scholarly journals T Helper Cell Lineage-Defining Transcription Factors: Potent Targets for Specific GVHD Therapy?

2022 ◽  
Vol 12 ◽  
Author(s):  
Julia Campe ◽  
Evelyn Ullrich
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Acute Gvhd ◽  
Cell Subset ◽  
Cell Transplant ◽  
T Helper ◽  
Hematopoietic Stem ◽  
Th Cell ◽  
Th17 Differentiation

Allogenic hematopoietic stem cell transplantation (allo-HSCT) represents a potent and potentially curative treatment for many hematopoietic malignancies and hematologic disorders in adults and children. The donor-derived immunity, elicited by the stem cell transplant, can prevent disease relapse but is also responsible for the induction of graft-versus-host disease (GVHD). The pathophysiology of acute GVHD is not completely understood yet. In general, acute GVHD is driven by the inflammatory and cytotoxic effect of alloreactive donor T cells. Since several experimental approaches indicate that CD4 T cells play an important role in initiation and progression of acute GVHD, the contribution of the different CD4 T helper (Th) cell subtypes in the pathomechanism and regulation of the disease is a central point of current research. Th lineages derive from naïve CD4 T cell progenitors and lineage commitment is initiated by the surrounding cytokine milieu and subsequent changes in the transcription factor (TF) profile. Each T cell subtype has its own effector characteristics, immunologic function, and lineage specific cytokine profile, leading to the association with different immune responses and diseases. Acute GVHD is thought to be mainly driven by the Th1/Th17 axis, whereas Treg cells are attributed to attenuate GVHD effects. As the differentiation of each Th subset highly depends on the specific composition of activating and repressing TFs, these present a potent target to alter the Th cell landscape towards a GVHD-ameliorating direction, e.g. by inhibiting Th1 and Th17 differentiation. The finding, that targeting of Th1 and Th17 differentiation appears more effective for GVHD-prevention than a strategy to inhibit Th1 and Th17 cytokines supports this concept. In this review, we shed light on the current advances of potent TF inhibitors to alter Th cell differentiation and consecutively attenuate GVHD. We will focus especially on preclinical studies and outcomes of TF inhibition in murine GVHD models. Finally, we will point out the possible impact of a Th cell subset-specific immune modulation in context of GVHD.

Genetic Disparity between Stem Cell Transplant Patients and Their Donors Assessed by High Density Single Nucleotide Polymorphism (SNP) Microarray Typing Is Seen in Specific Genomic Regions.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2127-2127
Author(s):  
Ephraim P. Hochberg ◽  
David Miklos ◽  
Emmanuel Zorn ◽  
Jesse Levin ◽  
Carol Reynolds ◽  
...  
Keyword(s):  
Stem Cell ◽  
T Cell ◽  
Acute Gvhd ◽  
High Density ◽  
Differential Sensitivity ◽  
Cell Transplant ◽  
Chromosome 6 ◽  
Allele Sharing ◽  
Hematopoietic Stem ◽  
Genomic Regions

Abstract Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality following allogeneic hematopoietic stem cell transplantation (HSCT). Advances in molecular typing of major histocompatibility complex (MHC) alleles have decreased but not eliminated the occurrence of acute GVHD. Minor histocompatibility antigen (mHA) disparities between patients and their molecularly HLA-identical donors are likely to represent the residual targets of donor immunity. The majority of mHAs are encoded by SNPs which are disparate between patient and donor. Identification of these immunogenic SNPs would provide both a clinical benefit as well as insight into the biology of GVHD. We used Affymetrix high-density arrays to assess 10,043 SNPs spanning the entire genome. We have examined genomic DNA from 36 patient/donor pairs (21 with acute GVHD grades II-IV and 15 without GVHD). All patients and donors were HLA and sex-matched siblings and all patients received T-cell depleted transplants. T cell depletion is important in this context as it removes the potential pharmacogenomic confounding factor of differential sensitivity to immunosuppressive agents. Because the donors and recipients are related, large areas of the genome are identical by descent. Similarly sized blocks of chromosome are disparately inherited. We hypothesized that genomic regions important to the immunopathogenesis of GVHD would be more likely to be disparately inherited in pairs with GVHD than in pairs without GVHD. As a control of our method we compared the area of chromosome 6 surrounding the MHC complex and demonstrated that there was minimal SNP disparity in either GVHD pairs or asymptomatic pairs. We also focused our attention on the region of chromosome 6 outside the MHC area, hypothesizing that disparities in this area would have to result from recombination events. Interestingly the only major area of disparity on Chr 6 was in the gene GMDS, which has been proposed to play a role in the extravasation of activated lymphocytes. We then examined other chromosomes for evidence of genomic regions that that were selectively disparate in GVHD pairs. We were able to identify several genomic regions that appeared to be associated with this outcome. The Lander-Green algorithm was used to estimate the allele sharing between the siblings for each SNP marker and then the disparity score was defined as the average allele sharing of the GVHD group - average allele sharing of the asymptomatic group. The five genomic regions with the highest disparity scores (ranked in order of score) and the gene closest to the region are: 11q14.3 (gene cysteine and histidine-rich domain (CHORD)-containing, zinc binding protein 1), 9p22.2 (gene SH3GL2), 5p15 (gene KIAA0947), 10p15.1 (gene aldo-keto reductase family 1, member C4), and 9p21.2 (Chr 9 ORF 72). The highly disparate regions ranged between 70 and 2200 kb in size. Interestingly the total number of disparate SNPs was not different between pairs with GVHD and pairs without GVHD, supporting the hypothesis that a limited number of SNPs are important immunologic targets. This technique of genome-wide disparity analysis is a promising addition to our ability to define important mHA.

