scholarly journals Interval of IL-2 Administration Has a Major Impact on Treatment Efficacy for Regulatory T Cell Expansion and Homeostasis

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1928-1928
Author(s):  
Yuriko Kishi ◽  
Yusuke Meguri ◽  
Miki Iwamoto ◽  
Takeru Asano ◽  
Takanori Yoshioka ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Regulatory T Cell ◽  
Dose Interval ◽  
Daily Administration ◽  
Optimum Dose ◽  
Spleen Cells ◽  
Ki 67 ◽  
Significant Difference

Abstract IL-2 has a critical role in the immune homeostasis by expansion and maintenance of regulatory T cell (Treg). We previously demonstrated that low-dose IL-2 administration preferentially increased Treg in patients with active chronic GVHD and resulted in clinical improvement of the symptoms. In these years, the tolerogenic effects of IL-2 have been tested in the setting of various autoimmune-based diseases by many clinical trials. However, the schedules of IL-2 administration in each trial are different and the optimal strategy for expansion and maintenance of Treg is still unclear. To tackle this issue, we examined the impact of IL-2 dose intervals on Treg and the optimization for the induction and maintenance therapy by using murine IL-2 therapy model. CD4+CD25+Foxp3+ Treg were analyzed comparing with CD4+CD25-Foxp3- conventional CD4 T cells (Tcon) and CD8+ T cells. The expressions of Ki-67 and Bcl-2 in each subset were also examined. First, we performed the experimental allogeneic BMT, in which model 1x10E6 spleen cells (CD45.2) and 5x10E6 T cell depleted bone marrow cells (CD45.1) from C57BL/6 donors were transplanted into lethally-irradiated B6D2F1 recipients (CD45.2). To explore the optimum dose-interval for the increase of Treg from the baseline level as the induction therapy, we administered 5000 IU of IL-2 to recipients subcutaneously once (Induction-A), twice (Induction-B), four (Induction -C) or seven (Induction-D) times a week from day 35 to 49 after transplant. On day 50 after transplant, peripheral blood and spleen cells of mice from each group were harvested and CD45.1+H-2Kd- Donor bone-marrow derived lymphocytes were evaluated. The absolute number and %Treg of CD4 T cells, and the Ki-67 and Bcl-2 expression in Treg were compared with each frequency of administration. To increase Treg from baseline, the daily administration (Induction-D) provided the best Treg response among the tested groups and there was significant difference between control group and group I-D (%Treg: 10.5 % vs. 16.7 %, p<0.05). Treg proliferation was positively related to the frequency of IL-2 administration (%Ki-67+ cells: 17.7%, 17.2 %, vs. 9.9%, 9.0% and 7.8%, respectively, p<0.05). Secondly, we investigated the optimum dose-interval for the maintenance of expanded Treg level after the initial inductive IL-2 administration. We expanded Treg by daily administration of IL-2 to B6 mice for 2 weeks, thereafter we administrated IL-2 twice (Maintenance-A), four (Maintenance-B), or seven (Maintenance-C) times a week from day 14 to 21 for the maintenance. Of interest, to maintain expanded Treg level after the induction IL-2 therapy, Treg in cohorts of Maintenance-B and Maintenance-C were significantly higher than in cohort Maintenance-A (mean 9.7%, 10.2% vs 7.1%, respectively) and there was no significant differences between cohorts Maintenance-B and Maintenance-C, suggesting daily administration is not necessary and intermittent administration within the threshold may work for the maintenance of expanded Treg level after the intensive IL-2 administration. Treg proliferation of cohort Maintenance-C was significantly higher than that of the other cohorts (mean %Ki-67: 19.8 %, 20.5 % vs. 23.9 %, p<0.05). There were no significant difference in Bcl-2 expression in Treg among these cohorts but seemed negatively related to the frequency of IL-2 administration. These data suggested that daily administration of IL-2 seems to be optimal for expansion of Treg for induction therapy. In contrast, to maintain the expanded Treg, daily administration may not be required but less frequent times of administration within the threshold could be preferable. Taken together, the interval of IL-2 administration should be a major factor for Treg homeostasis as well as IL-2 dosage. Our data provide important information for developing therapeutic strategies to modulate Treg homeostasis in vivo and promote immune tolerance after transplant. Disclosures No relevant conflicts of interest to declare.

