Publications by authors named "Antonio Pierini"

44 Publications

Novel Immune Cell-Based Therapies to Eradicate High-Risk Acute Myeloid Leukemia.

Front Immunol 2021 3;12:695051. Epub 2021 Aug 3.

Institute of Hematology and Centre of Haemato-Oncology Research (CREO), University and Hospital of Perugia, Perugia, Italy.

Adverse genetic risk acute myeloid leukemia (AML) includes a wide range of clinical-pathological entities with extremely poor outcomes; thus, novel therapeutic approaches are needed. Promising results achieved by engineered chimeric antigen receptor (CAR) T cells in other blood neoplasms have paved the way for the development of immune cell-based therapies for adverse genetic risk AML. Among these, adoptive cell immunotherapies with single/multiple CAR-T cells, CAR-natural killer (NK) cells, cytokine-induced killer cells (CIK), and NK cells are subjects of ongoing clinical trials. On the other hand, allogeneic hematopoietic stem cell transplantation (allo-HSCT) still represents the only curative option for adverse genetic risk AML patients. Unfortunately, high relapse rates (above 50%) and associated dismal outcomes (reported survival ~10-20%) even question the role of current allo-HSCT protocols and emphasize the urgency of adopting novel effective transplant strategies. We have recently demonstrated that haploidentical allo-HSCT combined with regulatory and conventional T cells adoptive immunotherapy (Treg-Tcon haplo-HSCT) is able to overcome disease-intrinsic chemoresistance, prevent leukemia-relapse, and improve survival of adverse genetic risk AML patients. In this , we briefly review the recent advancements with immune cell-based strategies against adverse genetic risk AML and discuss how such approaches could favorably impact on patients' outcomes.
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http://dx.doi.org/10.3389/fimmu.2021.695051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8368440PMC
August 2021

Rifaximin use favoured micafungin-resistant Candida spp. infections in recipients of allogeneic hematopoietic cell transplantation.

Ann Hematol 2021 Sep 28;100(9):2375-2380. Epub 2021 Jun 28.

Division of Hematology and Clinical Immunology, University of Perugia, Perugia, Italy.

Damage to gut mucosa following conditioning regimens may favour bacterial infections that can trigger graft versus host disease (GvHD) in patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT). Rifaximin, an oral and non-absorbable antibiotic, has been recently proposed as effective prophylaxis to reduce bacterial infections in the gut and consequently acute GvHD in this setting. The present study evaluated safety and outcomes of HSCT patients that were treated with rifaximin prophylaxis at Perugia University Hospital. Rifaximin prophylaxis was introduced as standard of care in HSCT patients in May 2018. We retrieved data from 118 consecutive transplants, and we compared the outcomes of rifaximin-treated patients with historical controls that did not receive antibiotic prophylaxis. While incidences of neutropenic fever, documented bacterial infections, and aGvHD were similar in the two groups, we found an increased frequency of invasive candidiasis and clinically relevant Candida spp. infections in rifaximin-treated patients (5 patients vs 1 patient, 25% [± 0.99%] vs 1% [± 0.01%], p < .0001). Three rifaximin-treated patients experienced life-threating candidemia (2 C. krusei, 1 C. orthopsilosis). Rifaximin was the only factor that increased the risk of Candida spp. infections. Rifaximin could have contributed to microbiome disruption which favoured an outbreak of life-threatening Candida infections. This important complication forced us to halt its use. Larger, prospective studies are needed to assess the impact of rifaximin prophylaxis on incidence of bacterial infections, aGvHD, and survival of HSCT patients.
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http://dx.doi.org/10.1007/s00277-021-04569-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8357665PMC
September 2021

First Multimodal, Three-Dimensional, Image-Guided Total Marrow Irradiation Model for Preclinical Bone Marrow Transplantation Studies.

Int J Radiat Oncol Biol Phys 2021 Jun 11. Epub 2021 Jun 11.

Department of Radiation Oncology, City of Hope Medical Center, Duarte, California; Beckman Research Institute of City of Hope, Duarte, California; Department of Radiation Oncology, University of Minnesota, Minneapolis, Minnesota. Electronic address:

Purpose: Total marrow irradiation (TMI) has significantly advanced radiation conditioning for hematopoietic cell transplantation in hematologic malignancies by reducing conditioning-induced toxicities and improving survival outcomes in relapsed/refractory patients. However, the relapse rate remains high, and the lack of a preclinical TMI model has hindered scientific advancements. To accelerate TMI translation to the clinic, we developed a TMI delivery system in preclinical models.

Methods And Materials: A Precision X-RAD SmART irradiator was used for TMI model development. Images acquired with whole-body contrast-enhanced computed tomography (CT) were used to reconstruct and delineate targets and vital organs for each mouse. Multiple beam and CT-guided Monte Carlo-based plans were performed to optimize doses to the targets and to vary doses to the vital organs. Long-term engraftment and reconstitution potential were evaluated by a congenic bone marrow transplantation (BMT) model and serial secondary BMT, respectively. Donor cell engraftment was measured using noninvasive bioluminescence imaging and flow cytometry.

Results: Multimodal imaging enabled identification of targets (skeleton and spleen) and vital organs (eg, lungs, gut, liver). In contrast to total body irradiation (TBI), TMI treatment allowed variation of radiation dose exposure to organs relative to the target dose. Dose reduction mirrored that in clinical TMI studies. Similar to TBI, mice treated with different TMI regimens showed full long-term donor engraftment in primary BMT and second serial BMT. The TBI-treated mice showed acute gut damage, which was minimized in mice treated with TMI.

