Autologous stem cell transplantation aids autoimmune patients by functional renewal and TCR diversification of regulatory T cells

Key Points Autologous HSCT induces functional renewal of regulatory T cells as well as a strong Treg TCR diversification in autoimmune patients. Adding regulatory T cells to the graft does not lead to additional clinical improvement but results in delayed donor T-cell reconstitution.

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AB0755 THE FIRST CASE OF AN AUTOLOGOUS STEM CELL TRANSPLANTATION FOR AN EXTREMELY REFRACTORY PROGRESSIVE SYSTEMIC JUVENILE IDIOPATHIC ARTHRITIS PATIENT IN BRASÍLIA, BRAZIL

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2021-eular.4132 ◽

2021 ◽

Vol 80 (Suppl 1) ◽

pp. 1405.2-1405

Author(s):

M. Rodrigues Pereira Almeida ◽

S. Veloso ◽

A. Siqueira Afonso

Keyword(s):

T Cells ◽

Stem Cell ◽

Juvenile Idiopathic Arthritis ◽

T Cell ◽

Stem Cell Transplantation ◽

Clinical Improvement ◽

Cell Transplantation ◽

Autologous Stem Cell Transplantation ◽

Systemic Juvenile Idiopathic Arthritis ◽

Sustained Remission

Background:Systemic juvenile idiopathic arthritis (sJIA) is one of the most perplexing diseases of childhood, with a wide range of presentation features and severity. Advances in treatment have improved the outcome, such as the use of modulators of proinflammatory cytokines and their receptors, but in a considerable number of children, especially in polyarticular or sJIA, refractoriness tends to be progressive. As of late, extreme cases have required Autologous Stem Cell Transplantation (ASCT) as a last resort.Objectives:To describe an extremely refractory clinical evolution in a sAIJ patient, the first one to be submitted to an ASCT for an autoinflammatory condition in Brasília, Brazil’s capital.Methods:Case ReportThe patient was classified according to the Pediatric International League of Associations for Rheumatology for sJIA.I.T.S.M, female, 15-years-old, was diagnosed with sJIA at 6 with the onset of polyarthritis, daily fever for 6 weeks, rash and enlargement of liver and spleen. In the initial investigation infectious and neoplastic causes were ruled out.The use of prednisone and methotrexate lead to a preliminary effective clinical control, however after an year, there was clinical deterioration and etanercept was associated, leading to a short term improvement. At that time, in addition to anti-tumour necrosis factor medications, no other biological drugs were available in Brazil.Despite some brief periods of clinical improvement, after different regimes, the patient manifested recurrent flares. Since the diagnosis she has already been treated with:[1]Methotrexate (2011 to 2014*, 2016 to 2020). *In 2015, it was replaced by Leflunomide (up to May 2016, discontinued due to intolerance);[2]Etanercept (2012);[3]Cyclosporine (August to December 2012);[4]Adalimumab (2013);[5]Tocilizumab (May 2014 to January 2015);[6]Canaquinumab (March 2015 to April 2016);[7]Mycophenolate Mofetil (February 2016, discontinued in May 2017 due to start of Cyclophosphamide);[8]Rituximab (May 2016 to April 2017);[9]Cyclophosphamide at a dose of 2 grams /m2 as well as Filgrastim for mobilization and collection of stem cells (May 2017, in a attempt of ASCT), but the parents were unable to reach a mutual agreement to consent to the continuation of this therapy;[10]Cyclophosphamide (500mg /m2) in a mensal scheme application for nine consecutive months since June 2017, once was observed a clinical improvement after the intensive immunosuppression previous to ASCT attempt;[11]Several courses of methylprednisolone and human immunoglobulin since the initial diagnosis;[12]Abatacept (November 2019 to October 2020);[13]ASCT was suggested again in 2020, this time with the consent of all family members.Results:An intensive immunosuppression [Cyclophosphamide and anti-thymocyte globulin (ATG)] followed by ASCT (October 2020) resulted in apparent sustained remission. The patient’s evolution since the transplant has been optimistic. She is currently on a low dose prednisone prescription. To ensure a less profound depletion of T cells, a better control of systemic disease and antimicrobial and antiviral prophylaxis after transplantation, slow tapering of corticosteroids were performed.Conclusion:The goal of hematopoietic stem cell transplantation in patients with autoimmune disease is to reprogram the immune system with the eradication of autoreactive cells, renew the population of regulatory T cells and restore the diversity of T cell receptor function.ASCT has been used in some refractory children with sJIA as well as hematologic malignancy and some progressive autoimmune diseases. However it is associated with significant morbidity and mortality, due to prolonged and severe depression of T cell immunity.References:[1]D.M.C. Brinkman et al. Arthritis Care & Research (2007)[2]Voltarelli Júlio C. et al. Rev. Bras. Hematol. Hemoter. (2010)[3]Angelo De Cata et al. Clin Exp Med. (2016)[4]Joost F. Swart et al. Nature Reviews Rheumatology (2017)Acknowledgements:We kindly thank the Hematology service of Hospital da Criança de Brasília, where the patient was subjected to the autologous stem-cell transplantation.Disclosure of Interests:None declared