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

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2450-2450
Author(s):  
Cathrin Klingeberg ◽  
Anna Lena Illert ◽  
Nicolas Schneider ◽  
Christian Peschel ◽  
Cornelius Miething ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Large Cell ◽  
Cre Recombinase ◽  
Double Negative ◽  
Ki 67 ◽  
The Impact

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

Requirements for the promotion of allogeneic engraftment by anti-CD154 (anti-CD40L) monoclonal antibody under nonmyeloablative conditions

Blood ◽  
2001 ◽  
Vol 98 (2) ◽  
pp. 467-474 ◽  
Author(s):  
Patricia A. Taylor ◽  
Christopher J. Lees ◽  
Herman Waldmann ◽  
Randolph J. Noelle ◽  
Bruce R. Blazar
Keyword(s):  
Monoclonal Antibody ◽  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Immune Suppression ◽  
Regulatory T Cell ◽  
Cd4 Cells ◽  
Major Goal ◽  
Cd8 Cells

The promotion of alloengraftment in the absence of global immune suppression and multiorgan toxicity is a major goal of transplantation. It is demonstrated that the infusion of a single modest bone marrow dosage in 200 cGy-irradiated recipients treated with anti-CD154 (anti-CD40L) monoclonal antibody (mAb) resulted in chimerism levels of 48%. Reducing irradiation to 100 or 50 cGy permitted 24% and 10% chimerism, respectively. In contrast, pan–T-cell depletion resulted in only transient engraftment in 200 cGy-irradiated recipients. Host CD4+ cells were essential for alloengraftment as depletion of CD4+ cells abrogated engraftment in anti-CD154–treated recipients. Strikingly, the depletion of CD8+ cells did not further enhance engraftment in anti-CD154 mAb–treated recipients in a model in which rejection is mediated by both CD4+ and CD8+ T cells. However, anti-CD154 mAb did facilitate engraftment in a model in which only CD8+ T cells mediate rejection. Furthermore, CD154 deletional mice irradiated with 200 cGy irradiation were not tolerant of grafts, suggesting that engraftment promotion by anti-CD154 mAb may not simply be the result of CD154:CD40 blockade. Together, these data suggest that a CD4+regulatory T cell may be induced by anti-CD154 mAb. In contrast to anti-CD154 mAb, anti-B7 mAb did not promote donor engraftment. Additionally, the administration of either anti-CD28 mAb or anti-CD152 (anti–CTLA-4) mAb or the use of CD28 deletional recipients abrogated engraftment in anti-CD154 mAb–treated mice, suggesting that balanced CD28/CD152:B7 interactions are required for the engraftment-promoting capacity of anti-CD154 mAb. These data have important ramifications for the design of clinical nonmyeloablative regimens based on anti-CD154 mAb administration.

EXTH-63. A NOVEL MOUSE MODEL OF DIFFUSE MIDLINE GLIOMA FOR TARGETED IMMUNOTHERAPY

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi177-vi177
Author(s):  
Maggie Seblani ◽  
Markella Zannikou ◽  
Joseph Duffy ◽  
Rebecca Levine ◽  
Qianli Liu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
De Novo ◽  
Constitutive Expression ◽  
Ki 67 ◽  
Durable Response ◽  
Microvascular Proliferation ◽  
Significant Difference ◽  
De Novo Tumors