Conclusions: A novel multimodal image guided preclinical TMI model is reported here. TMI conditioning maintained long-term engraftment with reconstitution potential and reduced organ damage. Therefore, this TMI model provides a unique opportunity to study the therapeutic benefit of reduced organ damage and BM dose escalation to optimize treatment regimens in BMT and hematologic malignancies.
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http://dx.doi.org/10.1016/j.ijrobp.2021.06.001DOI Listing
June 2021

Haploidentical age-adapted myeloablative transplant and regulatory and effector T cells for acute myeloid leukemia.

Blood Adv 2021 03;5(5):1199-1208

Division of Hematology and Clinical Immunology, Department of Medicine, University of Perugia, Perugia, Italy.

Allogeneic hematopoietic stem cell transplantation (HSCT) is the most effective treatment in eradicating high-risk acute myeloid leukemia (AML). Here, we present data from a novel HLA-haploidentical HSCT protocol that addressed the 2 remaining major unmet medical needs: leukemia relapse and chronic graft-versus-host disease (cGVHD). Fifty AML patients were enrolled in the study. The conditioning regimen included total body irradiation for patients up to age 50 years and total marrow/lymphoid irradiation for patients age 51 to 65 years. Irradiation was followed by thiotepa, fludarabine, and cyclophosphamide. Patients received an infusion of 2 × 106/kg donor regulatory T cells on day -4 followed by 1 × 106/kg donor conventional T cells on day -1 and a mean of 10.7 × 106 ± 3.4 × 106/kgpurified CD34+ hematopoietic progenitor cells on day 0. No pharmacological GVHD prophylaxis was administered posttransplantation. Patients achieved full donor-type engraftment. Fifteen patients developed grade ≥2 acute GVHD (aGVHD). Twelve of the 15 patients with aGVHD were alive and no longer receiving immunosuppressive therapy. Moderate/severe cGVHD occurred in only 1 patient. Nonrelapse mortality occurred in 10 patients. Only 2 patients relapsed. Consequently, at a median follow-up of 29 months, the probability of moderate/severe cGVHD/relapse-free survival was 75% (95% confidence interval, 71%-78%). A novel HLA-haploidentical HSCT strategy that combines an age-adapted myeloablative conditioning regimen with regulatory and conventional T-cell adoptive immunotherapy resulted in an unprecedented cGVHD/relapse-free survival rate in 50 AML patients with a median age of 53 years. This trial was registered with the Umbria Region Institutional Review Board Public Registry as identification code 02/14 and public registry #2384/14 and at www.clinicaltrials.gov as #NCT03977103.
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http://dx.doi.org/10.1182/bloodadvances.2020003739DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7948281PMC
March 2021

Infusion of Host-Derived Unlicensed NK Cells Improves Donor Engraftment in Non-Myeloablative Allogeneic Hematopoietic Cell Transplantation.

Front Immunol 2020 7;11:614250. Epub 2021 Jan 7.

Blood and Marrow Transplantation, Stanford University School of Medicine, Stanford, CA, United States.

Allogeneic hematopoietic cell transplantation (allo-HCT) is an efficacious and frequently the only treatment option for some hematological malignances. However, it often faces severe morbidities and/or mortalities due to graft host disease, and the severity of the conditioning regiment needed, that result in toxicity-related issues poorly tolerable for some patients. These shortcomings have led to the development of less aggressive alternatives like non-myeloablative (NMAC) or reduced-intensity conditioning regiments (RIC). However, these approaches tend to have an increase of cancer relapse and limited persistence of donor-specific chimerism. Thus, strategies that lead towards an accelerated and more durable donor engraftment are still needed. Here, we took advantage of the ability of host-derived unlicensed NK (UnLicNK) cells to favor donor cell engraftment during myeloablative allo-HCT, and evaluated if the adoptive transfer of this cell type can improve donor chimerism in NAMC settings. Indeed, the infusion of these cells significantly increased mixed chimerism in a sublethal allo-HCT mouse model, resulting in a more sustainable donor cell engraftment when compared to the administration of licensed NK cells or HCT controls. We observed an overall increase in the total number and proportion of donor B, NK and myeloid cells after UnLicNK cell infusion. Additionally, the extension and durability of donor chimerism was similar to the one obtained after the tolerogenic Tregs infusion. These results serve as the needed bases for the implementation of the adoptive transfer of UnLicNK cells to upgrade NMAC protocols and enhance allogeneic engraftment during HCT.
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http://dx.doi.org/10.3389/fimmu.2020.614250DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7817981PMC
July 2021

Clinical-Grade Expanded Regulatory T Cells Are Enriched with Highly Suppressive Cells Producing IL-10, Granzyme B, and IL-35.

Biol Blood Marrow Transplant 2020 12 19;26(12):2204-2210. Epub 2020 Sep 19.