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γδ T-cell reconstitution after HLA-haploidentical hematopoietic transplantation depleted of TCR-αβ+/CD19+ lymphocytes

Blood ◽

10.1182/blood-2014-09-599423 ◽

2015 ◽

Vol 125 (15) ◽

pp. 2349-2358 ◽

Cited By ~ 145

Author(s):

Irma Airoldi ◽

Alice Bertaina ◽

Ignazia Prigione ◽

Alessia Zorzoli ◽

Daria Pagliara ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

T Cell ◽

B Cells ◽

Stem Cell Transplantation ◽

Γδ T Cell ◽

Cytomegalovirus Reactivation ◽

T Cell Reconstitution ◽

Hematopoietic Transplantation ◽

Key Points

Key Points Vδ1 and Vδ2 T cells promptly reconstitute in children given haploidentical stem cell transplantation depleted of αβ+ T and CD19+ B cells. Vδ1 cells are expanded in patients experiencing cytomegalovirus reactivation; ZOL potentiates Vδ2 killing against leukemia blasts.

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Human herpesvirus 6 viremia affects T-cell reconstitution after allogeneic hematopoietic stem cell transplantation

Blood Advances ◽

10.1182/bloodadvances.2017012724 ◽

2018 ◽

Vol 2 (4) ◽

pp. 428-432 ◽

Cited By ~ 15

Author(s):

Coco de Koning ◽

Rick Admiraal ◽

Stefan Nierkens ◽

Jaap Jan Boelens

Keyword(s):

Stem Cell ◽

T Cell ◽

Hematopoietic Stem Cell Transplantation ◽

Stem Cell Transplantation ◽

Cell Transplantation ◽

Human Herpesvirus ◽

Human Herpesvirus 6 ◽

Hematopoietic Stem ◽

T Cell Reconstitution ◽

Key Points

Key PointsOnly high HHV6 viremia (>105 copies/mL) affects late but not early T-cell reconstitution after HCT. Antivirals improve T-cell reconstitution probability in the context of HHV6 viremia after HCT.

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Prediction of T-cell reconstitution by assessment of T-cell receptor excision circle before allogeneic hematopoietic stem cell transplantation in pediatric patients

Blood ◽

10.1182/blood-2004-04-1405 ◽

2005 ◽

Vol 105 (2) ◽

pp. 886-893 ◽

Cited By ~ 52

Author(s):