scholarly journals Discovery of TSC-101: A First-in-Class Natural HA-2-Specific TCR to Treat Leukemia Following Hematopoietic Stem Cell Transplant Therapy

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1704-1704
Author(s):  
Ribhu Nayar ◽  
Mollie M Jurewicz ◽  
Sonal Jangalwe ◽  
Hannah Bader ◽  
Kimberly M Cirelli ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Hematopoietic Stem Cell Transplant ◽  
Stem Cell Transplant ◽  
Surface Expression ◽  
Cell Transplant ◽  
Hematopoietic Stem ◽  
Genome Wide ◽  
Clinical Candidate

Abstract Background Approximately 50% of AML patients relapse following allogeneic hematopoietic stem cell transplant therapy, leaving them with very few treatment options (Rautenberg et al. (2019) Int. J. Mol. Sci. 20:228). Rare patients who naturally develop a minor antigen-specific graft-versus-leukemia T cell response show substantially lower relapse rates (Marijt et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100:2742-2747; Spierings et al. (2013) Biol. Blood Marrow Transplant. 19:1244-1253). HA-2 (YIGEVLVSV, genotype RS_61739531 C/C or T/C) is an HLA-A*02:01- and haematopoietically-restricted minor histocompatibility antigen derived from the class I myosin protein, MYO1G (Pierce et al. (2001) J. Immunol. 167:3223-3230). Patients receiving donor lymphocyte infusion from HA-2-mismatched donors who develop HA-2-specific T cells show a graft vs leukemia response and often experience long-term remission (Marijt et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100:2742-2747), making HA-2 an ideal candidate for TCR-engineered T cell immunotherapy of liquid tumors. Methods Using TScan's proprietary ReceptorScan platform, we discovered 1,302 HA-2-specific TCRs by screening 237 million naïve CD8 + T cells from 5 healthy HA-2-negative donors. We evaluated these TCRs using our proprietary DexScan platform to select the 15 TCRs with the highest surface expression and greatest affinity for the HA-2 peptide when transferred into primary human T cells. We further tested each TCR individually in our clinical vector backbone for surface expression, selective cytotoxicity, cytokine production, and proliferation using a panel of cell lines that express varying levels of HLA-A*02:01 and MYO1G. Finally, the top 5 TCRs were evaluated for alloreactivity using an array-based screen assessing 108 MHC-I molecules individually, and for off-target cross-reactivity using our proprietary genome-wide TargetScan platform. A lead TCR with limited alloreactivity and a narrow off-target profile was selected as our lead TSC-101 TCR. The avidity of TSC-101 for its putative off-targets was further measured in peptide-pulsed experiments to better appreciate the toxicity risks associated with our lead clinical candidate. Results and Conclusion Of the 1,302 HA-2-specific TCRs identified by our ReceptorScan platform, we identified TSC-101 as the most active TCR. TSC-101 displayed no alloreactivity to 107/108 HLAs tested and limited off-target risks in a genome-wide screens. Potential off-target peptides identified for TSC-101 displayed extremely weak avidities, predicting an absence of toxicity risks for our clinical candidate. Based on these results, TSC-101 has been advanced to IND-enabling activities to prepare for first-in-human testing in 2022. To our knowledge, this is the first clinical grade HA-2-specifc TCR being developed for immunotherapy for liquid tumors. Disclosures Macbeath: TScan Therapeutics: Current Employment, Current equity holder in publicly-traded company.

scholarly journals Prior Use of Mogamulizumab to Allogenic Hematopoietic Stem Cell Transplantation Induces Severe Acute Graft-Versus-Host Disease

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1940-1940 ◽  
Author(s):  
Takeshi Sugio ◽  
Koji Kato ◽  
Takatoshi Aoki ◽  
Takanori Ota ◽  
Noriyuki Saito ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Regulatory T Cells ◽  
Research Funding ◽  
Acute Gvhd ◽  
Cell Lymphoma ◽  
Hematopoietic Stem ◽  
Graft Versus Host