Department of Medicine and Aging Sciences, University of Chieti-Pescara, Pescara, Italy; Department of Oncology Hematology, Pescara Hospital, Pescara, Italy. Electronic address:

In the setting of T cell-depleted, full-haplotype mismatched transplantation, adoptive immunotherapy with regulatory T cells (Tregs) and conventional T cells (Tcons) can prevent graft-versus-host disease (GVHD) and improve post-transplantation immunologic reconstitution and is associated with a powerful graft-versus-leukemia effect. To improve the purity and the quantity of the infused Tregs, good manufacturing practices (GMP)-compatible expansion protocols are needed. Here we expanded Tregs using an automated, clinical-grade protocol. Cells were extensively characterized in vitro, and their efficiency was tested in vivo in a mouse model. Tregs were selected by CliniMacs (CD4CD25, 94.5 ± 6.3%; FoxP3, 63.7 ± 11.5%; CD127, 20 ± 3%; suppressive activity, 60 ± 7%), and an aliquot of 100 × 10 was expanded for 14 days using the CliniMACS Prodigy System, obtaining 684 ± 279 × 10 cells (CD4CD25, 99.6 ± 0.2%; FoxP3, 82 ± 8%; CD127, 1.1 ± 0.8%; suppressive activity, 75 ± 12%). CD39 and CTLA4 expression levels increased from 22.4 ± 12% to 58.1 ± 13.3% (P < .05) and from 20.4 ± 6.7% to 85.4 ± 9.8% (P < .01), respectively. TIM3 levels increased from .4 ± .05% to 29 ± 16% (P < .05). Memory Tregs were the prevalent population, whereas naive Tregs almost disappeared at the end of the culture. mRNA analysis displayed significant increases in CD39, IL-10, granzyme B, and IL-35 levels at the end of culture period (P < .05). Conversely, IFNγ expression decreased significantly by day +14. Expanded Tregs were sorted according to TIM3, CD39, and CD62L expression levels (purity >95%). When sorted populations were analyzed, TIM3 cells showed significant increases in IL-10 and granzyme B (P < .01) .When expanded Tregs were infused in an NSG murine model, mice that received Tcons only died of GVHD, whereas mice that received both Tcons and Tregs survived without GVHD. GMP grade expanded cells that display phenotypic and functional Treg characteristics can be obtained using a fully automated system. Treg suppression is mediated by multiple overlapping mechanisms (eg, CTLA-4, CD39, IL-10, IL-35, TGF-β, granzyme B). TIM3 cells emerge as a potentially highly suppressive population. © 2020 American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc.
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http://dx.doi.org/10.1016/j.bbmt.2020.08.034DOI Listing
December 2020

Editorial: The Immunobiology of HLA-Haploidentical Hematopoietic Cell Transplantation.

Front Immunol 2020 20;11:1031. Epub 2020 May 20.

Hematology and Clinical Immunology and Bone Marrow Transplant Program, Department of Medicine, University of Perugia, Perugia, Italy.

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http://dx.doi.org/10.3389/fimmu.2020.01031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7251076PMC
April 2021

Indirect Impact of PD-1/PD-L1 Blockade on a Murine Model of NK Cell Exhaustion.

Front Immunol 2020 11;11. Epub 2020 Feb 11.

Blood and Marrow Transplantation, Stanford University School of Medicine, Stanford, CA, United States.

The induction of exhaustion on effector immune cells is an important limiting factor for cancer immunotherapy efficacy as these cells undergo a hierarchical loss of proliferation and cytolytic activity due to chronic stimulation. Targeting PD-1 has shown unprecedented clinical benefits for many cancers, which have been attributed to the prevention of immune suppression and exhaustion with enhanced anti-tumor responses. In this study, we sought to evaluate the role of the PD-1/PD-L1 pathway in murine natural killer (NK) cell activation, function, and exhaustion. In an IL-2-dependent exhaustion mouse model, neutralization of the PD-1/PD-L1 pathway improved NK cell activation after chronic stimulation when compared to control-treated mice. These cells displayed higher proliferative capabilities and enhanced granzyme B production. However, the blockade of these molecules during long-term IL-2 stimulation did not alter the progression of NK cell exhaustion (NCE), suggesting an indirect involvement of PD-1/PD-L1 on NCE. Given the expansion of CD8 T cells and regulatory T cells (Tregs) observed upon acute and chronic stimulation with IL-2, either of these two populations could influence NK cell homeostasis after PD-L1/PD-1 therapy. Importantly, CD8 T cell activation and functional phenotype were indeed enhanced by PD-1/PD-L1 therapy, particularly with anti-PD-1 treatment that resulted in the highest upregulation of CD25 during chronic stimulation and granted an advantage for IL-2 over NK cells. These results indicate a competition for resources between NK and CD8 T cells that arguably delays the onset of NCE rather than improving its activation during chronic stimulation. Supporting this notion, the depletion of CD8 T cells reversed the benefits of PD-1 therapy on chronically stimulated NK cells. These data suggest a bystander effect of anti-PD1 on NK cells, resulting from the global competition that exists between NK and CD8 T cells for IL-2 as a key regulator of these cells' activation. Thus, achieving an equilibrium between these immune cells might be important to accomplish long-term efficacy during anti-PD-1/IL-2 therapy.
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http://dx.doi.org/10.3389/fimmu.2020.00007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026672PMC
February 2021

CD4FOXP3 Regulatory T Cell Therapies in HLA Haploidentical Hematopoietic Transplantation.

Front Immunol 2019 17;10:2901. Epub 2019 Dec 17.

Hematology and Clinical Immunology and Bone Marrow Transplant Program, Department of Medicine, University of Perugia, Perugia, Italy.