Abstract In children, diffuse midline gliomas retain poor outcomes, failing to have a durable response to conventional therapies. Immunotherapies hold promise, with the integration of the host's immune system fundamental to their design. Here, we describe a novel genetically engineered immunocompetent model that incorporates interleukin 13 receptor alpha 2 (IL13Rα2), a tumor-associated antigen, to evaluate the antitumor activity of IL13Rα2-CAR T cell and bispecific T cell engager (BiTE) therapies in preclinical studies. The RCAS-Tva delivery system was utilized to induce gliomagenesis through p53 loss and the constitutive expression of PDGFB and human IL13Rα2 in Nestin-Tva;p53fl/fl mice. Pups were injected with virus-producing DF1 cells, encoding either for RCAS-Cre and PDGFB+IL13Rα2 or RCAS-Cre and PDGFB. Kaplan-Meier survival curves established and compared tumor growth dynamics in both models. Tumor tissue was characterized through immunohistochemistry and H&E staining. Cell lines generated from tumor-bearing tissue were used for orthotopic injection and in vitro studies. Expression of PDGFB and IL13Rα2 was confirmed by flow cytometry and western blot. In both groups, de novo tumors developed without significant difference in median survival between RCAS:PDGFB (n=25, 40 days) and RCAS:PDGB+IL13Rα2 (n=32, 39 days). Tumors demonstrated characteristics of high-grade glioma such as infiltration, pseudopalisading necrosis, microvascular proliferation, high Ki-67 index, heterogenous IL13Rα2 expression, with notable presence of CD11b+ macrophages and low count of CD3+ T cells. Orthotopic tumors from developed cell lines were histologically similar to de novo tumors. Treatment of generated cell lines with IL13Rα2-targeting BiTE protein resulted in a loss of glioma cell viability and target-specific activation of T cells. Engineered de novo tumors possess histopathologic features common to diffused midline gliomas. IL13Rα2-positive cell lines derived from de novo tumors were responsive to targeted treatment, opening the opportunity for preclinical assessment of IL13Rα2-directed immunotherapies, with the potential for clinical translation.

scholarly journals Retroviral transformation in vitro of chicken T cells expressing either alpha/beta or gamma/delta T cell receptors by reticuloendotheliosis virus strain T.

1993 ◽  
Vol 177 (3) ◽  
pp. 647-656 ◽  
Author(s):  
M D Marmor ◽  
T Benatar ◽  
M J Ratcliffe
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Reticuloendotheliosis Virus ◽  
Spleen Cells ◽  
Gamma Delta ◽  
Alpha Beta ◽  
Gamma Delta T Cell

Exposure of normal juvenile chicken bone marrow cells to the replication defective avian reticuloendotheliosis virus strain T (REV-T) (chicken syncytial virus [CSV]) in vitro resulted in the generation of transformed cell lines containing T cells. The transformed T cells derived from bone marrow included cells expressing either alpha/beta or gamma/delta T cell receptors (TCRs) in proportions roughly equivalent to the proportions of TCR-alpha/beta and TCR-gamma/delta T cells found in the normal bone marrow in vivo. Essentially all TCR-alpha/beta-expressing transformed bone marrow-derived T cells expressed CD8, whereas few, if any, expressed CD4. In contrast, among TCR-gamma/delta T cells, both CD8+ and CD8- cells were derived, all of which were CD4-. Exposure of ex vivo spleen cells to REV-T(CSV) yielded transformed polyclonal cell lines containing &gt; 99% B cells. However, REV-T(CSV) infection of mitogen-activated spleen cells in vitro resulted in transformed populations containing predominantly T cells. This may be explained at least in part by in vitro activation resulting in dramatically increased levels of T cell REV-T(CSV) receptor expression. In contrast to REV-T(CSV)-transformed lines derived from normal bone marrow, transformed lines derived from activated spleen cells contained substantial numbers of CD4+ cells, all of which expressed TCR-alpha/beta. While transformed T cells derived from bone marrow were stable for extended periods of in vitro culture and were cloned from single cells, transformed T cells from activated spleen were not stable and could not be cloned. We have therefore dissociated the initial transformation of T cells with REV-T(CSV) from the requirements for long-term growth. These results provide the first demonstration of efficient in vitro transformation of chicken T lineage cells by REV-T(CSV). Since productive infection with REV-T(CSV) is not sufficient to promote long-term growth of transformed cells, these results further suggest that immortalization depends not only upon expression of the v-rel oncogene but also on intracellular factor(s) whose expression varies according to the state of T cell physiology and/or activation.

scholarly journals Long non-coding RNA MEG3 mediates the miR-149-3p/FOXP3 axis by reducing p53 ubiquitination to exert a suppressive effect on regulatory T cell differentiation and immune escape in esophageal cancer

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Qi-Rong Xu ◽  
Jian Tang ◽  
Hong-Ying Liao ◽  
Ben-Tong Yu ◽  
Xiang-Yuan He ◽  
...  
Keyword(s):  
T Cells ◽  
Esophageal Cancer ◽  
Cell Differentiation ◽  
T Cell ◽  
Regulatory T Cell ◽  
Immune Escape ◽  
T Cell Differentiation ◽  
Ki 67 ◽  
Non Coding Rna ◽  
Long Non Coding Rna

