Abstract
Establishing and maintaining tolerance to self-antigens or innocuous foreign antigens is vital for the preservation of organismal health. Within the thymus, medullary thymic epithelial cells ...(mTECs) expressing autoimmune regulator (AIRE) have a critical role in self-tolerance through deletion of autoreactive T cells and promotion of thymic regulatory T (T
reg
) cell development
1–4
. Within weeks of birth, a separate wave of T
reg
cell differentiation occurs in the periphery upon exposure to antigens derived from the diet and commensal microbiota
5–8
, yet the cell types responsible for the generation of peripheral T
reg
(pT
reg
) cells have not been identified. Here we describe the identification of a class of RORγt
+
antigen-presenting cells called Thetis cells, with transcriptional features of both mTECs and dendritic cells, comprising four major sub-groups (TC I–TC IV). We uncover a developmental wave of Thetis cells within intestinal lymph nodes during a critical window in early life, coinciding with the wave of pT
reg
cell differentiation. Whereas TC I and TC III expressed the signature mTEC nuclear factor AIRE, TC IV lacked AIRE expression and was enriched for molecules required for pT
reg
generation, including the TGF-β-activating integrin αvβ8. Loss of either major histocompatibility complex class II (MHCII) or ITGB8 by Thetis cells led to a profound impairment in intestinal pT
reg
differentiation, with ensuing colitis. By contrast, MHCII expression by RORγt
+
group 3 innate lymphoid cells (ILC3) and classical dendritic cells was neither sufficient nor required for pT
reg
generation, further implicating TC IV as the tolerogenic RORγt
+
antigen-presenting cell with an essential function in early life. Our studies reveal parallel pathways for the establishment of tolerance to self and foreign antigens in the thymus and periphery, respectively, marked by the involvement of shared cellular and transcriptional programmes.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Bone marrow transplantation (BMT) offers curative potential for patients with high-risk hematologic malignancies, but the post-transplantation period is characterized by profound immunodeficiency. ...Recent studies indicate that the intestinal microbiota not only regulates mucosal immunity, but can also contribute to systemic immunity and hematopoiesis. Using antibiotic-mediated microbiota depletion in a syngeneic BMT mouse model, here we describe a role for the intestinal flora in hematopoietic recovery after BMT. Depletion of the intestinal microbiota resulted in impaired recovery of lymphocyte and neutrophil counts, while recovery of the hematopoietic stem and progenitor compartments and the erythroid lineage were largely unaffected. Depletion of the intestinal microbiota also reduced dietary energy uptake and visceral fat stores. Caloric supplementation through sucrose in the drinking water improved post-BMT hematopoietic recovery in mice with a depleted intestinal flora. Taken together, we show that the intestinal microbiota contribute to post-BMT hematopoietic reconstitution in mice through improved dietary energy uptake.
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•Intestinal microbiota depletion impairs hematopoiesis after bone marrow transplantation•Intestinal flora depletion decreases energy harvest and reduces visceral adipose tissue•Caloric supplementation rescues impaired hematopoiesis in microbiota-depleted mice•The effects of intestinal flora disruption are dose dependent
Intestinal bacteria can exert effects on systemic hematopoiesis. Staffas et al. show that the intestinal flora contributes to hematopoietic recovery after bone marrow transplantation (BMT) through improved dietary energy uptake. The findings suggest possible clinical intervention strategies for improved BMT outcomes.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The transfer of T cell receptor (TCR) genes into patient T cells is a promising approach for the treatment of both viral infections and cancer. Although efficient methods exist to identify antibodies ...for the treatment of these diseases, comparable strategies to identify TCRs have been lacking. We have developed a high-throughput DNA-based strategy to identify TCR sequences by the capture and sequencing of genomic DNA fragments encoding the TCR genes. We establish the value of this approach by assembling a large library of cancer germline tumor antigen-reactive TCRs. Furthermore, by exploiting the quantitative nature of TCR gene capture, we show the feasibility of identifying antigen-specific TCRs in oligoclonal T cell populations from either human material or TCR-humanized mice. Finally, we demonstrate the ability to identify tumor-reactive TCRs within intratumoral T cell subsets without knowledge of antigen specificities, which may be the first step toward the development of autologous TCR gene therapy to target patient-specific neoantigens in human cancer.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
T cell reconstitution correlates with outcomes in patients undergoing stem cell transplantation (HCT). A functional thymus is needed for the maturation of T cells, but unfortunately, the thymus is ...highly sensitive to acute injury, such as cytoreductive conditioning given to HCT patients. To enhance T cell reconstitution, it is therefore imperative to explore therapies that can boost thymic regeneration and T cell output. Regulatory T cells (Tregs) are known to mediate homeostasis and tissue regeneration in a variety of tissues, but their role in thymic injury is not known. This study aims to shed light on the role of regulatory T cells (Tregs) in thymic regeneration.