Since their discovery CD4FOXP3 regulatory T cells (Tregs) represented a promising tool to induce tolerance in allogeneic hematopoietic cell transplantation. Preclinical models proved that adoptive transfer of Tregs or the use of compounds that can favor their function are effective for prevention and treatment of graft-vs.-host disease (GvHD). Following these findings, Treg-based therapies have been employed in clinical trials. Adoptive immunotherapy with Tregs effectively prevents GvHD induced by alloreactive T cells in the setting of one HLA haplotype mismatched hematopoietic transplantation. The absence of post transplant pharmacologic immunosuppression unleashes T-cell mediated graft-vs.-tumor (GvT) effect, which results in an unprecedented, almost complete control of leukemia relapse in this setting. In the present review, we will report preclinical studies and clinical trials that demonstrate Treg ability to promote donor engraftment, protect from GvHD and improve GvT effect. We will also discuss new strategies to further enhance efficacy of Treg-based therapies.
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http://dx.doi.org/10.3389/fimmu.2019.02901DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6927932PMC
November 2020

The Evolution of T Cell Depleted Haploidentical Transplantation.

Front Immunol 2019 27;10:2769. Epub 2019 Nov 27.

Division of Hematology and Clinical Immunology, Department of Medicine, University of Perugia, Perugia, Italy.

Work on bone marrow transplantation from haploidentical donor has been proceeding for over 20 years all over the world and new transplant procedures have been developed. To control both graft rejection and graft vs. host disease, some centers have preferred to enhance the intensity of the conditioning regimens and the post-transplant immune suppression in the absence of graft manipulation; others have concentrated on manipulating the graft in the absence of any additional post-transplant immune suppressive agent. Due to the current high engraftment rates, the low incidence of graft-vs.-host disease and regimen related mortality, transplantation from haploidentical donors have been progressively offered even to elderly patients. Overall, survivals compare favorably with reports on transplants from unrelated donors. Further improvements will come with successful implementation of strategies to enhance post-transplant immune reconstitution and to prevent leukemia relapse.
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http://dx.doi.org/10.3389/fimmu.2019.02769DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6890606PMC
November 2020

CMV Management with Specific Immunoglobulins: A Multicentric Retrospective Analysis on 92 Allotransplanted Patients.

Mediterr J Hematol Infect Dis 2019 1;11(1):e2019048. Epub 2019 Sep 1.

IRCCS San Raffaele Scientific Institute, Milano, Italy, Hematology and Bone Marrow Transplantation Unit.

CMV represents one of the most severe life-threatening complications of allogeneic stem cell transplantation (allo-SCT). Pre-emptive treatment is highly effective, but toxicity and repetitive reactivation of CMV represent a significant challenge in the clinical practice. The use of anti-CMV specific immunoglobulins (Megalotect) is controversial. We retrospectively collected data on 92 patients submitted to allo-SCT for hematological malignancies, in whom Megalotect was used either for prophylaxis (n=14) or with pre-emptive therapy, together with an anti-CMV specific drug (n=78). All the patients were considered at high-risk, due to the presence of at least one risk factor for CMV reactivation. The treatment was well tolerated, with no reported infusion reactions, nor other adverse events, none of the 14 cases treated with Megalotect as prophylaxis developed CMV reactivation. 51/78 (65%) patients who received Megalotect during pre-emptive treatment achieved complete clearance of CMV viremia, and 14/51 patients (29%) developed a breakthrough CMV infection. 7/78 patients (9%) developed CMV disease. The projected 1-year OS, 1-year TRM, and 1-year RR is 74%, 15%, and 19%, respectively. No differences were observed in terms of OS, TRM, and RR by comparing patients who achieved a complete response after treatment versus those who did not. These retrospective data suggest that Megalotect is safe and well-tolerated. When used as prophylaxis, no CMV reactivation was recorded. Further prospective trials are warranted to identify the best set of patients who can benefit from Megalotect alone or in addition to anti-CMV specific drugs.
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http://dx.doi.org/10.4084/MJHID.2019.048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6736170PMC
September 2019

The "ultimate" haploidentical transplantation for the elderly with high-risk acute myeloid leukemia.

Bone Marrow Transplant 2019 08;54(Suppl 2):803-805

Hematopoietic Stem Cell Transplantation Program, Division of Hematology and Clinical Immunology, Department of Medicine, University of Perugia, Perugia, Italy.

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http://dx.doi.org/10.1038/s41409-019-0618-xDOI Listing
August 2019

T cell depletion and no post transplant immune suppression allow separation of graft versus leukemia from graft versus host disease.

Bone Marrow Transplant 2019 08;54(Suppl 2):775-779

Blood and Marrow Transplantation Program, Division of Hematology and Clinical Immunology, Department of Medicine, University of Perugia, Perugia, Italy.

Allogeneic hematopoietic cell transplantation from a human leukocyte antigen (HLA) haplotype mismatched donor (haploidentical transplantation) was not feasible for the treatment of hematologic malignancies until the early 1990s, due to the high risk of rejection and graft-versus-host disease (GVHD). The first successful protocol of haploidentical transplantation was based on a highly myeloablative and immunosuppressive conditioning regimen and the infusion of a "mega-dose" of T-cell-depleted hematopoietic stem cells. More than 90% of patients engrafted and <10% developed GVHD. The protocol did not include post-transplant immunosuppression, which favored the graft-versus-tumor effect mediated by alloreactive NK cells and residual alloreactive T cells. However, donor post-transplant immune reconstitution was slow with a high risk of infection-related mortality. More recently, T-cell-depleted haploidentical transplantation has become the platform for innovative cell therapies that aim to enhance T-cell immunity while preventing adverse reactions against host tissues. One strategy is adoptive immunotherapy with conventional T cells and regulatory T cells. Preclinical studies and clinical trials have proven that regulatory T cells control GVHD caused by co-infused conventional T cells while the graft-versus-tumor effect is retained. The use of regulatory T cells in the absence of any other form of immune suppression allowed for a conventional T cell-mediated full eradication of disease in the vast majority of high-risk acute leukemia patients.
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http://dx.doi.org/10.1038/s41409-019-0597-yDOI Listing
August 2019

Multi-institutional evaluation of MVCT guided patient registration and dosimetric precision in total marrow irradiation: A global health initiative by the international consortium of total marrow irradiation.

