Chronic GvHD Is Characterized By Impaired B Cell Development in the Bone Marrow

Blood ◽

10.1182/blood.v126.23.1883.1883 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1883-1883

Author(s):

Oleg Kolupaev ◽

Michelle West ◽

Bruce R. Blazar ◽

Stephen Tilley ◽

James Coghill ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

B Cells ◽

B Cell ◽

Cd4 T Cells ◽

Critical Role ◽

Cell Development ◽

B Cell Development ◽

B Lymphopoiesis

Abstract Background. Chronic-graft-versus-host disease (cGvHD) continues to be a major complication following allogeneic hematopoietic stem cell transplantation (HSCT). Despite significant progress, mechanisms underlying development of the pathology are yet to be fully understood. Recent studies utilizing mouse models and patient samples have demonstrated a critical role for B cells in GvHD pathogenesis. Bone marrow (BM)-derived B cells can produce auto-reactive antibodies causing tissue fibrosis and multiorgan cGvHD. Impaired B cell homeostasis in the periphery, activation due to abnormally high levels of B cell-activating factor (BAFF), increased survival of auto-reactive B cells and aberrant BCR signaling are shown to be important for disease progression in cGvHD patients. Murine models also highlighted the critical role of germinal center reactions, particularly interactions between T follicular helper (Tfh) cells and B cells for generation of auto-antibodies which are responsible for triggering immune responses and cell-mediated toxicity. A growing body of evidence has emerged highlighting the fact that BM itself is a target organ during acute GvHD (aGvHD) with recent work suggesting a role for donor CD4+ T cells in BM specific aGvHD. Our group has shown that patients with higher numbers of BM B cell precursors were less likely to develop cGvHD after allogeneic HSCT (Fedoriw et al., 2012). These observations indicate clinical relevance of impaired BM B lymphopoiesis for cGvHD development. Methods. In order to investigate the effect of cGvHD on BM B cell development, we used the well-characterized major mismatch B6 into B10.BR model of systemic cGvHD. Recipient mice were treated with cyclophosphamide on day -3 and -2, irradiated with 700 cGy on day -1, and injected with 107 T cell depleted (TCD) BM with or without total splenic T cells (0.5-1x105). Mice were monitored for 30 days, and BM and spleen was harvested and analyzed using flow cytometry. Results. Consistent with patient data, we observed a decrease in the frequency and number of donor-derived uncommitted common lymphoid progenitors (CLP) and B cell progenitors in the BM+ allogeneic T cells group (CLP: 0.17±0.03% vs. 0.06±0.01%, p <0.01; pro B: 2.2 ± 0.5% vs. 0.7 ± 0.3%, p<0.05; pre B: 15.3±1.8% vs. 6.3±2.4%, p<0.05; immature B cells: 5.7±0.7% vs. 2.1±0.7%, p<0.01) (Fig.1). As previously reported for this model, we also found a decrease in the frequency of follicular (FO) B cells (Flynn et al., 2014). We hypothesized that during cGvHD the B cell progenitor BM niche is affected by donor CD4+ T cells leading to impaired B lymphopoiesis. Bone marrow from BM+T cell animals had a significantly higher frequency of CD4+ cells compared to the control group (0.45±0.06% vs. 0.2±0.02%). Depletion of CD4+ T cells using anti-CD4 antibody during the first two weeks after transplant improved pathology scores and prevented weight loss in BM+T cells mice. We also observedpartial recovery of B cell progenitors and Lin-CD45-CD31-CD51+ osteoblasts (OB) in animals treated with anti-CD4 antibodies (pre B 3.5±1.1% vs. 20.4±4.5%, p<0.05; immature B: 1.9±0.9% vs. 3.5±0.3%; OB: 0.8±0.1% vs.1.2±0.2%). A recent study showed that activation and proliferation of conventional T cells in aGvHD model can be prevented by in vivo expansion of regulatory T cells (Tregs) using αDR3 antibody (4C12). We adopted this approach to determine whether Tregs can suppress the cytotoxic effect of donor CD4+ T cells in BM in cGvHD model. Animals that received T cells from 4C12-treated donors had an increase in survival and lower cGvHD pathology scores. These mice also had higher frequency of pro B, pre B, and immature B cells compared to the mice infused with T cells from isotype-treated donors. Conclusions. These studies demonstrate that BM development of B lymphocytes is impaired in a mouse model of systemic cGvHD. Our data suggests that donor-derived CD4+ T cells are involved in the destruction of hematopoietic niches in BM, particularly OB, which support B lymphopoiesis. Moreover, depletion of CD4+ T cells and infusion with in vivo expanded Tregs reduced the severity of cGvHD. Thus, Treg therapy in patients with cGvHD may be important for BM B cell development, and improvement of clinical outcomes. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Ammonium Ions Derived from Spontaneous Decomposition of L-Glutamine Inhibit Lymphoid Differentiation from Hematopoietic Stem/Progenitor Cells.