Using a model of thymic injury and regeneration in mice receiving sublethal total body irradiation (SL-TBI, 550cGy), we observed a significant increase in Tregs ( Fig. 1A), located in both the medullary and cortical areas of the thymus on multiparameter immunohistochemistry. Employing a targeted ablation of Tregs at the time of injury using the FOXP3DTR mouse model, we could observe significant delay in thymic regeneration in mice depleted of Tregs (day 7 post SL-TBI, p=<0.0001, n=10, Fig. 1B).
RAG2GFP mice, where GFP is expressed under the RAG2 promoter, have been used to discern recirculating and de-novo produced Tregs. To characterize the nature of the Treg expansion, we treated RAG2GFP mice with SL-TBI and analyzed their thymus at multiple time points after injury. Interestingly, the increase in Tregs was mainly due to recirculating RAG2-GFP- Tregs but not de-novo produced RAG2+GFP+ Tregs after injury. Brdu administration after injury indicated proliferation of these RAG2-GFP- Tregs. Parabiotic experiments linking RAG2GFP mice expressing either the congenic marker CD45.1 or CD45.2 confirmed the recirculating nature of these cells, both at steady state and after injury. Furthermore, when adoptively transferring sorted Thymic Tregs from FOXP3RFP mice to mice receiving SL-TBI, we significantly enhanced thymic cellularity after injury and homing of these Tregs to the thymus.
To discern the transcriptional profile of these cells, we used single-cell sequencing of CD45+ RAG2- cells in 2- and 18-month-old RAG2GFP mice before and at day 1, 4, and 7 after sublethal irradiation. This enabled us to discover the age- and injury-associated changes in recirculating T cells. While the aged thymus had a similar surge of Treg numbers after injury, this response was blunted compared to their young counterparts.
Factor normalization methods enabled us to find two distinct gene regulatory networks highly specific to the RAG2-GFP-Tregs. One gene network was highly enriched for Zfp36l1, a poised RNA known to mediate cell stability. Selectively ablating Zfp36l1 in Tregs using the Foxp3Cre ZFP36L1 flox/flox model impaired regeneration significantly after SL-TBI. The other distinct RAG2-GFP- Treg specific gene network was enriched for the regenerative factor Amphiregulin ( Areg). Amphiregulin was seen to be increased in thymic supernatants, and its receptor Egfr was seen to be transcriptionally upregulated in thymic epithelial cells after injury. In parallel, the most significantly upregulated pathway using cell-to-cell interaction prediction models on single-cell data indicated a strong likelihood of Treg to thymic epithelial cell interactions through amphiregulin to Egfr. Functional inhibition of amphiregulin with an antibody enabled us to observe delayed thymic regeneration, indicating amphiregulin as a factor in the regenerative capacity of thymic recirculating Tregs.
Mapping human gene orthologs of the gene regulatory program provided by factor normalization in murine RAG2GFP- Tregs on single cell data of human pediatric thymic samples from children undergoing open heart surgeries enabled us to distinguish a putatively analogous human population of recirculating thymic Tregs. This population was found to be amphiregulin positive. And human thymic epithelial cell cultures with recombinant amphiregulin enhanced the growth of human thymic epithelial cells, as measured by life imaging, and promoted signalling through MAPK ERK1/2.