Xiaohua Chen ◽

Raymond Barfield ◽

Ely Benaim ◽

Wing Leung ◽

James Knowles ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

T Cell ◽

Hematopoietic Stem Cell Transplantation ◽

Stem Cell Transplantation ◽

T Cell Receptor ◽

Cell Receptor ◽

Hematopoietic Stem ◽

Thymic Function ◽

T Cell Reconstitution

Abstract The extent and rapidity with which T cells are regenerated from graft-derived precursor cells directly influences the incidence of infection and the T-cell–based graft-versus-tumor effect. Measurement of T-cell receptor excision circles (TRECs) in peripheral blood is a means of quantifying recent thymic T-cell production and has been used after transplantation in many studies to estimate thymus-dependent T-cell reconstitution. We hypothesized that the quality of thymic function before transplantation affects thymus-dependent T-cell reconstitution after transplantation. We used real-time polymerase chain reaction (PCR) to quantify signal-joint TRECs (sjTRECs) before and after transplantation. T-cell reconstitution was evaluated by T-cell receptor β (TCRβ) CDR3 size spectratyping. We tested 77 healthy sibling donors and 244 samples from 26 pediatric recipients of allogeneic hematopoietic stem cell transplantation (AHSCT). Blood from the healthy donors contained 1200 to 155 000 sjTREC copies/mL blood. Patients who had greater than 1200 copies/mL blood before transplantation showed early recovery of sjTREC numbers and TCRβ repertoire diversity. In contrast, patients who had fewer than 1200 copies/mL blood before transplantation demonstrated significantly slower restoration of thymus-dependent T cells. We conclude that the rate of reconstitution of thymus-dependent T cells is dependent on the competence of thymic function in the recipients before transplantation. Therefore, pretransplantation measurement of sjTREC may provide an important tool for predicting thymus-dependent T-cell reconstitution after transplantation.

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Donor T Cells Maintain Myeloma-Immune Equilibrium after Autologous Stem Cell Transplantation and Concurrent Immunotherapy Promotes Cure

Blood ◽

10.1182/blood-2018-99-118058 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2031-2031

Author(s):

Simone A Minnie ◽

David Smith ◽

Kate H Gartlan ◽

Thomas S Watkins ◽

Kate A Markey ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

T Cell ◽

Stem Cell Transplantation ◽

Autologous Stem Cell Transplantation ◽

Median Survival ◽

Cd8 T Cells ◽

Cd8 T Cell ◽

Post Transplant

Abstract Autologous stem cell transplantation (ASCT) remains an important consolidation treatment for multiple myeloma (MM) patients, even in the era of novel agents. The prolongation of plateau-phase induced by ASCT is generally attributed to intensive cytoreduction. However, ASCT generates inflammation and profound lymphodepletion, which may result in hitherto unexpected immunological effects. To investigate potential immunological contributions to myeloma control after ASCT, we developed preclinical models of transplantation for MM using Vk*MYC myeloma that generates bony lytic lesions, a serum M band and marrow plasmacytosis that are hallmarks of clinical disease. Myeloma-bearing B6 recipients underwent myeloablative conditioning and were transplanted with naïve B6 bone marrow (BM) grafts with or without T cells from donors that were myeloma-naïve (SCT) or had low M bands at the time of harvest to mimic ASCT. Surprisingly, we demonstrate the broad induction of T cell-dependent myeloma control with enhanced median survival in recipients of grafts containing T cells compared to T cell depleted (TCD) BM alone (SCT= 91 days and ASCT > 100 days post-transplant vs TCD BM alone= 44 days; p<0.0001). Myeloma was most efficiently controlled when recipients were transplanted with memory T cells (CD44+) from autologous grafts (median survival: ASCT-CD44+ T cells >90 days post-transplant vs. CD44─ T cells = 50 days; p = 0.0006). Importantly, T cells adoptively transferred from recipients surviving > 120 days (MM-primed) protected secondary recipients compared to T cells from naïve donors (median survival: MM-primed > 120 days post-transplant vs 65 days naïve T cells; p = 0.0003). Furthermore, MM-primed CD8 T cells were restricted in TCR repertoire and provided protection in a myeloma clone-specific fashion, indicative of a tumor-specific T cell response. Despite this immune-mediated control of myeloma after SCT, progression still occurred in the majority of recipients. We phenotyped CD8+ T cells from the BM of MM-relapsed, MM-controlled and MM-free (that had never seen myeloma) mice 8 weeks after SCT. Expression of the inhibitory receptors, programmed cell death protein 1 (PD-1) and T cell immunoreceptor with Ig and ITIM domains (TIGIT) on BM CD8+ T-cells strongly correlated with myeloma cell number (r = 0.729, p<0.0001 and r = 0.796, p<0.0001 respectively). Additionally, the co-stimulatory/adhesion receptor CD226 (DNAM-1) was markedly downregulated as myeloma progressed (r = - 0.865, p<0.0001), as was interferon-γ secretion (r = - 0.76, p = 0.0022). t-SNE analysis confirmed an irreversible exhaustion signature at myeloma progression, characterized by the absence of DNAM-1 and co-expression of PD-1, TIM-3, TIGIT together with CD101 and CD38. Immune-checkpoint inhibition (CPI) early post-SCT, using antibodies against PD-1 or TIGIT facilitated long-term myeloma control (median survival in both treatment arms > 120 days post-SCT vs. 60 and 68 days respectively; p <0.05). Furthermore, TIGIT blockade limited CD8+ T cell exhaustion, increased CD107a and IFNγ secretion and expanded a memory CD8+ T cell population in the BM. Genetic deletion of either IFNγ or the IFNγ receptor from the donor graft resulted in dramatic myeloma progression after SCT. Consequently, treatment with a CD137 (4-IBB) agonist early after SCT profoundly augmented CD8+IFNγ+GranzymeB+ T-cell expansion in the BM, such that majority of treated animals eliminated myeloma and survived long-term. These data provide insights into an unappreciated mechanism of action of ASCT whereby myeloma immune-equilibrium is established and suggest that combination with immunotherapeutic strategies is a rational approach to generate long term disease control. Disclosures Smyth: Bristol Myers Squibb: Other: Research agreement; Tizona Therapeutics: Research Funding.