Blood ◽

10.1182/blood.v110.11.2244.2244 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2244-2244

Author(s):

Gerald J. Spangrude ◽

Birgitta Johnson ◽

Scott Cho ◽

Xiaosong Huang ◽

L. Jeanne Pierce

Keyword(s):

Bone Marrow ◽

T Cell ◽

B Cell ◽

Progenitor Cells ◽

Stromal Cell ◽

T Cell Development ◽

Cell Development ◽

Ammonium Ions ◽

Hematopoietic Stem ◽

Lymphocyte Differentiation

Abstract The ability to study lymphocyte differentiation in culture has been greatly advanced by the availability of the OP9 bone marrow stromal cell line, which was derived from an op/op mouse and thus lacks M-CSF. As a result, the normal default myeloid differentiation from bone marrow-derived stem and progenitor cells does not occur, and lymphocyte differentiation is favored. Introduction of the Notch ligand Delta-like 1 into OP9 cells results in promotion of T cell development and parallel suppression of B cell development. While the OP9-DL1 model of T cell development works quite well when fetal liver-derived progenitors are cultured, the success of T cell development from adult bone marrow-derived progenitors has been more difficult to reproduce. We have undertaken a systematic analysis of variables that can prevent efficient T cell development in OP9-DL1 cultures, and have found that one limiting factor that impacts the efficiency of differentiation of both T and B cell lineages is the accumulation of ammonium ions as a result of the spontaneous decomposition of l-glutamine. L-glutamine, which is present at 2 to 4 mM in standard tissue culture media, is unstable and will spontaneously degrade to form ammonium ions and pyroglutamic acid at a rate of 1%/day at 4°C and at a 10-fold higher rate at 37°C. To evaluate the effects of the two major products of l-glutamine decomposition on lymphoid differentiation, we added each product to differentiation cultures at 3 mM in the presence of a stable source of l-glutamine (l-alanyl-l-glutamine). Cultures were established in 1 ml containing 4×104 stromal cells (OP9 for B cell differentiation, OP9-DL1 for T cell differentiation), 1×103 bone marrow-derived lymphoid progenitors enriched by phenotype (c-kit+LinnegSca-1+Thy-1.1neg), and 5 ng/ml Flt3L plus 5 ng/ml IL-7. Every 3 to 4 days, cultures were harvested and passaged onto fresh stromal cell monolayers; lymphoid cells were counted and evaluated for surface antigen expression at each passage. While addition of pyroglutamic acid had no inhibitory effect on lymphocyte growth or differentiation, addition of ammonium chloride slowed growth and prevented differentiation of both T and B lymphocytes. Growth of the stromal cell monolayers was not affected by ammonium chloride at the concentrations utilized in these studies. We conclude that freshly-prepared culture medium, preferably containing a stabilized form of l-glutamine, is a critical aspect contributing to the success of lymphocyte differentiation cultures established from adult bone marrow cells. We also found that decreasing IL-7 concentrations to 1 ng/ml resulted in more rapid differentiation of T cells and a more balanced representation of CD4 and CD8 single positive cells. Our studies help define optimal conditions for differentiation of bone marrow-derived lymphoid progenitor cells into T and B lineages in vitro, and provide evidence that hematopoietic differentiation displays variable degrees of sensitivity to ammonium ions derived from decomposition of l-glutamine. These results will help define optimal conditions for expansion and differentiation of hematopoietic stem and progenitor cells in vitro.