Abstract [Introduction] Adult T-cell leukemia/lymphoma (ATL) is an aggressive peripheral T-cell lymphoma (PTCL) with a dismal prognosis. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative treatment in ATL patients. Mogamulizumab, a humanized anti-CC chemokine receptor 4 (CCR4) monoclonal antibody, is a novel immunotherapeutic agent, effective in treating patients with PTCL such as ATL, PTCL-not specified, and cutaneous T-cell lymphoma. However, in allo-HSCT setting, we should be careful to use mogamulizumab because CCR4 is expressed in regulatory T cells: The mogamulizumab treatment may accelerate GVHD by eradicating regulatory T cells in allo-HSCT patients. Here, we retrospectively analyzed the effect of mogamulizumab on GVHD development in ATL patients treated with mogamulizumab prior to allo-HSCT. [Patients and Methods] Data from the Fukuoka Bone Marrow Transplantation Group were retrospectively analyzed after the approval of mogamulizumab use in Japan. [Results] A total of 24 patients with ATL received mogamulizumab prior to allo-HSCT between April 2012 and April 2015 in our group. The median age at allo-HSCT was 58.5 years (range, 32-72). The median intervals from the last administration of mogamulizumab to allo-HSCT were 25 days (range, 9-126). The median total dose of mogamulizumab was 3 mg/kg (range, 1-8 mg/kg). After treatment with mogamulizumab, 18 patients (75%) had achieved in remission (CR in 4 patients and PR in 14) at allo-HSCT. Ten patients received unrelated bone marrow, 5 received related peripheral blood, and 9 received cord blood as stem cell sources. Eleven patients were treated with full-intensity conditioning and 13 received reduced-intensity conditioning. Graft-versus-host disease (GVHD) prophylaxis consisted of calcineurin inhibitors (cyclosporine or tacrolimus) with short-term methotrexate in 14 patients and mycophenolate mofetil in 9. The cumulative incidence (CI) of acute GVHD at 100 days was 66.6% in grade 2-4 and 33.3% in grade 3-4. The involved organs of acute GVHD were skin in 14 patients, gut in 10, and liver in 4. Among 14 patients who developed grade 2-4 acute GVHD, 5 had severe fluid retention such as pleural effusion or ascites associated with GVHD. Chronic GVHD was observed in 6 patients, and 5 of them were extensive disease. The CI of transplant-related mortality (TRM) and relapse at 1-year were 53.2% (95%CI, 29.3-72.3%) and 29.6% (95%CI, 12.6-48.9%), respectively. The leading cause of death was GVHD (n = 7). The 1-year overall survival and progression-free survival were 19.2% (95%CI, 5.7-38.8%) and 17.2% (95%CI, 4.9-35.7%), respectively. [Discussion] Use of mogamulizumab prior to transplantation in allo-HSCT patients has a merit to decrease the burden of ATL cells. However, it was associated with an increase of TRM due to severe GVHD. Although most of ATL patients achieved better disease status at allo-HSCT through mogamulizumab and the survival rate was expected to be 50% based on the previous data, the survival in the present study was ~20%. These data suggest that mogamulizumab administered before transplantation may have retained until an early phase of post-transplantation, and the donor or host-derived regulatory T cells might be eliminated, allowing the GVHD T-cell clone to expand. Since mogalizumab is a potent anti-ATL agent, we need to develop new treatment protocols integrating mogalizumab at a suitable dose or administration timing, to minimize the unwanted GVHD development in future studies. Disclosures Akashi: Asahi Kasei: Research Funding, Speakers Bureau; Shionogi: Research Funding, Speakers Bureau; Astellas: Research Funding, Speakers Bureau; Celgene: Research Funding, Speakers Bureau; Chugai: Research Funding, Speakers Bureau; Bristol-Myers Squibb: Research Funding, Speakers Bureau; Novartis Pharma K.K.: Consultancy, Research Funding, Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Consultancy, Research Funding, Speakers Bureau.

scholarly journals Generation of BCR-ABL Reactive CD4+ T Helper Cells By Reprograming and Redifferentiation

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3424-3424
Author(s):  
Norihiro Ueda ◽  
Yasusi Uemura ◽  
Rhong Zhang ◽  
Shuichi Kitayama ◽  
Yutaka Yasui ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Tyrosine Kinase ◽  
Research Funding ◽  
T Helper ◽  
Th Cells ◽  
Hematopoietic Stem ◽  
Chugai Pharmaceutical ◽  
Dc Maturation