Abstract Background Long non-coding RNA (lncRNA) maternally expressed gene 3 (MEG3) has been implicated in the progression of esophageal cancer (EC). However, the specific mechanism of the involvement of MEG3 in EC development in relation to the regulation of immune escape remains uncertain. Thus, the aim of the current study was to investigate the effect of MEG3 on EC via microRNA-149-3p (miR-149-3p). Methods Gain- and loss-of-function experiments were initially performed in EC cells in addition to the establishment of a 4-nitroquinoline 1-oxide-induced EC mouse model aimed at evaluating the respective roles of forkhead box P3 (FOXP3), MEG3, miR-149-3p, mouse double minute 2 homolog (MDM2) and p53 in T cell differentiation and immune escape observed in EC. Results EC tissues were found to exhibit upregulated FOXP3 and MDM2 while MEG3, p53 and miR-149-3p were all downregulated. FOXP3 was confirmed to be a target gene of miR-149-3p with our data suggesting it reduced p53 ubiquitination and degradation by means of inhibiting MDM2. P53 was enriched in the promoter of miR-149-3p to upregulate miR-149-3p. The overexpression of MEG3, p53 or miR-149-3p or silencing FOXP3 was associated with a decline in CD25+FOXP3+CD4+ T cells, IL-10+CD4+ T cells and IL-4+CD4+ T cells in spleen tissues, IL-4, and IL-10 levels as well as C-myc, N-myc and Ki-67 expression in EC mice. Conclusion Collectively, MEG3 decreased FOXP3 expression and resulted in repressed regulatory T cell differentiation and immune escape in EC mice by upregulating miR-149-3p via MDM2-mediated p53.

scholarly journals Bone Marrow-Derived Mesenchymal Stem Cells Inhibit CD8+ t Cell Immune Responses Via PD-1/PD-L1 Pathway in Multiple Myeloma

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 53-54
Author(s):  
Zhaoyun Liu ◽  
Fu Mi ◽  
Mei Han ◽  
Mengyue Tian ◽  
Hui Liu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
T Cells ◽  
Mesenchymal Stem Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Conflicts Of Interest ◽  
Significant Difference ◽  
Normal Controls

High expression of the inhibitory receptor programmed death ligand 1 (PD-L1) on tumor cells and tumor stromal cells have been found play a key role in tumor immune evasion in several human malignancies. However, the expression of PD-L1 on bone marrow mesenchymal stem cells (BMSCs) and whether the PD-1/PD-L1 signal pathway is involved in the BMSCs versus T cell immune response in Multiple Myeloma (MM) remain poorly defined. In this study, we explored the expression of PD-L1 on BMSCs from newly diagnosed MM (NDMM) patients and the role of PD-1/PD-L1 pathway in BMSCs-mediated regulation of CD8+T cells. The data showed that the expression of PD-L1 on BMSCs in NDMM patients was significantly increased than that in normal controls (NC) (18.81±1.61% vs. 2.78±0.70 %; P&lt;0.001). Furthermore, the PD-1 expression on CD8+T cells with NDMM patients was significantly higher than that in normal controls (43.22±2.98% vs. 20.71±1.08%; P&lt;0.001). However, there was no significant difference in PD-1 expression of CD4+ T cells and NK cells between NDMM group and NC group. Additionally, the co-culture assays revealed that BMSCs significantly promoted CD8+ T cells apoptosis and suppressed CD8+ T cells function. However, PD-L1 inhibitor effectively reversed BMSCs-mediated suppression in CD8+ T cells. We also found that the combination of PD-L1 inhibitor and pomalidomide can further enhance the killing effect of CD8+ T cells on MM cells. In summary, our findings demonstrated that BMSCs in patients with MM may induce apoptosis of CD8+T cells through the PD-1/PD-L1 axis and inhibit the release of perforin and granzyme B from CD8+ T cells so as to promote the immune escape of MM. Disclosures No relevant conflicts of interest to declare.