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Vaccination with DC/Multiple Myeloma Fusions in Conjunction with Stem Cell Transplantation.

Blood ◽

10.1182/blood.v110.11.578.578 ◽

2007 ◽

Vol 110 (11) ◽

pp. 578-578

Author(s):

David Avigan ◽

Jacalyn Rosenblatt ◽

Baldev Vasir ◽

Zekui Wu ◽

Adam Bissonnette ◽

...

Keyword(s):

Multiple Myeloma ◽

T Cells ◽

Stem Cell ◽

T Cell ◽

Regulatory T Cells ◽

Stem Cell Transplantation ◽

Cell Transplantation ◽

Tumor Immunity ◽

Myeloma Cells ◽

Post Transplant

Abstract Autologous transplantation results in the transient reversal of tumor mediated tolerance due to the reduction in disease bulk, the depletion of regulatory T cells, and in the increased presence of tumor reactive lymphocytes during the period of lymphopoietic reconstitution. As a result, cancer vaccines are being explored as a means of targeting residual myeloma cells following stem cell transplant. We have developed a cancer vaccine in which patient derived tumor cells are fused with autologous dendritic cells (DCs). In this way multiple tumor antigens are presented in the context of DC mediated costimulation. We are conducting a study in which patients with multiple myeloma (MM) undergo stem cell transplantation followed by vaccination with 3 doses of DC/MM fusions. DCs were generated from adherent mononuclear cells cultured with GM-CSF and IL-4 for 5–7 days and matured with TNFa. DCs strongly expressed costimulatory and maturation markers. Myeloma cells were isolated from bone marrow aspirates and were identified by their expression of CD38, CD138, and/or MUC1. DC and MM cells were fused with polyethylene glycol as previously described and fusion cells were quantified by determining the percentage of cells that coexpress unique DC and myeloma antigens. To date, 19 patients have been enrolled and 18 have completed vaccine generation. Mean yield of the DC and myeloma preparations was 1.84 × 108 and 8.3 × 107 cells, respectively. Mean fusion efficiency was 40% and the mean cell dose was 4.3 × 106 fusion cells. As a measure of their potency as antigen presenting cells, fusion cells prominently stimulated allogeneic T cell proliferation in vitro. Mean stimulation indexes were 12, 57, and 31 for T cells stimulated by myeloma cells, DCs, and fusion cells, respectively. Adverse events judged to be potentially vaccine related included injection site reactions, pruritis, myalgias, fever, chills, and tachycardia. Six patients have completed the follow up period and 3 patients are currently undergoing vaccination. All patients achieved a partial response to transplant. Three patients demonstrated resolution of post-transplant paraprotein levels following vaccination. One patient with highly aggressive disease who experienced disease progression in the early post-transplant period, demonstrated initial response and then stabilization of disease with vaccination. We are examining the effect of transplant and vaccination on measures of cellular immunity, anti-tumor immunity and levels or activated as compared to regulatory T cells. T cell response to PHA mitogen was transiently depressed post-transplant. In contrast, a transient increase was noted post-transplant in mean T cell expression of IFNγ in response to autologous myeloma cell lysate. In preliminary studies, a relative increase in the ratio of activated (CD4/CD25low) to regulatory (CD4/CD25high) T cells was observed. To date, all evaluable patients demonstrated evidence of vaccine stimulated anti-tumor immunity as manifested by a rise in IFNγ expression by CD4 and/or CD8+ T cells following ex vivo exposure to autologous tumor lysate. In this ongoing study, fusion cell vaccination in conjunction with stem cell transplantation has been well tolerated, induced anti-tumor immunity and clinical responses in patients with multiple myeloma.

