Adoptive cellular therapies with T cells are increasingly used to treat a variety of conditions. For instance, in a recent phase 1/2 trial, we prophylactically administered multivirusspecific T... Show moreAdoptive cellular therapies with T cells are increasingly used to treat a variety of conditions. For instance, in a recent phase 1/2 trial, we prophylactically administered multivirusspecific T-cell products to protect recipients of T-cell-depleted allogeneic stem cell grafts against viral reactivation. To establish treatment efficacy, it is important to determine the fate of the individual transferred T-cell populations. However, it is difficult to unequivocally distinguish progeny of the transferred T-cell products from recipient- or stem cell graft-derived T cells that survived T-cell depletion during conditioning or stem cell graft manipulation. Using messenger RNA sequencing of the T-cell receptor beta-chains of the individual virus-specific T-cell populations within these T-cell products, we were able to track the multiple clonal virus-specific subpopulations in peripheral blood and distinguish recipient- and stem cell graft-derived virus-specific T cells from the progeny of the infused T-cell products. We observed in vivo expansion of virus-specific T cells that were exclusively derived from the T-cell products with similar kinetics as the expansion of virus-specific T cells that could also be detected before the T-cell product infusion. In addition, we demonstrated persistence of virus-specific T cells derived from the T-cell products in most patients who did not show viral reactivation. This study demonstrates that virus-specific T cells from prophylactically infused multiantigen-specific T-cell products can expand in response to antigen encounter in vivo and even persist in the absence of early viral reactivation. Show less
Since multiple different T-cell receptor (TCR) sequences can bind to the same peptide-MHC combination and the number of TCR-sequences that can theoretically be generated even exceeds the number of... Show moreSince multiple different T-cell receptor (TCR) sequences can bind to the same peptide-MHC combination and the number of TCR-sequences that can theoretically be generated even exceeds the number of T cells in a human body, the likelihood that many public identical (PUB-I) TCR-sequences frequently contribute to immune responses has been estimated to be low. Here, we quantitatively analyzed the TCR-repertoires of 190 purified virus-specific memory T-cell populations, directed against 21 epitopes of Cytomegalovirus, Epstein-Barr virus and Adenovirus isolated from 29 healthy individuals, and determined the magnitude, defined as prevalence within the population and frequencies within individuals, of PUB-I TCR and of TCR-sequences that are highly-similar (PUB-HS) to these PUB-I TCR-sequences. We found that almost one third of all TCR nucleotide-sequences represented PUB-I TCR amino-acid (AA) sequences and found an additional 12% of PUB-HS TCRs differing by maximally 3 AAs. We illustrate that these PUB-I and PUB-HS TCRs were structurally related and contained shared core-sequences in their TCR-sequences. We found a prevalence of PUB-I and PUB-HS TCRs of up to 50% among individuals and showed frequencies of virus-specific PUB-I and PUB-HS TCRs making up more than 10% of each virus-specific T-cell population. These findings were confirmed by using an independent TCR-database of virus-specific TCRs. We therefore conclude that the magnitude of the contribution of PUB-I and PUB-HS TCRs to these virus-specific T-cell responses is high. Because the T cells from these virus-specific memory TCR-repertoires were the result of successful control of the virus in these healthy individuals, these PUB-HS TCRs and PUB-I TCRs may be attractive candidates for immunotherapy in immunocompromised patients that lack virus-specific T cells to control viral reactivation. Show less
HLA-matched allogeneic stem cell transplantation (alloSCT) is a potential curative therapy for patients with high-risk hematologic malignancies. The therapeutic effect of alloSCT is mediated by T... Show moreHLA-matched allogeneic stem cell transplantation (alloSCT) is a potential curative therapy for patients with high-risk hematologic malignancies. The therapeutic effect of alloSCT is mediated by T cells derived from the stem cell donor that have the potential to recognize and eliminate malignant cells of patient origin, better known as the graft-versus-leukemia effect. Besides, donor-derived T cells are of importance for the protection against viruses early after alloSCT. On the other hand, T cells of donor origin can also initiate harmful graft-versus-host disease if healthy cells of the patient are recognized. Therefore, the major challenge in the field of alloSCT is to find a balance between graft-versus-leukemia effect and viral protection versus graft-versus-host disease with the aim to reduce complications early after alloSCT. In this thesis, the manipulation of donor-derived T cells at different stages during the process of HLA-matched alloSCT was investigated: from in vitro T-cell depletion of the graft before infusion into the patient until adoptive T-cell therapy early after alloSCT to support anti-viral immunity and graft-versus-leukemia effect. Show less
Roex, M.C.J.; Wijnands, C.; Veld, S.A.J.; Egmond, E. van; Bogers, L.; Zwaginga, J.J.; ... ; Jedema, I. 2021