Abstract Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell disorder caused by BCR-ABL fusion protein that has constitutively active tyrosine kinase activity. Although the prognosis of the patient with CML in chronic phase has markedly improved by the advent of tyrosine kinase inhibiters, the management of the patients with CML in advanced phase remains to be the major challenge. Immunotherapy is considered to be one of the promising treatment strategies for refractory CML. BCR-ABL fusion region, b3a2 peptide, represents a neo-epitope that can induce CML-specific immune responses. The activation of b3a2 peptide-specific CD4+ T helper (Th) cells and their interaction with dendritic cells (DCs) can induce a robust cytotoxic T lymphocyte (CTL)-mediated anti-leukemic immunity through epitope spreading. However, current vaccination strategies cannot effectively induce the proliferation of antigen-specific Th cells in vivo, presumably due to the tumor-induced immunosuppressive milieu. In addition, ex vivo expansion of antigen-specific Th cells attenuates their effector functions by expansion-related cell senescence, and the procedure to establish antigen-specific Th cells for each patient's treatment is too complicated for the clinical application. The purpose of the present study is to establish a method to generate large amounts of functional b3a2-specific CD4+ Th cells enough for the treatment of the patients with refractory CML by using induced pluripotent stem cell (iPSC) technology. First, we established b3a2-specific CD4+ Th clone from peripheral blood mononuclear cells of a healthy donor positive for HLA-DRB1*09:01 and HLA-A*24:02. The Th clone recognized b3a2 peptide in the context of HLA-DR9 and exhibited a Th1 profile. Second, we established iPSCs from the Th clone and differentiated them into T cell lineage by coculture with OP9 stromal cells expressing Notch ligand Delta-like 1. The iPSC-derived T cells (b3a2-iPS-T cells) expressed the same T cell antigen receptor (TCR) as the original Th clone but not CD4 molecule. Because CD4 acts as a co-receptor in the TCR-mediated Th responses, we transduced b3a2-iPS-T cells with CD4 gene. The CD4-expressing b3a2-iPS-T cells (CD4+ b3a2-iPS-T cells) recognized b3a2 peptide in the context of HLA-DR9 as the original Th clone. Moreover, CD4+ b3a2-iPS-T cells activated by b3a2 peptide induced DC maturation, as indicated by the upregulation of CD86 on DCs. In the additional presence of HLA-A24-restricted Wilms tumor 1 (WT1) peptide, the mature DCs stimulated primary expansion of WT1-specific CTLs. The CTLs exerted cytotoxicity against WT1 peptide-loaded HLA-A24 positive cell lines. These data suggest that the CD4+ b3a2-iPS-T cells have a potential to induce effective anti-leukemic immunity via DC maturation and subsequent CTL responses. The current approach enable to provide large amounts of b3a2 specific CD4+ Th-like cells that would augment CTL-mediated anti-leukemic responses via DC maturation, which may contribute to the treatment of patients with refractory CML. Disclosures Kiyoi: Yakult Honsha Co.,Ltd.: Research Funding; FUJIFILM Corporation: Patents & Royalties, Research Funding; Eisai Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Consultancy, Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Novartis Pharma K.K.: Research Funding; Mochida Pharmaceutical Co., Ltd.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; FUJIFILM RI Pharma Co.,Ltd.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Alexion Pharmaceuticals: Research Funding; MSD K.K.: Research Funding; Japan Blood Products Organization: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Pfizer Inc.: Research Funding; Bristol-Myers Squibb: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Taisho Toyama Pharmaceutical Co., Ltd.: Research Funding; Teijin Ltd.: Research Funding. Naoe:Celgene K.K.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Toyama Chemical CO., LTD.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Patents & Royalties, Research Funding; Chugai Pharmaceutical Co., Ltd.: Patents & Royalties; FUJIFILM Corporation: Patents & Royalties, Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Pfizer Inc.: Research Funding; Astellas Pharma Inc.: Research Funding. Kaneko:AsTlym Co., Ltd: Other: founder, shareholder and scientific adviser.

Safety and Preliminary Efficacy of "Ready to Administer" Cytomegalovirus (CMV)-Specific T Cells for the Treatment of Patients with Refractory CMV Infection

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 388-388 ◽  
Author(s):  
Ifigeneia Tzannou ◽  
Kathryn S. Leung ◽  
Caridad Martinez ◽  
Swati Naik ◽  
Stephen Gottschalk ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Stem Cell Transplant ◽  
Fixed Dose ◽  
Effector Memory ◽  
Cell Transplant ◽  
Decision Tool ◽  
Widespread Application ◽  
Hematopoietic Stem