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Altered T-Lymphocyte Biology Following High-Dose Melphalan and Autologous Stem Cell Transplantation With Implications for Adoptive T-Cell Therapy

Frontiers in Oncology ◽

10.3389/fonc.2020.568056 ◽

2020 ◽

Vol 10 ◽

Author(s):

Thomas Mika ◽

Swetlana Ladigan-Badura ◽

Abdelouahid Maghnouj ◽

Bakr Mustafa ◽

Susanne Klein-Scory ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

T Cell ◽

Stem Cell Transplantation ◽

Cell Transplantation ◽

Autologous Stem Cell Transplantation ◽

Cell Phenotype ◽

High Dose ◽

Car T ◽

The Impact

In relapsed and refractory multiple myeloma (MM), adoptive cell therapies (ACT) including CAR-T-cells are under clinical investigation. However, relapse due to T-cell exhaustion or limited persistence is an obstacle. Before ACT are considered in MM, high-dose (HD) melphalan followed by autologous stem-cell transplantation (autoSCT) has been administered in most clinical situations. Yet, the impact of HD chemotherapy on T-cells in MM with respect to ACT is unclear. In this study, T-lymphocytes’ phenotypes, expansion properties, lentiviral transduction efficacy, and gene expression were examined with special respect to patients following HD melphalan. Significant impairment of T-cells’ expansion and transduction rates could be demonstrated. Expansion was diminished due to inherent disadvantages of the predominant T-cell phenotype but restored over time. The quantitative fraction of CD27−/CD28− T-cells before expansion was predictive of T-cell yield. Following autoSCT, the transduction efficacy was reduced by disturbed lentiviral genome integration. Moreover, an unfavorable T-cell phenotype after expansion was demonstrated. In initial analyses of CD107a degranulation impaired T-cell cytotoxicity was detected in one patient following melphalan and autoSCT. The findings of our study have potential implications regarding the time point of leukapheresis for CAR-T-cell manufacturing. Our results point to a preferred interval of more than 3 months until patients should undergo cell separation for CAR-T therapy in the specific situation post-HD melphalan/autoSCT. Monitoring of CD27−/CD28− T-cells, has the potential to influence clinical decision making before apheresis in MM.

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Human Double-Negative Regulatory T Cells Modulate Effector Functions of Conventional T Cells By Selectively Blocking mTOR Signaling

Blood ◽

10.1182/blood-2018-99-112943 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2410-2410

Author(s):