Radiother Oncol 2019 12 14;141:275-282. Epub 2019 Aug 14.

Department of Radiation Oncology, Beckman Research Institute, City of Hope, Duarte, USA. Electronic address:

Purpose: Total marrow irradiation (TMI) is a highly conformal treatment of the human skeleton structure requiring a high degree of precision and accuracy for treatment delivery. Although many centers worldwide initiated clinical studies using TMI, currently there is no standard for pretreatment patient setup. To this end, the accuracy of different patient setups was measured using pretreatment imaging. Their impact on dose delivery was assessed for multiple institutions.

Methods And Materials: Whole body imaging (WBI) or partial body imaging (PBI) was performed using pretreatment megavoltage computed tomography (MVCT) in a helical Tomotherapy machine. Rigid registration of MVCT and planning kilovoltage computed tomography images were performed to measure setup error and its effect on dose distribution. The entire skeleton was considered the planning target volume (PTV) with five sub regions: head/neck (HN), spine, shoulder and clavicle (SC), and one avoidance structure, the lungs. Sixty-eight total patients (>300 images) across six institutions were analyzed.

Results: Patient setup techniques differed between centers, creating variations in dose delivery. Registration accuracy varied by anatomical region and by imaging technique, with the lowest to the highest degree of pretreatment rigid shifts in the following order: spine, pelvis, HN, SC, and lungs. Mean fractional dose was affected in regions of high registration mismatch, in particular the lungs.

Conclusions: MVCT imaging and whole body patient immobilization was essential for assessing treatment setup, allowing for the complete analysis of 3D dose distribution in the PTV and lungs (or avoidance structures).
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http://dx.doi.org/10.1016/j.radonc.2019.07.010DOI Listing
December 2019

Regulation of murine NK cell exhaustion through the activation of the DNA damage repair pathway.

JCI Insight 2019 06 18;5. Epub 2019 Jun 18.

Blood and Marrow Transplantation, Stanford University School of Medicine, Stanford, California, USA.

NK cell exhaustion (NCE) due to sustained proliferation results in impaired NK cell function with loss of cytokine production and lytic activity. Using murine models of chronic NK cell stimulation, we have identified a phenotypic signature of NCE characterized by up-regulation of the terminal differentiation marker KLRG1 and by down-regulation of eomesodermin and the activating receptor NKG2D. Chronic stimulation of mice lacking NKG2D resulted in minimized NCE compared to control mice, thus identifying NKG2D as a crucial mediator of NCE. NKG2D internalization and downregulations on NK cells has been previously observed in the presence of tumor cells with high expression of NKG2D ligands (NKG2DL) due to the activation of the DNA damage repair pathways. Interestingly, our study revealed that during NK cell activation there is an increase of MULT1, and NKG2DL, that correlates with an induction of DNA damage. Treatment with the ATM DNA damage repair pathway inhibitor KU55933 (KU) during activation reduced NCE by improving expression of activation markers and genes involved in cell survival, by sustaining NKG2D expression and by preserving cell functionality. Importantly, NK cells expanded ex vivo in the presence of KU displayed increased anti-tumor efficacy in both NKG2D-dependent and -independent mouse models. Collectively, these data demonstrate that NCE is caused by DNA damage and regulated, at least in part, by NKG2D. Further, the prevention of NCE is a promising strategy to improve NK cell-based immunotherapy.
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http://dx.doi.org/10.1172/jci.insight.127729DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6675585PMC
June 2019

TNFR2 signaling modulates immunity after allogeneic hematopoietic cell transplantation.

Cytokine Growth Factor Rev 2019 06 15;47:54-61. Epub 2019 May 15.

Hematology and Clinical Immunology and Bone Marrow Transplant Program, Department of Medicine, University of Perugia, Perugia, 06132, Italy. Electronic address:

Tumor necrosis factor-α (TNF-α) signaling through TNF receptor 2 (TNFR2) plays a complex immune regulatory role in allogeneic hematopoietic cell transplantation (HCT). TNF-α is rapidly released in the circulation after the conditioning regimen with chemotherapy and/or radiotherapy. It activates the function of donor alloreactive T cells and donor Natural Killer cells and promotes graft versus tumor effects. However, donor alloreactive T cells also attack host tissues and cause graft versus host disease (GVHD), a life-threatening complication of HCT. Indeed, anti-TNF-α therapy has been used to treat steroid-refractory GVHD. Recent studies have highlighted another role for TNFR2 signaling, as it enhances the function of immune cells with suppressive properties, in particular CD4Foxp3 regulatory T cells (Tregs). Various clinical trials are employing Treg-based treatments to prevent or treat GVHD. The present review will discuss the effects of TNFR2 signaling in the setting of allogeneic HCT, the implications for the use of anti-TNF-α therapy to treat GVHD and the clinical perspectives of strategies that specifically target this pathway.
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http://dx.doi.org/10.1016/j.cytogfr.2019.05.001DOI Listing
June 2019

The Effect of TNF-α on Regulatory T Cell Function in Graft-versus-Host Disease.

Front Immunol 2018 28;9:356. Epub 2018 Feb 28.