Download Full-text

Gene Targeting of RhoA Reveals Its Essential Role In Lymphopoiesis

Blood ◽

10.1182/blood.v116.21.282.282 ◽

2010 ◽

Vol 116 (21) ◽

pp. 282-282

Author(s):

Shuangmin Zhang ◽

Yi Zheng ◽

Richard Lang ◽

Fukun Guo

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

B Cell ◽

Cell Development ◽

B Cell Development ◽

Brdu Incorporation ◽

Hematopoietic Stem ◽

Cell Functions

Abstract Abstract 282 RhoA GTPase is an intracellular signal transducer capable of regulating a wide range of cell functions including cytoskeleton dynamics, proliferation, and survival. In lymphocytes, studies by using dominant negative mutant or C3 transferase expressing transgenic mice suggest that RhoA is involved in TCR and BCR signaling and related T cell functions such as polarization, migration, survival, and proliferation. To date, the physiological role of RhoA in lymphocyte development remains unclear. In this study, we have achieved T cell, B cell, and hematopoietic stem cell-specific deletion of RhoA by conditional gene targeting with CD2, CD19 and Mx1 promoter-driven Cre expression, respectively, in the RhoAloxP/loxP mice. First, we found that RhoA gene disruption in early T cells caused a drastic decrease in thymocyte cellularity, with the numbers of CD4−CD8− double negative (DN), CD4+CD8+ double positive (DP), CD4+CD8− single positive (SP), and CD4−CD8+ SP T cells decreased by 88.8% ± 6.0%, 99.4% ± 1.0%, 99.3% ± 1.2%, and 98.6% ± 2.0%, respectively. Among DN subpopulations, CD44+CD25− (DN1), CD44+CD25+ (DN2), CD44−CD25+ (DN3), and CD44−CD25− (DN4) cells were reduced by 91.7% ± 6.0%, 54.9% ± 27.7%, 50.9% ± 33.3%, and 96.7% ± 3.4%, respectively. Further, RhoA knockout led to a significant loss of DP thymocytes at the initial stage (CD69highTCRint) of positive selection, suggesting that RhoA is required for positive selection. The decreased thymocyte cellularity in mutant mice is associated with increased apoptosis of all thymic T lineages. RhoA deficiency also resulted in a perturbation in thymocyte cell cycle progression as manifested by increased BrdU incorporation in DN1 and DN2 cells and decreased BrdU incorporation in DN4 and DP cells. Concomitantly, RhoA-deficient thymocytes showed a 59.8% ± 26.3% reduction in proliferative potential in response to TCR crosslinking. Western blot analysis revealed that the activities of ZAP70, LAT, Akt, Erk, and p38 were impaired in RhoA-/- thymocytes. In periphery, spleens of the RhoA null mice contained 7.4% ± 8.0% of CD4+ T cells and 3.7% ± 2.7% of CD8+ T cells compared with that of wild type (WT) mice. Loss of peripheral mature T cells in mutant mice is reflected by a marked reduction of naive T cells, whereas effector and memory phenotype cells were marginally affected by RhoA deficiency. RhoA-deficient naïve T cells were more susceptible to apoptosis, suggesting that homeostatic defect of naïve T cells in RhoA-/- mice is attributed to impaired cell survival. Abrogation of RhoA caused an increased in vivo BrdU incorporation in naïve T cell compartments. Thus, RhoA deficiency induces naïve T cell homeostatic proliferation, possibly due to a compensatory effect of lymphopenia. In contrast to that in thymocytes, Erk was constitutively activated in RhoA-deficient splenic T cells. These observations implicate RhoA in the multiple stages of T cell development and the proper assembly of early TCR signaling complex. Second, deletion of RhoA in pre-proB cells had no effect on early B cell development in bone marrow but significantly inhibited late B cell development in spleen, resulting in 78.2% ± 13.6%, 78.6% ± 16.9%, and 93.2% ± 3.4% reduction in transitional, follicular, and marginal zone B cells, respectively. Plasma cells in spleen were decreased by 50.9 % ± 25.9% in RhoA null mice. However, we did not detect any changes in survival of in vivo RhoA-/- B cells or RhoA-/- B cells cultured in vitro with survival factor BAFF. Distinct from previously characterized Cdc42 knockout mice, BAFF-R expression was not altered in RhoA-/- B cells. Moreover, RhoA-/- B cells appeared to be normal in proliferation and Akt and Erk activation in response to BCR crosslinking. These data suggest that RhoA is important for late B cell development through regulation of differentiation but not cell survival or proliferation. Finally, deletion of RhoA from hematopoietic stem cells did not affect common lymphoid progenitor production, indicating that RhoA is not required for early lymphoid progenitor commitment. Taken together, these lineage-specific mouse genetic studies demonstrate that RhoA critically regulates T and B cell development by distinct cellular mechanisms at multiple stages of lymphopoiesis. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