Tabea Haug ◽

Michael Aigner ◽

Heiko Bruns ◽

Moritz Peuser ◽

Dorothea Gebhardt ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

T Cell ◽

Regulatory T Cells ◽

Stem Cell Transplantation ◽

Cell Transplantation ◽

Mtor Signaling ◽

Regulatory Function ◽

Acid Uptake ◽

Double Negative

Abstract Regulatory T cells play an important role in the maintenance of immune tolerance to self-antigens and are involved in modulating immune responses to promote resolution of inflammation. The population of TCRαβ+ CD4-/CD8- (double-negative, DN) T cells has attracted growing attention as a result of their potent immune regulatory function. In murine models, DN T cells were able to prevent rejection of allogeneic and xenogeneic organ grafts by effectively suppressing reactive T cells. In addition, DN T cells possess the capacity to resolve various inflammatory conditions, including graft-versus-host disease (GvHD) after allogeneic hematopoietic stem cell transplantation. Notably, first clinical studies in patients after stem cell transplantation demonstrated an inverse correlation between the frequency of circulating DN T cells and the severity of acute GvHD, suggesting a therapeutic potential of human DN T cells. To gain a better understanding of the molecular mechanism of suppression, we investigated whether human DN T cells modulate distinct signaling processes in conventional T cells. We found that DN T cells selectively block mTOR signaling but not activation of mitogen-activated protein kinases. Enforced activation of the mTOR pathway by a chemical activator rendered conventional T cells unsusceptible to DN T cell-mediated suppression, confirming the critical function of mTOR signaling. Given that mTOR is a major regulator of cellular metabolism, we further determined the impact of DN T cells on the metabolic framework of conventional T cells. Of interest, DN T cells diminished upregulation of the glycolytic machinery and glucose uptake in conventional T cells, whereas fatty acid uptake was not modified. Next, we investigated the fate and function of effector cells after DN T cell co-culture. Of importance, DN T cells suppressed proliferation but also altered expression of differentiation markers, transcription factors and homing receptors. Further analyses demonstrated that CD4+ T cells failed to produce effector cytokines IL-17 and IFN-γ after coculture with DN T cells, whereas IL-2 secretion was amplified. The selective modification was induced by a direct cell-cell contact dependent mechanism between CD4 and DN T cells and not as a consequence of competition for nutrients or growth factors. Together, our findings expand the understanding of DN T cell functionality and support that human DN T cells represent an interesting opportunity to limit and modulate T-cell reactivity. Disclosures No relevant conflicts of interest to declare.

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Increased Immune-Regulatory Receptor Expression on Effector T Cells as Early Indicators of Relapse Following Autologous Stem Cell Transplantation for Multiple Myeloma

Frontiers in Immunology ◽

10.3389/fimmu.2021.618610 ◽

2021 ◽

Vol 12 ◽

Author(s):

Lydia Lee ◽

Nouf Alrasheed ◽

Garima Khandelwal ◽

Evelyn Fitzsimons ◽

Huw Richards ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

T Cell ◽

Stem Cell Transplantation ◽

Cell Transplantation ◽

Autologous Stem Cell Transplantation ◽

Effector T Cells ◽

Early Relapse ◽

Tcr Repertoire ◽

Pre Treatment

The benefit of autologous stem cell transplantation (ASCT) in newly diagnosed myeloma patients, apart from supporting high dose chemotherapy, may include effects on T cell function in the bone marrow (BM). We report our exploratory findings on marrow infiltrating T cells early post-ASCT (day+100), examining phenotype and T cell receptor (TCR) repertoire, seeking correlations with timing of relapse. Compared to healthy donors (HD), we observed an increase in regulatory T cells (CD4+FoxP3+, Tregs) with reduction in CD4 T cells, leading to lower CD4:8 ratios. Compared to paired pre-treatment marrow, both CD4 and CD8 compartments showed a reduction in naïve, and increase in effector memory subsets, suggestive of a more differentiated phenotype. This was supported by increased levels of several immune-regulatory and activation proteins (ICOS, PD-1, LAG-3, CTLA-4 and GzmB) when compared with HD. Unsupervised analysis identified a patient subgroup with shorter PFS (p=0.031) whose BM contained increased Tregs, and higher immune-regulatory markers (ICOS, PD-1, LAG-3) on effector T cells. Using single feature analysis, higher frequencies of marrow PD-1+ on CD4+FoxP3- cells and Ki67+ on CD8 cells were independently associated with early relapse. Finally, studying paired pre-treatment and post-ASCT BM (n=5), we note reduced abundance of TCR sequences at day+100, with a greater proportion of expanded sequences indicating a more focused persistent TCR repertoire. Our findings indicate that, following induction chemotherapy and ASCT, marrow T cells demonstrate increased activation and differentiation, with TCR repertoire focusing. Pending confirmation in larger series, higher levels of immune-regulatory proteins on T cell effectors at day+100 may indicate early relapse.