Hematology and Clinical Immunology and Bone Marrow Transplant Program, Department of Medicine, University of Perugia, Perugia, Italy.

FoxP3 regulatory T cells (Tregs) are a subset of CD4 T cells that can suppress proliferation and effector functions of T cells, B cells, NK cells, and antigen-presenting cells. Treg deficiency causes dramatic immunologic disease in both animal models and humans. As they are capable to suppress the function and the proliferation of conventional CD4 and CD8 T cells, Treg-based cell therapies are under evaluation for the treatment of various autoimmune diseases and are currently employed to prevent graft-versus-host disease (GvHD) in clinical trials of hematopoietic stem cell transplantation. Even though tumor necrosis factor-α (TNF-α) is well known for its pro-inflammatory role, recent studies show that it promotes Treg activation and suppressive function. In the present review, we discuss the role of TNF-α in Treg function and the possible implications on the actual treatments for immune-mediated diseases, with a particular attention to GvHD.
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http://dx.doi.org/10.3389/fimmu.2018.00356DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5835761PMC
April 2019

T cells expressing chimeric antigen receptor promote immune tolerance.

JCI Insight 2017 10 19;2(20). Epub 2017 Oct 19.

Division of Blood and Marrow Transplantation, Stanford University School of Medicine, Stanford, California, USA.

Cellular therapies based on permanent genetic modification of conventional T cells have emerged as a promising strategy for cancer. However, it remains unknown if modification of T cell subsets, such as Tregs, could be useful in other settings, such as allograft transplantation. Here, we use a modular system based on a chimeric antigen receptor (CAR) that binds covalently modified mAbs to control Treg activation in vivo. Transient expression of this mAb-directed CAR (mAbCAR) in Tregs permitted Treg targeting to specific tissue sites and mitigated allograft responses, such as graft-versus-host disease. mAbCAR Tregs targeted to MHC class I proteins on allografts prolonged islet allograft survival and also prolonged the survival of secondary skin grafts specifically matched to the original islet allograft. Thus, transient genetic modification to produce mAbCAR T cells led to durable immune modulation, suggesting therapeutic targeting strategies for controlling alloreactivity in settings such as organ or tissue transplantation.
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http://dx.doi.org/10.1172/jci.insight.92865DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5846896PMC
October 2017

Clinical-Grade-Expanded Regulatory T Cells Prevent Graft-versus-Host Disease While Allowing a Powerful T Cell-Dependent Graft-versus-Leukemia Effect in Murine Models.

Biol Blood Marrow Transplant 2017 Nov 17;23(11):1847-1851. Epub 2017 Jul 17.

Department of Hematology, Transfusion Medicine and Biotechnologies, Ospedale Civile, Pescara, Italy; Department of Medicine and Aging Sciences, University of Chieti-Pescara, Italy. Electronic address:

We developed a good manufacturing practices-compatible expansion protocol to improve number and purity of regulatory T cells (Tregs) available for clinical trials. Six clinical-grade separation procedures were performed, followed by expansion with high-dose interleukin (IL)-2, anti-CD3/anti-CD28 TCR stimulation, and rapamycin for 19 days achieving a median of 8.5-fold (range, 6.25 to 13.7) expansion. FOXP3 expression was stably maintained over the culture period, while the percentage of CD127 was significantly reduced. The in vitro suppression assay showed a strong Mixed Lymphocytes Reaction inhibition. In vitro amplification did not induce any karyotypic modification. To evaluate the graft-versus-host disease (GVHD)/graft-versus-leukemia (GVL) bifunctional axis, expanded Tregs and conventional T cells (Tcons) were tested in NOD/SCID/IL2Rgnull mice injected with primary acute myeloid leukemia (AML) cells, AML cell line, acute lymphoid leukemia Philadelphia cell line, or Burkitt-like lymphoma cell line. All mice that received leukemia cells together with expanded Tregs and Tcons were rescued from leukemia and survived without GVHD, showing that Treg expansion procedure did not compromise GVHD control and the strong Tcon-mediated GVL activity. This report might represent the basis for treating high-risk leukemia and/or relapsed/refractory leukemia patients with high-dose Treg/Tcons.
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http://dx.doi.org/10.1016/j.bbmt.2017.07.009DOI Listing
November 2017

Foxp3 regulatory T cells maintain the bone marrow microenvironment for B cell lymphopoiesis.

Nat Commun 2017 05 9;8:15068. Epub 2017 May 9.

Department of Medicine, Division of Blood and Marrow Transplantation, Stanford University, 269W. Campus Drive, Stanford, California 94305, USA.

Foxp3 regulatory T cells (Treg cells) modulate the immune system and maintain self-tolerance, but whether they affect haematopoiesis or haematopoietic stem cell (HSC)-mediated reconstitution after transplantation is unclear. Here we show that B-cell lymphopoiesis is impaired in Treg-depleted mice, yet this reduced B-cell lymphopoiesis is rescued by adoptive transfer of affected HSCs or bone marrow cells into Treg-competent recipients. B-cell reconstitution is abrogated in both syngeneic and allogeneic transplantation using Treg-depleted mice as recipients. Treg cells can control physiological IL-7 production that is indispensable for normal B-cell lymphopoiesis and is mainly sustained by a subpopulation of ICAM1 perivascular stromal cells. Our study demonstrates that Treg cells are important for B-cell differentiation from HSCs by maintaining immunological homoeostasis in the bone marrow microenvironment, both in physiological conditions and after bone marrow transplantation.
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http://dx.doi.org/10.1038/ncomms15068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5436085PMC
May 2017

DR3 signaling modulates the function of Foxp3+ regulatory T cells and the severity of acute graft-versus-host disease.