Abstract Despite advances in antiviral drugs, Cytomegalovirus (CMV) infections remain a significant cause of morbidity and mortality in immunocompromised individuals. We have recently demonstrated in hematopoietic stem cell transplant (HSCT) recipients that adoptively-transferred virus-specific T cells, generated from healthy 3rd party donors and administered as an "ready to administer" product, can be curative, even in patients with drug-refractory CMV infections. However, broader implementation has been hindered by the postulated need for extensive panels of T cell lines representing a diverse HLA profile, as well as the complexities of large scale manufacturing for widespread clinical application. To address these potential issues, we have developed a decision tool that identified a short list of donors who provide HLA coverage for >90% of the stem cell transplant population. Furthermore, to generate banks of CMV-specific T cells from these donors, we have created a simple, robust, and linearly scalable manufacturing process. To determine whether these advances would enable the widespread application of "ready to administer" T cells, we generated CMV cell banks (Viralym-C™) from 9 healthy donors selected by our decision tool, and initiated a fixed-dose (2x107 cells/m2) Phase I clinical trial for the treatment of drug-refractory CMV infections in pediatric and adult HSCT recipients. To generate the Viralym-C™ banks, we stimulated donor peripheral blood mononuclear cells (PBMCs) with overlapping peptide libraries spanning the immunodominant CMV antigens pp65 and IE1. Cells were subsequently expanded in a G-Rex device, resulting in a mean fold expansion of 103±12. The lines were polyclonal, comprising both CD4+ (21.3±6.7%) and CD8+ (74.8±6.9%) T cells, and expressed central CD45RO+/CD62L+ (58.5±4.2%) and effector memory markers CD45RO+/CD62L- (35.3±12.2%). Furthermore, the lines generated were specific for the target antigens (IE1: 419±100; pp65 1070±31 SFC/2x105, n=9). To date, we have screened 12 patients for study participation, and from our bank of just 9 lines we have successfully identified a suitable line for all patients within 24 hours. Of these, 6 patients have been infused; 5 received a single infusion and 1 patient required 2 infusions for sustained benefit. There were no immediate infusion-related toxicities; and despite the HLA disparity between the Viralym-C lines and the patients infused, there were no cases of de novo or recurrent graft versus host disease (GvHD). One patient developed a transient fever a few hours post-infusion, which spontaneously resolved. Based on viral load, measured by quantitative PCR, or symptom resolution (in patients with disease), Viralym-C™ cells controlled active infections in all 5 evaluable patients; 4 patients had complete responses, and 1 patient had a partial response within 4 weeks of cell infusion. One patient with CMV retinitis had complete resolution of symptoms following Viralym-C™ infusion. In conclusion, our results demonstrate the feasibility, preliminary safety and efficacy of "ready to administer" Viralym-C™ cells that have been generated from a small panel of healthy, eligible CMV seropositive donors identified by our decision support tool. These data suggest that cost-effective, broadly applicable T cell anti-viral therapy may be feasible for patients following HSCT and potentially other conditions. Disclosures Tzannou: ViraCyte LLC: Consultancy. Leen:ViraCyte LLC: Equity Ownership, Patents & Royalties. Kakarla:ViraCyte LLC: Employment.

Multivirus-Specific T Cell Immunotherapy to Prevent or Treat Infections of Stem Cell Transplant Recipients.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2207-2207
Author(s):  
Ulrike Gerdemann ◽  
Anne Christin ◽  
Carlos A. Ramos ◽  
Yuriko Fujita ◽  
Juan F. Vera ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Cell Lines ◽  
Stem Cell Transplant ◽  
Acute Infection ◽  
Functional Characterization ◽  
Cell Transplant ◽  
Activated T Cells ◽  
Hematopoietic Stem

Abstract Viral infections caused by community viruses frequently cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. Antiviral drugs are costly, often have severe adverse effects, and are frequently ineffective. Treatment of the underlying problem, namely lack of antigen-specific T cells, should offer effective and longterm protection. Our group has produced trivirus-reactive T cells targeting EBV, CMV, and adenoviruses (Adv) using monocytes and EBV-transformed lymphoblastoid cell lines (EBV-LCL) expressing pp65 from an adenoviral vector as antigen-presenting cells to present CMV, Adv and EBV antigens. As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs post-infusion into HSCT recipients and appeared to protect the recipients against all three viruses. Despite these encouraging clinical results, the broader implementation of the approach is limited by (i) the infectious virus material (EBV/Adv) required for CTL generation and (ii) the prolonged culture required to produce the EBV-LCL, increase viral specificity and reduce alloreactivity (3 months) which means T cells must be produced “speculatively” for all patients. Finally, (iii) “antigenic competition” between multiple viruses limits the extension of the approach to additional problematic pathogens. To overcome these limitations we have developed an approach to rapidly produce multivirus-specific CTL with broad spectrum specificity without using adenoviral vectors or EBV-LCL. Using the Amaxa system to nucleofect monocyte-derived DCs we consistently detected GFP transgene expression in 39% (median; range 30–58%) of cells 24hrs posttransfection. Viability was ~70% and the DC maturation state, as measured by CD80, 83, 86, and HLA-DR expression, was unaffected by the transfection. To show that nucleofected DCs reactivated virus-specific T cells in vitro, we cocultured p-Shuttle-pp65-GFP-transfected DCs from CMV seropositive donors with PBMCs at a responder:stimulator ratio of 20:1. After nine days, phenotypic and functional characterization of the responder T cell lines showed higher or comparable frequencies of pp65-specific T cells in IFN-g ELIspot and minimal alloreactivity when compared to pp65-specific T cells lines generated from the same donors using our standard protocol with Ad5f35pp65-transduced DCs as APCs. Pentamer analysis of pShuttle-pp65-generated CTL lines also showed a higher frequency of pp65 pentamer-directed T cells than the Ad5f35pp65-transduced counterparts (median 2.05 fold higher frequency of HLA-A2 NLV-directed T cells; range 1.34–3.35 fold) (n=4 donors). Importantly, this protocol could also be used to reactivate T cells against multiple viruses for which high (EBV), intermediate (BK), and low (Adv) frequencies of reactive memory T cells circulate. Using a panel of p-Shuttle plasmids encoding LMP2 and BZLF1 (EBV), Large T (BK), and Hexon and Penton (Adv), we amplified CTLs from seropositive donors, using as stimulators DCs transfected with each construct. This modification overcomes the need for EBV-LCL generation. Furthermore, we demonstrated that by pooling transfected DCs prior to coculture with PBMC, we could reproducibly generate multivirus-specific CTL lines with specificity for all the stimulating antigens, irrespective of the circulating memory T cell frequency. To further shorten the CTL production process, we established that virus-activated T cells could be specifically selected by IFN-g capture 24 hours after DC stimulation and that the selected cells were highly specific for the stimulating antigens as measured by IFN-g ELIspot, proliferation and cytotoxicity assay. In summary, we have established a GMP-applicable protocol for the rapid generation (<10 days) of two different CTL products without using infectious viral material. In 10 days we can generate virus-specific CTLs with broad specificity which can be administered prophylactically to high risk SCT recipients. However by combining DC transfection with IFN-g selection we can also rapidly generate mono- or multivirus-specific CTL products for treatment of acute infection. We demonstrate the feasibility of generating CTL lines targeting 6 different antigens from 4 common viruses without using infectious viral material. Future studies will extend our approach to additional viral, fungal, and bacterial antigens.