ALL Progression Induces PD1 on T Cells and Blockade of PD1 Enhances Adoptive Immunotherapy

Blood ◽

10.1182/blood.v118.21.2587.2587 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2587-2587 ◽

Cited By ~ 1

Author(s):

Chad R Burk ◽

William Fix ◽

Haiying Qin ◽

Terry J Fry

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

B Cell ◽

Poor Response ◽

Cell Phenotype ◽

Early Progression ◽

Pd1 Blockade

Abstract Abstract 2587 Background: Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy and, despite tremendous success in therapy over the past 3 decades, remains a primary cause of cancer-related mortality in children. Enthusiasm for the use cellular immunotherapy for ALL has been tempered by the poor response to donor lymphocyte infusions following allogeneic hematopoietic stem cell transplantation. However, ALL blasts are susceptible to T cell and NK cell mediated lysis in vitro suggesting that poor response to in vivo immune interventions may be due to events occurring during the priming of the immune response. Using a murine model of precursor B cell ALL we examined the impact of leukemia progression on T cells in vivo. Methods: We developed a transplantable syngeneic model of pediatric ALL derived from transgeneic mice expressing human E2aPBX1, a recurring translocation present in 5% of pediatric leukemia (Bijl et al, Genes and Development, 2005). This murine line displays a precursor B cell phenotype and results in 100% lethality following injection of 100,000 cells (Qin et al, ASH, 2010). Using congenic (CD45.1) B6 recipients, we tracked the early progression of ALL in vivo and examined the T cells in the leukemia-containing compartments by flow cytometry and PCR. Results: Using congenic markers, ALL cells can be detected in bone marrow as early as 3 days following intravenous injection of 1,000,000 cells with a sensitivity of 0.01%. Spleen and lymph node involvement was seen later (10 days) followed by the detection of circulating blasts by 2 weeks. E2aPBX1 cells express variable levels of costimulatory molecules in vitro with no change in expression during in vivo progression. Notably, PDL1 and PDL2 are expressed both in vitro and in vivo at higher levels than on non-malignant precursor B cells in leukemia-bearing mice. Remarkably, although PD1+ T cells are not seen in the bone marrow of non-leukemia-bearing mice, PD1 expression on bone marrow T cells was markedly increased during progression such that 60–80% of all bone marrow CD4 and CD8 T cells were positive by 2 weeks following leukemia injection (figure). In addition to expression of PD1, these T cells also co-expressed Tim3, a phenotype associated with T cell exhaustion. Blockade of PD1 or PDL1 starting 3 days following leukemia injection had no impact on leukemia progression. However, combining PD1 blockade with the adoptive transfer of T cells from leukemia-primed donors resulted in improved survival compared to primed T cells alone (p=0.0004). Conclusions: Early progression of ALL results in the induction of PD1 and Tim3 on T cells in vivo. Combination of PD1 blockade plus adoptive T cell therapy results in therapeutic benefit suggesting that this axis may be an attractive target in ALL. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Impaired bone marrow B-cell development in mice with a bronchiolitis obliterans model of cGVHD

Blood Advances ◽

10.1182/bloodadvances.2017014977 ◽

2018 ◽

Vol 2 (18) ◽

pp. 2307-2319 ◽

Cited By ~ 1

Author(s):