Integrated Immunological Analysis of the Bone Marrow Tumor Microenvironment in Myeloproliferative Neoplasms to Determine Potential Efficacy of Immune Checkpoint Blockade

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2766-2766
Author(s):  
Robert Orlowski ◽  
Alexander Huang ◽  
Mercy Gohil ◽  
James Mangan ◽  
Marissa Vignali ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Tumor Microenvironment ◽  
Cd8 T Cells ◽  
Immune Checkpoint ◽  
Employment Equity ◽  
Healthy Donors ◽  
Significant Difference ◽  
Equity Ownership

Abstract BACKGROUND: Immune checkpoint blockadewith anti-PD-1/PD-L1 therapyhas demonstrated remarkable efficacy in multiple tumor types. Biomarker candidates for predicting likelihood of response to targeted immunotherapy are being actively investigated including inhibitory or activating receptors on CD8+ lymphocytes, corresponding ligands on tumor or antigen-presenting cells (APCs), T-cell functionality, and the T-cell receptor (TCR) repertoire found within a tumor microenvironment. Myelofibrosis (MF) and Chronic Myeloid Leukemia (CML) are tumors responsive to immunotherapy, most notably allogeneic transplantation (alloSCT), and donor lymphocyte infusion. Although tyrosine kinase inhibitors can improve patient outcomes, a potentially curative therapeutic option other than alloSCT is needed. PURPOSE: To determine the immune profile of the bone marrow tumor microenvironment in patients with CML and MF compared to healthy donors in order to assess the rationale and potential efficacy of novel immune checkpoint therapies. METHODS: Cryopreserved bone marrow aspirate mononuclear cells (MNCs) from healthy donors (HDs) (n=11), untreated CML (n=9) or MF (n= 12) were analyzed by flow cytometry. CD3+ CD8+ lymphocytes were divided into naïve, central memory (CM), effector memory (EM), and terminal effector (TEMRA) subsets for analysis. Expression of immune checkpoint receptors including PD-1, 4-1BB, TIM3, LAG3, and TIGIT were evaluated on each population. Known corresponding ligands including PD-L1 and PD-L2 were assessed in CML samples on blasts, plasmacytoid dendritic cells (pDCs), myeloid dendritic cells (mDCs), and monocytes. T-cell function was evaluated by cytokine production, cytotoxicity, and proliferation in CD3+ CD8+ PD1+ or PD1- populations. To assess the TCR repertoire found within the tumor microenvironment, non-naive CD8+ T-cells were sorted into PD-1+ and PD-1- populations, and then CDR3 region of the TCRB gene, together with sufficient flanking sequence to identify most V, D, and J genes was sequenced using the immunoSEQ platform from Adaptive Biotechnologies. RESULTS: There was a significant difference in the CML CD3+ CD8+ subset distribution compared to HDs with EM% increased at 60.01% vs. 41.25% (p =0.0137), and TEMRA 44.51% vs. 20.64% (p=0.0004). CM% trended downwards (32.15% to 21.58%, p=0.118) while naïve% was equivalent in CML and HDs (22.13% vs. 20.87%). The percentage of PD-1+ non-naïve CD8+ T-cells (EM, TEMRA, CM combined) was significantly increased in CML samples at 55.14% (range 31-69%) compared to HDs at 38.98% (range 34.8% to 55.5%; p=0.0050). PD-1 expression was consistently increased across all subgroups in CML (CM: 67.06% vs 53.22%, EM: 60.01% vs. 41.25%, TEMRA: 44.51% vs 20.64% p <0.05 for all). There was no statistically significant difference in CML compared to HDs for secondary receptors including TIGIT, TIM3, LAG3, or 4-1BB. Fewer than 5% of CML blasts were positive for the PD-L1 or PD-L2 ligands, however PD-L1 expression was increased on mDCs compared to HD samples (53.08% vs 24.63%; p=0.0015). In contrast to these findings in CML there was no significant proportional difference in CD8+ subsets, PD-1 status, or other receptors between MF and HDs. Anti-CD3/28 stimulation did not induce differential IFN-γ/TNF-alpha production, granzyme production, or proliferation (Ki67+) among the CD8+ PD-1+ or PD-1- T-cells from CML samples. To begin to estimate T cell clonality in the bone marrow tumor microenvironment, TCRβ sequencing of sorted non-naïve CD8- T-cells showed several clones markedly overrepresented in the diseased PD-1+ compartment. Conclusions: The CML tumor microenvironment is enriched in CD8+ T-cells expressing the inhibitory receptor PD-1 while APC subsets express increased PD-L1. This represents a potential axis of tumor driven immunosuppression amenable to immune checkpoint blockade. This is in contrast to MF, where the immunoprofile was not detectably different from healthy donors. These findings may reflect differences in tumor immunogenicity, cytokine mileu, or the APC types present. In-vivo testing using murine models for both diseases is underway to gain a better understanding of the role of immune checkpoint therapies. Disclosures Mangan: Incyte Corporation: Membership on an entity's Board of Directors or advisory committees. Vignali:Adaptive Biotechnologies: Employment, Equity Ownership. Emerson:Adaptive Biotechnologies: Employment, Equity Ownership. Robins:Adaptive Biotechnologies: Consultancy, Equity Ownership, Patents & Royalties. Yusko:Adaptive Biotechnologies: Employment, Equity Ownership.