Blood 2016 12 19;128(24):2846-2858. Epub 2016 Oct 19.

Division of Blood and Marrow Transplantation, Stanford University, Stanford, CA.

CD4Foxp3 regulatory T cells (Treg) are a subpopulation of T cells, which regulate the immune system and enhance immune tolerance after transplantation. Donor-derived Treg prevent the development of lethal acute graft-versus-host disease (GVHD) in murine models of allogeneic hematopoietic stem cell transplantation. We recently demonstrated that a single treatment of the agonistic antibody to DR3 (death receptor 3, αDR3) to donor mice resulted in the expansion of donor-derived Treg and prevented acute GVHD, although the precise role of DR3 signaling in GVHD has not been elucidated. In this study, we comprehensively analyzed the immunophenotype of Treg after DR3 signal activation, demonstrating that DR3-activated Treg (DR3-Treg) had an activated/mature phenotype. Furthermore, the CD25Foxp3 subpopulation in DR3-Treg showed stronger suppressive effects in vivo. Prophylactic treatment of αDR3 to recipient mice expanded recipient-derived Treg and reduced the severity of GVHD, whereas DR3 activation in mice with ongoing GVHD further promoted donor T-cell activation/proliferation. These data suggest that the function of DR3 signaling was highly dependent on the activation status of the T cells. In conclusion, our data demonstrated that DR3 signaling affects the function of Treg and T-cell activation after alloantigen exposure in a time-dependent manner. These observations provide important information for future clinical testing using human DR3 signal modulation and highlight the critical effect of the state of T-cell activation on clinical outcomes after activation of DR3.
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http://dx.doi.org/10.1182/blood-2016-06-723783DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5159706PMC
December 2016

TNF-α priming enhances CD4+FoxP3+ regulatory T-cell suppressive function in murine GVHD prevention and treatment.

Blood 2016 08 30;128(6):866-71. Epub 2016 Jun 30.

Division of Blood and Marrow Transplantation, Department of Medicine, Stanford University School of Medicine, Stanford, CA; and.

CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) have been shown to effectively prevent graft-versus-host disease (GVHD) when adoptively transferred in murine models of hematopoietic cell transplantation and in phase 1/2 clinical trials. Critical limitations to Treg clinical application are the paucity of cells and limited knowledge of the mechanisms of in vivo function. We hypothesized that inflammatory conditions in GVHD modify Treg characteristics and activity. We found that peripheral blood of recipient animals during acute GVHD (aGVHD) induces Treg activation and enhances their function. The serum contains high levels of tumor necrosis factor-α (TNF-α) that selectively activates Tregs without impacting CD4(+)FoxP3(-) T cells. TNF-α priming induces Treg in vivo proliferation, whereas it limits the ability of CD4 and CD8 conventional T cells (Tcons) to proliferate and induce GVHD. TNF-α-primed Tregs prolong animal survival as compared with unprimed Tregs when used at an unfavorable Treg:Tcon ratio, demonstrating enhanced in vivo efficacy of TNF-α-primed Tregs. Because TNF-α is produced by several immune cells during inflammation, our work elucidates aspects of the physiologic mechanisms of Treg function. Furthermore, TNF-α priming of Tregs provides a new tool to optimize Treg cellular therapies for GVHD prevention and treatment.
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http://dx.doi.org/10.1182/blood-2016-04-711275DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4982455PMC
August 2016

New mechanism of lymphoma-induced bone marrow aplasia.

Ann Hematol 2016 May 9;95(6):1013-5. Epub 2016 Mar 9.

Division of Hematology and Clinical Immunology, Department of Medicine, University of Perugia, Perugia, Italy.

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http://dx.doi.org/10.1007/s00277-016-2632-zDOI Listing
May 2016

NK Cell and CD4+FoxP3+ Regulatory T Cell Based Therapies for Hematopoietic Stem Cell Engraftment.

Stem Cells Int 2016 5;2016:9025835. Epub 2016 Jan 5.

Blood and Marrow Transplantation, Stanford University School of Medicine, Stanford, CA 94305, USA.

Allogeneic hematopoietic cell transplantation (HCT) is a powerful therapy to treat multiple hematological diseases. The intensive conditioning regimens used to allow for donor hematopoietic stem cell (HSC) engraftment are often associated with severe toxicity, delayed immune reconstitution, life-threatening infections, and thus higher relapse rates. Additionally, due to the high incidence of graft versus host disease (GvHD), HCT protocols have evolved to prevent such disease that has a detrimental impact on antitumor and antiviral responses. Here, we analyzed the role of host T and natural killer (NK) cells in the rejection of donor HSC engraftment as well as the impact of donor regulatory T cells (Treg) and NK cells on HSC engraftment. We review some of the current strategies that utilize NK or Treg to improve allogeneic HCT therapy in order to accomplish better HSC engraftment and immune reconstitution and achieve a lower incidence of cancer relapse, opportunistic infections, and GvHD.
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http://dx.doi.org/10.1155/2016/9025835DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4736409PMC
February 2016

Regulatory T Cell Immunotherapy in Immune-Mediated Diseases.

Curr Stem Cell Rep 2015 Dec 12;1(4):177-186. Epub 2015 Sep 12.

Blood and Marrow Transplantation, Stanford University School of Medicine, Stanford, CA, USA.