Oleg V. Kolupaev ◽

Trisha A. Dant ◽

Hemamalini Bommiasamy ◽

Danny W. Bruce ◽

Kenneth A. Fowler ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

B Cell ◽

Bronchiolitis Obliterans ◽

Critical Role ◽

Cell Development ◽

B Cell Development ◽

Allogeneic Bone ◽

B Cell Precursors

Abstract Chronic graft-versus-host disease (cGVHD) causes significant morbidity and mortality in patients after allogeneic bone marrow (BM) or stem cell transplantation (allo-SCT). Recent work has indicated that both T and B lymphocytes play an important role in the pathophysiology of cGVHD. Previously, our group showed a critical role for the germinal center response in the function of B cells using a bronchiolitis obliterans (BO) model of cGVHD. Here, we demonstrated for the first time that cGVHD is associated with severe defects in the generation of BM B lymphoid and uncommitted common lymphoid progenitor cells. We found an increase in the number of donor CD4+ T cells in the BM of mice with cGVHD that was negatively correlated with B-cell development and the frequency of osteoblasts and Prrx-1–expressing perivascular stromal cells, which are present in the B-cell niche. Use of anti-DR3 monoclonal antibodies to enhance the number of donor regulatory T cells (Tregs) in the donor T-cell inoculum ameliorated the pathology associated with BO in this model. This correlated with an increased number of endosteal osteoblastic cells and significantly improved the generation of B-cell precursors in the BM after allo-SCT. Our work indicates that donor Tregs play a critical role in preserving the generation of B-cell precursors in the BM after allo-SCT. Approaches to enhance the number and/or function of donor Tregs that do not enhance conventional T-cell activity may be important to decrease the incidence and severity of cGVHD in part through normal B-cell lymphopoiesis.

Download Full-text

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.

Download Full-text

B cell adaptor for PI3-kinase (BCAP) modulates CD8+ effector and memory T cell differentiation

Journal of Experimental Medicine ◽

10.1084/jem.20171820 ◽

2018 ◽

Vol 215 (9) ◽

pp. 2429-2443 ◽

Cited By ~ 10

Author(s):

Mark D. Singh ◽

Minjian Ni ◽

Jenna M. Sullivan ◽

Jessica A. Hamerman ◽

Daniel J. Campbell

Keyword(s):

T Cells ◽

Cell Differentiation ◽

T Cell ◽

B Cell ◽

Cd8 T Cells ◽

T Cell Development ◽

Cell Development ◽

T Cell Differentiation ◽

Pi3k Signaling ◽

Memory T Cell

CD8+ T cells respond to signals via the T cell receptor (TCR), costimulatory molecules, and immunoregulatory cytokines by developing into diverse populations of effector and memory cells. The relative strength of phosphoinositide 3-kinase (PI3K) signaling early in the T cell response can dramatically influence downstream effector and memory T cell differentiation. We show that initial PI3K signaling during T cell activation results in up-regulation of the signaling scaffold B cell adaptor for PI3K (BCAP), which further potentiates PI3K signaling and promotes the accumulation of CD8+ T cells with a terminally differentiated effector phenotype. Accordingly, BCAP-deficient CD8+ T cells have attenuated clonal expansion and altered effector and memory T cell development following infection with Listeria monocytogenes. Thus, induction of BCAP serves as a positive feedback circuit to enhance PI3K signaling in activated CD8+ T cells, thereby acting as a molecular checkpoint regulating effector and memory T cell development.

Download Full-text

Adoptive Transfer of In Vitro Generated T Cell Precursors Enhances Donor T Cell Reconstitution and Graft-Versus-Tumor Activity in Allogeneic Hematopoietic Stem Cell Transplantation Recipients.

Blood ◽

10.1182/blood.v106.11.63.63 ◽

2005 ◽

Vol 106 (11) ◽

pp. 63-63 ◽

Cited By ~ 1

Author(s):