A Calcineurin Inhibitor Does Not Affect the Post-Transplantation Cyclophosphamide -Induced Regulatory T Cell Expansion after Bone Marrow Transplantation in a Murine Chronic Gvhd Model

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3351-3351
Author(s):  
Kyosuke Saeki ◽  
Yoshinobu Maeda ◽  
Keisuke Seike ◽  
Katsuma Tani ◽  
Taiga Kuroi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Bone Marrow Transplantation ◽  
T Cell ◽  
Calcineurin Inhibitor ◽  
Chronic Gvhd ◽  
Effector T Cells ◽  
Post Transplantation ◽  
Significant Difference ◽  
Ifn Γ

Abstract Chronic graft-versus-host disease (GVHD) remains a major late complication of allogeneic hematopoietic stem cell transplantation (HSCT). Prolonged administration of a calcineurin inhibitor, such as cyclosporin A (CsA), does not decrease the risk of chronic GVHD. Indeed, we and others have reported that long-term use of CsA impairs reconstitution of bone marrow-derived regulatory T cells (Tregs) and increases susceptibility to chronic GVHD. Recently, the administration of post-transplantation cyclophosphamide (PTCY) has been used widely in GVHD prophylaxis and lower incidences of acute and chronic GVHD have been reported. Here, we evaluated the effects of PTCY and CsA on Tregs and cytokine production in effector T cells using the well-defined chronic GVHD mouse model of B10.D2 (H2d) cells into BALB/c (H2d) mice. Sublethally-irradiated recipient BALB/c mice were transplanted with 8.0 × 106 T cell-depleted bone marrow cells and 2.0 × 106 T cell-replete spleen cells from donor B10.D2 mice. PTCY was administered by intraperitoneal injection at days 3 and 4 post bone marrow transplantation (BMT). CsA was injected peritoneally once daily, starting on day 0 of BMT until the day of analysis. Administration of PTCY, with or without CsA, improved the clinical chronic GVHD score significantly at day 50 versus the controls and the CsA-alone group (controls: 2.8 ± 0.34, PTCY: 0.5 ± 0.0, p < 0.01; CsA alone: 1.8 ± 0.73, PTCY-CsA: 0.5 ± 0.0, p < 0.01). There was no significant difference in the GVHD score between the PTCY and PTCY-CsA groups. Histopathological examinations of the skin at day 50 after BMT also showed significantly reduced chronic GVHD damage in the PTCY group versus the controls and the CsA-alone group (controls vs. PTCY, p < 0.001; CsA alone vs. PTCY, p < 0.01). Flow cytometry analysis of the cells isolated from the peripheral lymph nodes 28 days after BMT revealed that the proportions of Tregs to CD4+ T cells in the PTCY (25.8 ± 2.1%) and PTCY-CsA (29.5 ± 2.2%) groups were remarkably higher than the controls (8.3 ± 2.7%) and CsA-alone (6.7 ± 1.6%) groups (p < 0.05). There was no significant difference in the Treg ratio between the PTCY and PTCY-CsA groups. Intracellular staining showed that IFN-γ single-positive and IFN-γ/IL-17 double-positive CD4 T cells were well suppressed in the PTCY and PTCY-CsA groups versus the controls and CsA-alone group (controls: 38.2 ± 3.5%, PTCY: 11.9 ± 3.1%, p < 0.01; CsA alone: 46.9 ± 2.2%, PTCY-CsA: 17.9%, p < 0.01). Our data suggest that PTCY treatment attenuated chronic GVHD via enhanced reconstitution of donor Tregs and suppression of IFN-γ/IL-17-producing effector T cells. Moreover, CsA does not appear to hinder this suppressive effect of PTCY. This result seems helpful for understanding how PTCY prevents chronic GVHD in clinical settings. Disclosures Maeda: Mundipharma KK: Research Funding.