Prospective simultaneous quantification of human cytomegalovirus-specific CD4+ and CD8+ T-cell reconstitution in young recipients of allogeneic hematopoietic stem cell transplants

Blood ◽  
2006 ◽  
Vol 108 (4) ◽  
pp. 1406-1412 ◽  
Author(s):  
Daniele Lilleri ◽  
Giuseppe Gerna ◽  
Chiara Fornara ◽  
Laura Lozza ◽  
Rita Maccario ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Hematopoietic Stem Cell ◽  
Human Cytomegalovirus ◽  
Immune Reconstitution ◽  
Hcmv Infection ◽  
Cd8 T Cell ◽  
Cell Transplant ◽  
Hematopoietic Stem

AbstractWe investigated immune reconstitution against human cytomegalovirus (HCMV) in 57 hematopoietic stem cell transplant (HSCT) recipients, aged 1 to 24 years, through a novel method combining T-cell stimulation by HCMV-infected autologous dendritic cells with simultaneous cytometric quantification of HCMV-specific, IFNγ-producing CD4+ and CD8+ T cells. Lymphoproliferative response (LPR) to HCMV antigens was also determined. Patients were stratified into 2 groups according to HCMV serostatus, comprising 39 HCMV-seropositive (R+) and 18 HCMV-seronegative (R–) patients who received a transplant from a sero-positive donor. Recovery of both HCMV-specific CD4+ and CD8+ T-cell immunity occurred in all 39 R+ patients within 6 months and in 6 (33%) of 18 R– patients within 12 months. In R+ patients, the median numbers of HCMV-specific CD8+ and CD4+T cells were significantly higher than those of healthy controls, starting from days +60 and +180, respectively. In R– patients, the median numbers of HCMV-specific T cells were consistently lower than in R+ patients. LPR was delayed compared with reconstitution of IFNγ-producing T cells. Patients with delayed specific immune reconstitution experienced recurrent episodes of HCMV infection. HCMV seropositivity of young HSCT recipients is the major factor responsible for HCMV-specific immune reconstitution, irrespective of donor serostatus, and measurement of HCMV-specific T cells appears useful for correct management of HCMV infection.

The Graft-vs-Host Hematopoietic Effect Generated after Nonmyeloablative Allogeneic Stem Cell Transplantation (NST) Cures Patients with Severe PNH.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 811-811
Author(s):  
Yoshiyuki Takahashi ◽  
S. Chakrabarti ◽  
R. Srinivasan ◽  
T. Igarashi ◽  
A. Lundqvist ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Cell Transplantation ◽  
Conditioning Regimen ◽  
Cell Transplant ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
Donor T Cells

Abstract Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal disorder of hematopoietic stem cells characterized by RBC susceptibility to complement-mediated lysis. Infections related to neutropenia, bleeding associated with thrombocytopenia, and thrombosis all contribute to morbidity and mortality. Although allogeneic hematopoietic cell transplantation (HCT) can be curative, the high-risk of treatment-related mortality with myeloablative HCT precludes this approach for most patients with severe disease. We previously reported in vitro and in vivo data showing PNH cells could be killed by allo-reactive donor T-cells recognizing minor histocompatibility antigens expressed on both normal and GPI negative cells. Here we present updated data on a cohort of 11 patients with severe PNH who received a NST at the NHLBI from 5/99 through 6/2004. Eligibility included a diagnosis of PNH associated with one or more of the following :1) Transfusion dependence (n=9) 2) Prior thrombotic episodes (n=4) 3) Recurrent debilitating hemolytic crisis (n=7). Patients received a T-cell replete G-CSF mobilized blood stem cell transplant from an HLA-matched family donor following nonmyeloablative conditioning with cyclophosphamide (120mg/kg) and fludarabine (125mg/m2). Patients with a significant transfusion history had horse ATG (40mg/kg/day x 4) added to the conditioning regimen (n=9). CSA either alone (n=1) or combined with either MMF (n=4) or mini-dose methotrexate (n=6) was used as GVHD prophylaxis. The median % of GPI anchored protein negative neutrophils pre-transplant was 83% (range 13%–99%). Blood samples obtained post-transplant were analyzed by FACS to determine the percentage of persisting GPI negative neutrophils (CD15+/CD66b−/CD16−). Chimerism was also assessed post-transplant in T-cell and myeloid fractions by PCR assay of polymorphic short tandem repeats (STR). Neutrophil recovery occurred at a median 15 days (range 10–19). STR analysis revealed donor engraftment occurred in both myeloid and T-cell lineages in all patients. Self-limiting febrile hemolytic reactions associated with ATG administration (6/9 patients) and grade II-IV acute GVHD (n=5) were the most common complications associated with transplantation. With a median follow-up of 458 days (range 31–1917), all patients survive either in remission (n=8) or with declining GPI negative populations (n=3); GPI negative neutrophils were detected in all patients at engraftment but gradually declined until no longer detectable (<0.1%) in all 8 patients evaluable more than 100 days after transplantation, while 3 with shorter follow-up (days + 37, +51, +78) have persistent albeit rapidly declining PNH populations. The observation that GPI negative neutrophils populations decrease and ultimately disappear when myeloid chimerism transitions from mixed to full donor chimerism is consistent with PNH cells being eradicated through a graft-vs-host hematopoietic effect. None of the 7 patients with more than 1 year follow-up have had reoccurrence of their PNH clone. Conclusion: Alloreactive donor T-cells mediating graft-vs-host hematopoietic effects can immunologically eradicate PNH following NST. NST should be considered a viable and potentially curative option for patients with severe PNH.