Johannes L. Zakrzewski ◽

Adam A. Kochman ◽

Sidney X. Lu ◽

Theis H. Terwey ◽

Theo D. Kim ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

Stem Cell ◽

Growth Factors ◽

T Cell ◽

Hematopoietic Stem Cell Transplantation ◽

Hematopoietic Stem ◽

T Cell Reconstitution

Abstract Allogeneic hematopoietic stem cell transplantation (HSCT) is associated with a varying period of immunoincompetence that particularly affects he T cell lineage resulting in significant morbidity and mortality from opportunistic infections. Recent studies have shown that murine T cells and their precursors can be generated from hematopoietic stem cells (HSC) in vitro using a OP9-DL1 coculture system consisting of OP9 bone marrow stromal cells expressing the Notch 1 ligand Delta-like 1 and growth factors (interleukin 7 and fms-like tyrosine kinase-3 ligand). In this study we determined the effects of adoptively transferred in vitro generated T cell precursors on T cell reconstitution after allogeneic HSCT. We selected HSC (Lin- Sca-1hi c-kithi) from bone marrow (BM) of C57BL/6 mice and cultured these cells on a monolayer of OP9-DL1 cells in the presence of growth factors. These HSC expanded 2,000–5,000-fold within 3–4 weeks and consisted of >95% CD4-CD8-double negative (DN) T cell precursors after 16–28 days of culture. We infused these cells (8x106) with T cell depleted (TCD) BM (5x106) or purified HSC into allogeneic recipients using minor antigen mismatched and MHC class I/II mismatched transplant models. Control mice received TCD BM or purified HSC only. Progeny of OP9-DL1 derived T cell precursors were found in thymus and spleen increasing thymic cellularity and significantly improving thymic and splenic donor T cell chimerism. This effect was even more pronounced when purified HSC instead of whole BM were used as allograft. T cell receptor repertoire and proliferative response to foreign antigen (determined by third party MLR) of in vivo differentiated OP9-DL1 derived mature T cells were intact. Administration of in vitro generated T cell precursors did not induce graft-versus-host disease (GVHD) but mediated significant graft-versus-tumor (GVT) activity (determined by in vivo bioluminescence imaging) resulting in a subsequent significant survival benefit. This advantage was associated with better cytokine responses (IL-2, INF-g, TNF-a) in T cells originating from OP9-DL1 derived T cell precursors compared to BM donor derived T cells. We conclude that the adoptive transfer of OP9-DL1 derived T cell precursors significantly enhances post-transplant T cell reconstitution and GVT activity in the absence GVHD.

Download Full-text

CD40 Activity on Mesenchymal Cells Negatively Regulates OX40L to Maintain Bone Marrow Immune Homeostasis Under Stress Conditions

Frontiers in Immunology ◽

10.3389/fimmu.2021.662048 ◽

2021 ◽

Vol 12 ◽

Author(s):

Barbara Bassani ◽

Claudio Tripodo ◽

Paola Portararo ◽

Alessandro Gulino ◽

Laura Botti ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

B Cell ◽

Inflammatory Cytokines ◽

Conditioning Regimen ◽

Regulatory Elements ◽

Cell Development ◽

B Cell Development ◽

B Cell Precursors

BackgroundWithin the bone marrow (BM), mature T cells are maintained under homeostatic conditions to facilitate proper hematopoietic development. This homeostasis depends upon a peculiar elevated frequency of regulatory T cells (Tregs) and immune regulatory activities from BM-mesenchymal stem cells (BM-MSCs). In response to BM transplantation (BMT), the conditioning regimen exposes the BM to a dramatic induction of inflammatory cytokines and causes an unbalanced T-effector (Teff) and Treg ratio. This imbalance negatively impacts hematopoiesis, particularly in regard to B-cell lymphopoiesis that requires an intact cross-talk between BM-MSCs and Tregs. The mechanisms underlying the ability of BM-MSCs to restore Treg homeostasis and proper B-cell development are currently unknown.MethodsWe studied the role of host radio-resistant cell-derived CD40 in restoring Teff/Treg homeostasis and proper B-cell development in a murine model of BMT. We characterized the host cellular source of CD40 and performed radiation chimera analyses by transplanting WT or Cd40-KO with WT BM in the presence of T-reg and co-infusing WT or - Cd40-KO BM-MSCs. Residual host and donor T cell expansion and activation (cytokine production) and also the expression of Treg fitness markers and conversion to Th17 were analyzed. The presence of Cd40+ BM-MSCs was analyzed in a human setting in correlation with the frequency of B-cell precursors in patients who underwent HSCT and variably developed acute graft-versus-host (aGVDH) disease.ResultsCD40 expression is nearly undetectable in the BM, yet a Cd40-KO recipient of WT donor chimera exhibited impaired B-cell lymphopoiesis and Treg development. Lethal irradiation promotes CD40 and OX40L expression in radio-resistant BM-MSCs through the induction of pro-inflammatory cytokines. OX40L favors Teff expansion and activation at the expense of Tregs; however, the expression of CD40 dampens OX40L expression and restores Treg homeostasis, thus facilitating proper B-cell development. Indeed, in contrast to dendritic cells in secondary lymphoid organs that require CD40 triggers to express OX40L, BM-MSCs require CD40 to inhibit OX40L expression.ConclusionsCD40+ BM-MSCs are immune regulatory elements within BM. Loss of CD40 results in uncontrolled T cell activation due to a reduced number of Tregs, and B-cell development is consequently impaired. GVHD provides an example of how a loss of CD40+ BM-MSCs and a reduction in B-cell precursors may occur in a human setting.

Download Full-text