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WT1-Specific T Cell Responses Are Frequent after Allogeneic Stem Cell Transplantation for Acute and Chronic Myeloid Leukemia.

Blood ◽

10.1182/blood.v106.11.598.598 ◽

2005 ◽

Vol 106 (11) ◽

pp. 598-598

Author(s):

Chiara Gentilini ◽

Urte Hilbers ◽

Susanne Ganepola ◽

Carmen Scheibenbogen ◽

Katy Freyberg ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

T Cell ◽

Regulatory T Cells ◽

Stem Cell Transplantation ◽

Allogeneic Stem Cell Transplantation ◽

T Cell Responses ◽

Cell Responses ◽

Hla A2.1 ◽

Wt1 Protein

Abstract The transcription factor WT1 is often overexpressed in leukemic cells and thus represents a tumor specific antigen which has already been used as a target for immunotherapeutic approaches. To investigate its role in graft-versus-leukemia effects after allogeneic stem cell transplantation (SCT), the frequency of T cells specific for WT1 was determined in 52 patients with myeloid malignancies before and after allogeneic SCT. Methods: Using unseparated PBMCs, T cell frequencies were assessed by the IFN-gamma ELISPOT assay at continuous intervals after dose reduced (n=15) or conventional transplantation (n=37). In case of HLA-A2.1-positive pts, a panel of 4 peptides (Db126126-134, WH187187-195, WH242240-250 and WT1 3737-45), known to represent HLA-A2.1-restricted epitopes of the WT1 protein was applied. In addition, we made use of a pool of overlapping peptides derived from the WT1 protein to assess responses in HLA-A2.1-negative patients and against other epitopes. HIV-specific (ILKEPVHGV) and CMVpp65-specific (NLVPMVATV) peptides served as negative and positive controls. In case of positive results in the ELISPOT, cells were further analyzed in tetramer binding assays. In order to detect the expression of the WT1 gene in tumor cells, a quantitative RT-PCR was carried out. Results: 11 patients with CML and 41 patients with AML/MDS were analyzed. In contrast to 17 conventionally treated patients with CML who showed no WT-1-specific reactivity, antigen-specific T cells were present in high frequency (up to 356:100.000) in 7 of 11 pts with CML up to 780 days after allogeneic transplantation. Further, T cell-responses against WT-1 peptides were found in 18 of 41 patients with AML after transplant with frequencies between 21 and 322 spots/100 000 PBMC (median 70) for Db126 and 20-356 spots/100 000 PBMC (median 93) for WH187. In contrast, only 20% of patients pre allo SCT (4/20) had a positive response against Db126 (median 144, range 111–160) and WH187 (median 151, range 122–174). Only 2 out of 17 normal subjects showed Db126 and WH187 specific CTLs with very weak frequencies (maximal T cell response 40 and 41 spots /100 000 PBMC). Reactivity against WT1 37 or WH 242 was exclusively observed following transplantation (in 2 and 5 out of 16 patients). In HLA A2.1 positive as well as negative recipients, the pool of overlapping WT1 peptides allowed us to detect WT1-reactive T cells in 3 of 9 patients. The number of WT1-reactive T cells increases soon after transplantation, and is maintained at different levels over a longer period of time. Concomitantly, the proportion and number of CD3+/Foxp3+ regulatory T cells was shown to be significantly reduced in all patients tested so far. Conclusion: T cell responses against WT1 are elicited short after allogeneic SCT. We speculate that the lack of regulatory T cells during the early phase after HSCT could be responsible for this phenomenon. To further enhance WT1 specific immune reactivity during this phase after HSCT, we have started a phase II clinical trial, in which patients with AML and MDS are vaccinated with the Db126 peptide.

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