Phase I Clinical Study of Donor Lymphocyte Infusion Depleted of Alloreactive T Cells after Haplotype Mismatched Myeloablative Stem Cell Transplantation To Limit Infections and Malignant Relapse without Causing GVHD.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 309-309 ◽  
Author(s):  
Denis-Claude Roy ◽  
Sandra Cohen ◽  
Lambert Busque ◽  
Douglas Fish ◽  
Thomas Kiss ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
High Risk ◽  
Phase I ◽  
Acute Gvhd ◽  
Hematologic Malignancies ◽  
Infectious Complications ◽  
Cell Transplant ◽  
Post Transplant

Abstract Patients with very high risk hematologic malignancies who cannot find an HLA-matched related or unrelated donor can benefit from haplo-mismatched transplantation. The latter is, however, complicated by frequent and severe infectious complications and disease relapse due to delayed immune reconstitution. We have previously reported that photodynamic therapy (PDT) could selectively deplete donor alloreactive populations while preserving lymphocytes for immune responses. Indeed, the dibromorhodamine derivative TH9402 (Celmed BioSciences) has a propensity to accumulate in activated but not resting T cells. We present results of an ongoing Phase I clinical trial of haplo-mismatched allogeneic stem cell transplant (SCT) supplemented with donor lymphocyte infusions (DLIs) PDT depleted of host-reactive T cells. Nine high-risk patients with hematologic malignancies (5 AML relapsed or refractory, 2 MDS, 1 NHL relapsing after autologous SCT, 1 refractory CLL) entered the trial, 7 are evaluable for acute GVHD and reconstitution. Patients (4 M, 3 F) underwent transplantation with donor cells mismatched at 3 HLA Ags: 2 patients; 2Ags: 4 pts, and DR only: 1 pt). Donor mononuclear cells (MNCs) were incubated with recipient MNCs for 4 days, exposed to TH9402 PDT, stored frozen, and administered on day 30±3 after transplant at 3 graded DLI dose levels: 1×104 (1pt), 5×104 (3pts), and 1.3 x105 (3pts) CD3+ cells/kg. Anti-host cytotoxic T lymphocyte precursors (CTLp) were depleted from DLIs by approximately 1.5 logs, and flow cytometry showed greater than 90% elimination of activated T cells (CD4+CD25+ and CD8+CD25+) by TH9402 PDT. All stem cell grafts underwent in vitro immunomagnetic T cell depletion using CD34+ positive cell selection (Miltenyi). Median age at SCT was 57 years (range: 40–58). Five patients were in partial remission or had progressive disease, and 2 patients were in complete remission at the time of SCT. The myeloablative regimen consisted of TBI (1200 cGy), thiotepa (5 mg/kg) and fludarabine (40 mg/m2/day for 5 days) followed by infusion of CD3 depleted HSC grafts. A median of 9.2×106 CD34+ cells/kg were infused on day 0. No GVHD prophylaxis was administered. Evaluable patients showed durable hematologic engraftment: median time to >0.5×109 granulocytes/L was 10.5 days (8–20), and to >20×109 platelets/L without transfusion, 12 days (9–137) and achieved complete donor chimerism. No patient developed acute GVHD (grade II–IV), while 3 patients developed signs of chronic GVHD. Two patients died: one (cohort 1) of a post-transplant lymphoproliferative disease, and one (cohort 2), of relapsed AML. No other patient relapsed. Two pts (1 in cohort 2 and 1 in cohort 3) recovered greater than 0.3×109 CD3+ and CD4+ cells/L at 2 and 5 months post-DLI, and 4 pts had >0.2×109 CD3+ cells/L at 6 mo post-DLI. Although 4 patients developed infectious complications (HSV, CMV, Nocardia, Aspergillus), all resolved rapidly with appropriate therapy. The overall disease-free-survival and survival are 57% at 1 year (median follow-up: 9.4 mo). Our results indicate that the post-transplant infusion of a PDT treated DLI is feasible, does not induce acute GVHD, and may accelerate T cell reconstitution. This PDT strategy could represent an appealing alternative for patients in the higher age range who are at high risk for GVHD.