Broad clinical interest rapidly followed the recent discovery of different subpopulations of T cells that have immune regulatory properties and a number of studies have been conducted aiming to dissect the translational potential of these promising cells. In this review we will focus on forkhead box P3 (FoxP3) positive regulatory T cells, T regulatory type 1 cells and invariant natural killer T cells (iNKT). We will analyze their ability to correct immune dysregulation in animal models of immune mediated diseases and we will examine the first clinical approaches where these cells have been directly or indirectly employed. We will discuss successes, challenges and limitations that rose in the road to the clinical use of regulatory T cells.
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http://dx.doi.org/10.1007/s40778-015-0025-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4712720PMC
December 2015

Freeze and Thaw of CD4+CD25+Foxp3+ Regulatory T Cells Results in Loss of CD62L Expression and a Reduced Capacity to Protect against Graft-versus-Host Disease.

PLoS One 2015 22;10(12):e0145763. Epub 2015 Dec 22.

Division of Blood and Marrow Transplantation, Department of Medicine, Stanford University School of Medicine, Stanford, CA, United States of America.

The adoptive transfer of CD4+CD25+Foxp3+ regulatory T cells (Tregs) in murine models of allogeneic hematopoietic cell transplantation (HCT) has been shown to protect recipient mice from lethal acute graft-versus-host disease (GVHD) and this approach is being actively investigated in human clinical trials. Here, we examined the effects of cryopreservation on Tregs. We found that freeze and thaw of murine and human Tregs is associated with reduced expression of L-selectin (CD62L), which was previously established to be an important factor that contributes to the in vivo protective effects of Tregs. Frozen and thawed murine Tregs showed a reduced capacity to bind to the CD62L binding partner MADCAM1 in vitro as well as an impaired homing to secondary lymphoid organs in vivo. Upon adoptive transfer frozen and thawed Tregs failed to protect against lethal GVHD compared with fresh Tregs in a murine model of allogeneic HCT across major histocompatibility barriers. In summary, the direct administration of adoptively transferred frozen and thawed Tregs adversely affects their immunosuppressive potential which is an important factor to consider in the clinical implementation of Treg immunotherapies.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0145763PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4691201PMC
June 2016

Treatment with agonistic DR3 antibody results in expansion of donor Tregs and reduced graft-versus-host disease.

Blood 2015 Jul 10;126(4):546-57. Epub 2015 Jun 10.

Department of Medicine, Division of Blood and Marrow Transplantation, Stanford University, Stanford, CA;

The paucity of regulatory T cells (Tregs) limits clinical translation to control aberrant immune reactions including graft-versus-host disease (GVHD). Recent studies showed that the agonistic antibody to DR3 (αDR3) expanded CD4(+)FoxP3(+) Tregs in vivo. We investigated whether treating donor mice with a single dose of αDR3 could alleviate acute GVHD in a MHC-mismatched bone marrow transplantation model. αDR3 induced selective proliferation of functional Tregs. CD4(+) T cells isolated from αDR3-treated mice contained higher numbers of Tregs and were less proliferative to allogeneic stimuli. In vivo GVHD studies confirmed that Tregs from αDR3-treated donors expanded robustly and higher frequencies of Tregs within donor CD4(+) T cells were maintained, resulting in improved survival. Conventional T cells derived from αDR3-treated donors showed reduced activation and proliferation. Serum levels of proinflammatory cytokines (IFNγ, IL-1β, and TNFα) and infiltration of donor T cells into GVHD target tissues (gastrointestinal tract and liver) were decreased. T cells from αDR3-treated donors retained graft-vs-tumor (GVT) effects. In conclusion, a single dose of αDR3 alleviates acute GVHD while preserving GVT effects by selectively expanding and maintaining donor Tregs. This novel strategy will facilitate the clinical application of Treg-based therapies.
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http://dx.doi.org/10.1182/blood-2015-04-637587DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4513255PMC
July 2015

Donor Requirements for Regulatory T Cell Suppression of Murine Graft-versus-Host Disease.

J Immunol 2015 Jul 20;195(1):347-55. Epub 2015 May 20.

Blood and Marrow Transplantation, Stanford University School of Medicine, Stanford, CA 94305; and

Adoptive transfer of freshly isolated natural occurring CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg) prevents graft-versus-host disease (GVHD) in several animal models and following hematopoietic cell transplantation (HCT) in clinical trials. Donor-derived Treg have been mainly used, as they share the same MHC with CD4(+) and CD8(+) conventional T cells (Tcon) that are primarily responsible for GVHD. Third party-derived Treg are a promising alternative for cellular therapy, as they can be prepared in advance, screened for pathogens and activity, and banked. We explored MHC disparities between Treg and Tcon in HCT to evaluate the impact of different Treg populations in GVHD prevention and survival. Third-party Treg and donor Treg are equally suppressive in ex vivo assays, whereas both donor and third-party but not host Treg protect from GVHD in allogeneic HCT, with donor Treg being the most effective. In an MHC minor mismatched transplantation model (C57BL/6 → BALB/b), donor and third-party Treg were equally effective in controlling GVHD. Furthermore, using an in vivo Treg depletion mouse model, we found that Treg exert their main suppressive activity in the first 2 d after transplantation. Third-party Treg survive for a shorter period of time after adoptive transfer, but despite the shorter survival, they control Tcon proliferation in the early phases of HCT. These studies provide relevant insights on the mechanisms of Treg-mediated protection from GVHD and support for the use of third-party Treg in clinical trials.
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http://dx.doi.org/10.4049/jimmunol.1402861DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4475671PMC
July 2015
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