Background aims: To reduce the risk of graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (alloSCT), T-cell depletion (TCD) of grafts can be performed by the addition of... Show moreBackground aims: To reduce the risk of graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (alloSCT), T-cell depletion (TCD) of grafts can be performed by the addition of alemtuzumab (ALT) "to the bag" (in vitro) before transplantation. In this prospective study, the authors analyzed the effect of in vitro incubation with 20 mg ALT on the composition of grafts prior to graft infusion. Furthermore, the authors assessed whether graft composition at the moment of infusion was predictive for T-cell reconstitution and development of GVHD early after TCD alloSCT.Methods: Sixty granulocyte colony-stimulating factor-mobilized stem cell grafts were obtained from >= 9/10 HLA-matched related and unrelated donors. The composition of the grafts was analyzed by flow cytometry before and after in vitro incubation with ALT. T-cell reconstitution and incidence of severe GVHD were monitored until 12 weeks after transplantation.Results: In vitro incubation of grafts with 20 mg ALT resulted in an initial median depletion efficiency of T-cell receptor (TCR) alpha/beta T cells of 96.7% (range, 63.5-99.8%), followed by subsequent depletion in vivo. Graft volumes and absolute leukocyte counts of grafts before the addition of ALT were not predictive for the efficiency of TCR alpha/beta T-cell depletion. CD4(pos) T cells were depleted more efficiently than CD8(pos) T cells, and naive and regulatory T cells were depleted more efficiently than memory and effector T cells. This differential depletion of T-cell subsets was in line with their reported differential CD52 expression. In vitro depletion efficiencies and absolute numbers of (naive) TCR alpha/beta T cells in the grafts after ALT incubation were not predictive for T cell reconstitution or development of GVHD postalloSCT.Conclusions: The addition of ALT to the bag is an easy, fast and generally applicable strategy to prevent GVHD in patients receiving alloSCT after myeloablative or non-myeloablative conditioning because of the efficient differential depletion of donor-derived lymphocytes and T cells. (C) 2020 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved. Show less
Roex, M.C.J.; Hageman, L.; Veld, S.A.J.; Egmond, E. van; Hoogstraten, C.; Stemberger, C.; ... ; Jedema, I. 2020
Tumor-associated antigens (TAAs) are monomorphic self-antigens that are proposed as targets for immunotherapeutic approaches to treat malignancies. We investigated whether T cells with sufficient... Show moreTumor-associated antigens (TAAs) are monomorphic self-antigens that are proposed as targets for immunotherapeutic approaches to treat malignancies. We investigated whether T cells with sufficient avidity to recognize naturally overexpressed self-antigens in the context of self-HLA can be found in the T-cell repertoire of healthy donors. Minor histocompatibility antigen (MiHA)-specific T cellswere used as a model, as the influence of thymic selection on the T-cell repertoire directed against MiHA can be studied in both self (MiHA(pos) donors) and non-self (MiHA(neg) donors) backgrounds. T-cell clones directed against the HLA*02:01-restricted MiHA HA-1H were isolated from HA-1H(neg)/HLA-A*02:01(pos) and HA1H(pos)/HLA-A*02:01(pos) donors. Of the 16 uniqueHA-1H-specificT-cell clones, five T-cell clones derived from HA-1H(neg)/HLA-A*02:01(pos) donors and one T-cell clone derived from an HA1H(pos)/HLA-A*02:01(pos) donor showed reactivity against HA-1H(pos) target cells. In addition, in total, 663 T-cell clones (containing at least 91 unique clones expressing different T-cell receptors) directed against HLA*02:01-restricted peptides of TAA WT1-RMF, RHAMM-ILS, proteinase-3-VLQ, PRAME-VLD, and NY-eso-1-SLL were isolated from HLA-A*02:01(pos) donors. Only 3 PRAME-VLD-specific and one NY-eso-1-SLL-specific T-cell clone provoked interferon-g production and/or cytolysis upon stimulation with HLA-A*02:01(pos) malignant cell lines (but not primary malignant samples) naturally overexpressing the TAA. These results show that self-HLA-restricted T cells specific for selfantigens such as MiHA in MiHA(pos) donors and TAAs are present in peripheral blood of healthy individuals. However, clinical efficacy would require highly effective in vivo priming by peptide vaccination in the presence of proper adjuvants or in vitro expansion of the low numbers of self-antigen-specific T cells of sufficient avidity to recognize endogenously processed antigen. (Blood. 2020;136(4):455-467) Show less
Roex, M.C.J.; Balen, P. van; Germeroth, L.; Hageman, L.; Egmond, E. van; Veld, S.A.J.; ... ; Falkenburg, J.H.F. 2020
Prophylactic infusion of selected donor T cells can be an effective method to restore specific immunity after T-cell-depleted allogeneic stem cell transplantation (TCD-alloSCT). In this phase I/II... Show moreProphylactic infusion of selected donor T cells can be an effective method to restore specific immunity after T-cell-depleted allogeneic stem cell transplantation (TCD-alloSCT). In this phase I/II study, we aimed to reduce the risk of viral complications and disease relapses by administrating donor-derived CD8(pos) T cells directed against cytomegalovirus (CMV), Epstein-Barr virus (EBV) and adenovirus antigens, tumor-associated antigens (TAA) and minor histocompatibility antigens (MiHA). Twenty-seven of thirty-six screened HLA-A*02:01(pos) patients and their CMVpos and/or EBVpos donors were included. Using MHC-I-Streptamers, 27 T-cell products were generated containing a median of 5.2 x 10(6) cells. Twenty-four products were administered without infusion-related complications at a median of 58 days post alloSCT. No patients developed graft-versus-host disease during follow-up. Five patients showed disease progression without coinciding expansion of TAA/MiHA-specific T cells. Eight patients experienced CMV- and/or EBV-reactivations. Four of these reactivations were clinically relevant requiring antiviral treatment, of which two progressed to viral disease. All resolved ultimately. In 2/4 patients with EBV-reactivations and 6/8 patients with CMV-reactivations, viral loads were followed by the expansion of donor-derived virus target-antigen-specific T cells. In conclusion, generation of multi-antigen-specific T-cell products was feasible, infusions were well tolerated and expansion of target-antigen-specific T cells coinciding viral reactivations was illustrated in the majority of patients. Show less
Roex, M.C.J.; Balen, P. van; Germeroth, L.; Hageman, L.; Egmond, E. van; Veld, S.A.J.; ... ; Falkenburg, J.H.F. 2018