Taken together, our findings reveal a novel role for injury-resistant recirculating thymic regulatory T cells existing in both the human and murine thymus and may enable the development of new therapeutic targets for thymic regeneration for patients undergoing cytoreductive therapies.
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IJS, IMTLJ, KISLJ, NLZOH, NUK, SBCE, SBJE, UL, UM, UPUK, ZRSKP
Human leukocyte antigen (HLA) class I molecules generally present peptides (p) of 8 to 11 amino acids (aa) in length. Although an increasing number of examples with lengthy (>11 aa) peptides, ...presented mostly by HLA-B alleles, have been reported. Here we characterize HLA-A*02:01 restricted, in addition to the HLA-B*0702 and HLA-B*4402 restricted, lengthy peptides (>11 aa) arising from the B-cell ligandome. We analyzed a number of 15-mer peptides presented by HLA-A*02:01, and confirmed pHLA-I formation by HLA folding and thermal stability assays. Surprisingly the binding affinity and stability of the 15-mer epitopes in complex with HLA-A*02:01 were comparable with the values observed for canonical length (8 to 11 aa) HLA-A*02:01-restricted peptides. We solved the structures of two 15-mer epitopes in complex with HLA-A*02:01, within which the peptides adopted distinct super-bulged conformations. Moreover, we demonstrate that T-cells can recognize the 15-mer peptides in the context of HLA-A*02:01, indicating that these 15-mer peptides represent immunogenic ligands. Collectively, our data expand our understanding of longer epitopes in the context of HLA-I, highlighting that they are not limited to the HLA-B family, but can bind the ubiquitous HLA-A*02:01 molecule, and play an important role in T-cell immunity.
Background: The impact of long epitopes on T-cell immunity remains unclear.
Results: We identified and characterized 15-mer epitopes restricted to HLA-A*02:01.
Conclusion: HLA-A*02:01, in addition to the HLA-B family, can bind long epitopes that represent new antigenic targets for CD8+ T-cells.
Significance: The characterization of 15-mer epitopes restricted to HLA-A*02:01 expands our knowledge of the HLA-ligandome.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The graft vs. leukemia (GvL) effect as a method of preventing relapse is well described after allogeneic hematopoietic cell transplantation (HCT), but the mechanisms to this effect and how tumor ...sometimes develops resistance to GvL are just beginning to be understood. This article reviews and expands upon data presented at the Third International Workshop on Biology, Prevention and Treatment of Relapse after Stem Cell Transplantation held in Hamburg, Germany, in November 2016. We first discuss in detail the role that T-cell impairment early after HCT plays in relapse by looking at data from T cell-depleted approaches as well as the clear role that early T-cell recovery has shown in improving outcomes. We then review key findings regarding the role of specific KIR donor/recipient pairings that contribute to relapse prevention after HCT for several tumor types. Finally, we discuss a unique mouse model following the development of tumor resistance to GvL. Detailed molecular characterization of events marking the development of tumor resistance to the immunotherapy of GvL may help in developing future strategies to overcome immune escape.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
The thymus is extremely sensitive to exogenous insults but has a remarkable capacity to regenerate which is lost with age. Reactive oxygen species (ROS) accumulate early after tissue damage and ...despite their toxic potential, ROS and their byproducts (such as lipid peroxidation products-LPPs) can act as regeneration signals by activating membrane or intracellular sensors and subsequent stress-response signalling pathways. Using Sublethal Total Body Irradiation (SL-TBI) as a model of acute thymic injury, we found a rapid accumulation of thymic ROS as well as lipid peroxidation products on cell membranes after SLTBI (Figure 1A&B).