Haploidentical Transplantation In Adults ≥ 66 Years of Age

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3529-3529
Author(s):  
Dolores Grosso ◽  
Onder Alpdogan ◽  
Emmanuel C. Besa ◽  
Matthew Carabasi ◽  
Beth Colombe ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Older Patients ◽  
Karnofsky Performance Score ◽  
Cell Transplant ◽  
Hematopoietic Stem ◽  
Cell Dose ◽  
Co Morbidity ◽  
Donor T Cells

Abstract Abstract 3529 Hematologic malignancies in older patients are often characterized by resistant phenotypes which would ideally be treated by allogeneic hematopoietic stem cell transplant (HSCT). However, allogeneic HSCT in this age group is associated with greater regimen-related toxicity than in younger patients limiting its application. In addition, older patients have a smaller matched related donor pool due to the age and comorbidities of their siblings, but often have haploidentical offspring donor options. In order to extend the application of HSCT to older patients (>66), we developed a reduced intensity regimen utilizing haploidentical donors. In this approach, donor T cells and CD 34+ cells are infused at 2 separate times so that the dose and timing of each cell type can be independently controlled. This “break apart” method allows the tolerization of donor T cells by cyclophosphamide (CY) without exposure of the donor HSCs to the drug. Seventeen patients ages 66–77 with AML (9), advanced MDS (2), biphenotypic leukemia (1), CLL (2), NHL (2), and myelofibrosis (1) were treated. All but 2 patients had persistent disease at the time of HSCT. Ten of 17 had a Karnofsky Performance Score (KPS) of >90%, while 7 patients' KPS was 70–80%. The hematopoietic cell transplantation comorbidity index (HCT CI) for the group was between 0 and 5 points. Time from first treatment to HSCT was widely variable. The patients received fludarabine 30 mg/m2/d and cytarabine 2 Gms/m2/d on days -11 through -8. Thiotepa 5 mg/m2/d on days -11 to -9 was substituted for cytarabine in patients with active myeloid malignancies at HSCT. TBI (2 Gy) was given on d-6 immediately followed by 2 ×10e8/kg of the donor's T cells (DLI step). This T cell dose was associated with consistent engraftment and acceptable rates of GVHD in our prior studies. CY 60 mg/kg/d was given on days -3 and -2. A CD34 selected donor product was infused on day 0 (HSC step). T cells were collected from the donors prior to the start of G-CSF for stem cell mobilization. Mycophenolate Mofetil and Tacrolimus were started on d-1. All patients received the targeted T cell dose of 2×10e8 cells/kg, and within 24 hours developed fevers (median peak temperature 103.8f), and in many cases diarrhea and rash. All of the symptoms from this alloreaction resolved after the 2nd dose of CY. The median CD34+/kg dose was 3.34 × 10e6/kg (range 1.4–8.94 × 10e6/kg). The median amount of residual (non-tolerized) T cells in the HSC product was 0.24 × 10e4/kg (range 0.03–4.5 × 10e4/kg). One patient developed hypotension during the alloreaction requiring steroids. There were no other unexpected infusion-related reactions. Five patients developed decreases in ejection fraction (EF), not always associated with clinical heart failure, and 5 patients developed atrial dysrhythmias. Two early deaths occurred from sepsis and decreased EF on days +2 and +11, both in patients with KPS of 70–80% but with HCT-CI scores of <3 at the time of transplant. Fourteen of 17 (82%) patients were discharged to home and one (6%) to a skilled nursing facility. After discharge, one patient experienced a late rejection and died of toxicity from a second HSCT. Two patients died from severe GVHD (liver and gut) on days +161 and +90. The remaining patients had grade 0 to II GVHD controlled either with steroids or steroids plus photopheresis. One patient died from pneumonia on day +177. Four patients died from relapsed disease on days +55, +186, +198, and +510. Seven total patients (41%) are alive and without evidence of disease 1 to 30 months (median 19 months) after HSCT. All but 1 of these patients had a KPS of > 90% at transplant and all surviving patients had HCT CI scores of <3. Three of 3 patients with HCT CI scores >3 have died. The only 2 patients without active disease at HSCT are both alive, 19 and 26 months post HSCT. Except for the most recently treated patient who is not yet evaluable, all of these older patients have resumed their baseline activities of daily living. Haploidentical RIC using this 2 Step protocol is a viable treatment option for older adults. Rates of significant GVHD were acceptable and the majority of patients were able to tolerate the transplant procedures and be discharged to their homes. Based on the outcomes of this small group of patients, this type of therapy should be offered to fit senior adults with a low co-morbidity index especially if they have achieved a complete remission with up front therapy but are at high risk for relapse in the absence of HSCT. Disclosures: No relevant conflicts of interest to declare.

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