scholarly journals SPLENIC REPLENISHMENT OF SYNERGISTIC ABILITY TO BONE MARROW AND THYMIC CELLS OF NEONATALLY SPLENECTOMIZED CBA MICE

1972 ◽  
Vol 136 (4) ◽  
pp. 761-768 ◽  
Author(s):  
R. A. Bucsi ◽  
F. Borek ◽  
J. R. Battisto
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Red Blood Cells ◽  
Cell Population ◽  
Blood Cells ◽  
Spleen Cells ◽  
Cba Mice ◽  
Adult Mice ◽  
Collaborative Capacity

Bone marrow (B) and thymic (T) cells taken from adult mice that had been splenectomized within 24 hr of birth showed an inability to cooperate in the IgM response to sheep red blood cells. The defect in collaborative capacity was apparent in both sets of cells, but appeared to be more pronounced in the T cell population. Splenectomy performed at various neonatal intervals indicated that if removal of the spleen were delayed until 6 days after birth, B and T cells of the adult showed a 60% restoration in cooperation. Replenishment of the synergistic ability after neonatal splenectomy could be achieved by injecting spleen cells immediately after spleen removal or 2 months postsplenectomy.

scholarly journals Delayed infusion of immunocompetent donor cells after bone marrow transplantation breaks graft-host tolerance allows for persistent antileukemic reactivity without severe graft-versus-host disease

Blood ◽  
1995 ◽  
Vol 85 (11) ◽  
pp. 3302-3312 ◽  
Author(s):  
BD Johnson ◽  
RL Truitt
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Bone Marrow Transplantation ◽  
T Cell ◽  
Spleen Cells ◽  
Graft Versus Host ◽  
Donor Cells ◽  
Donor T Cells ◽  
Antileukemic Effect ◽  
Host Tolerance

The development of graft-host tolerance after bone marrow transplantation (BMT) is crucial to avoid the problems of graft-versus-host disease (GVHD) and graft rejection. GVHD can be eliminated by depleting mature donor T cells from the BM inoculum, thereby facilitating the development of graft-host tolerance. However, T-cell depletion often results in an increased incidence of graft rejection and an increased frequency of leukemia relapse. Thus, although graft-host tolerance is a desirable outcome, it can pose a significant threat to leukemia-bearing hosts. Using a major histocompatability complex (MHC)-matched allogeneic model of BMT (B10.BR into AKR), we found that irradiated recipients given donor BM alone displayed mixed T-cell chimerism and did not develop GVHD. Graft-host tolerance developed by 8 weeks after BMT in these chimeras, and they were susceptible to low-dose leukemia challenge. When sufficient numbers of donor spleen cells, as a source of T-cells, were added to the BM graft, AKR hosts developed severe and lethal GVHD. Antihost reactive donor T cells persisted in chimeras undergoing GVHD, indicating that graft-host tolerance did not develop. When administration of the spleen cells was delayed for 7 to 21 days after BMT, there was significantly less mortality because of GVHD. Day 21 was the optimal time for infusion of cells without development of GVHD. Graft-host tolerance was broken by the delayed infusion of donor cells, as indicated by the persistence of antihost-reactive donor T cells in these chimeras in T-cell receptor cross-linking and mixed lymphocyte reaction assays. Importantly, the persistence of antihost-reactive donor T cells correlated with along-term antileukemic effect that was still present at 100 days after transplant. Multiple infusions of immunocompetent donor cells could be administered without increasing the risk for GVHD if delayed until 21 days post-BMT. Delayed infusions of donor spleen cells also resulted in a long-term antileukemic effect in the absence of GVHD in an MHC-haplotype-mismatched model of BMT (SJL into [SJL x AKR]F1). Although delayed infusion of normal donor cells did not induce GVHD, spleen cells from donors previously sensitized to host alloantigens induced GVHD when infused 21 days after BMT. Thus, the ability of previously activated cells to induce GVHD was not inhibited in the same manner as naive cells. Results from limiting dilution analysis assays indicated that alloactivated interleukin-2-secreting CD4+ T cells were preferentially inhibited over cytolytic T cells.(ABSTRACT TRUNCATED AT 400 WORDS)

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