The damage-sensing ion channel Transient Receptor Potential cation channel family A member 1 (TRPA1) represents one of the major damage sensing receptors that can mediate cellular responses to oxidative stress mediators, such as LPPs. Using immunofluorescence (IF) microscopy we found that TRPA1 is enriched in the thymic medulla. Interestingly, although TRPA1 has been classically identified in nociceptive fibers, the major TRPA1 expressing structures in the thymus were not nerve fiber terminals, but primarily thymic endothelial cells (Figure1C), fibroblasts and subsets of epithelial cells. We have recently demonstrated that thymic endothelial cells can regulate regeneration through secretion of BMP-4, which can enhance Foxn1 expression and proliferation of thymic epithelial cells. In order to assess the functional role of TRPA1 in thymic regeneration after injury, we utilized TRPA1 knockout (TRPA1-/-) mice and quantified thymic reconstitution after SL-TBI. TRPA1-/- mice had significantly lower thymic cellularity compared to their age- and sex-matched WT controls, suggesting an association between TRPA1 deficiency and delayed endogenous thymic recovery (Figure 1D). The major deficit in thymocyte counts primarily affected double negative-4 (DN4), double positive (DP) and CD4+ single positive (SP-CD4+) thymocyte numbers. The thymic stroma of TRPA1-/- mice had lower endothelial cell and fibroblast counts (Figure 1D). In accordance with these findings drinking water administration of the TRPA1 agonist Allyl-Isothiocyanate (AITC), resulted in enhanced thymic regeneration after radiation exposure. Besides its positive effects on thymocyte counts, AITC significantly augmented endothelial cell counts after irradiation (Figure 1E).
In conclusion these results suggest that TRPA1 plays a non-redundant role in thymic regeneration and that exogenous TRPA1 stimulation can enhance immune recovery after damage.
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van den Brink:Seres: Research Funding; Jazz Pharmaceuticals: Consultancy; PureTech Health: Consultancy; Therakos Institute: Other: Speaking engagement.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Immunotherapy for hematological malignancies or solid tumors by administration of monoclonal antibodies or T cells engineered to express chimeric antigen receptors or T-cell receptors (TCRs) has ...demonstrated clinical efficacy. However, antigen-loss tumor escape variants and the absence of currently targeted antigens on several malignancies hamper the widespread application of immunotherapy. We have isolated a TCR targeting a peptide of the intracellular B cell–specific transcription factor BOB1 presented in the context of HLA-B*07:02. TCR gene transfer installed BOB1 specificity and reactivity onto recipient T cells. TCR-transduced T cells efficiently lysed primary B-cell leukemia, mantle cell lymphoma, and multiple myeloma in vitro. We also observed recognition and lysis of healthy BOB1-expressing B cells. In addition, strong BOB1-specific proliferation could be demonstrated for TCR-modified T cells upon antigen encounter. Furthermore, clear in vivo antitumor reactivity was observed of BOB1-specific TCR-engineered T cells in a xenograft mouse model of established multiple myeloma. Absence of reactivity toward a broad panel of BOB1− but HLA-B*07:02+ nonhematopoietic and hematopoietic cells indicated no off-target toxicity. Therefore, administration of BOB1-specific TCR-engineered T cells may provide novel cellular treatment options to patients with B-cell malignancies, including multiple myeloma.
•Isolation and characterization of a high-affinity TCR targeting the intracellular B cell–specific transcription factor BOB1.•T cells expressing a BOB1-specific TCR lysed and eradicated primary multiple myeloma and other B-cell malignancies in vitro and in vivo.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
CAR T cell therapy has shown great promise for the treatment of B cell malignancies. However, antigen-negative escape variants often cause disease relapse, necessitating the development of ...multi-antigen-targeting approaches. We propose that a T cell receptor (TCR)-based strategy would increase the number of potential antigenic targets, as peptides from both intracellular and extracellular proteins can be recognized. Here, we aimed to isolate a broad range of promising TCRs targeting multiple antigens for treatment of B cell malignancies. As a first step, 28 target genes for B cell malignancies were selected based on gene expression profiles. Twenty target peptides presented in human leukocyte antigen (HLA)-A∗01:01, -A∗24:02, -B∗08:01, or -B∗35:01 were identified from the immunopeptidome of B cell malignancies and used to form peptide-HLA (pHLA)-tetramers for T cell isolation. Target-peptide-specific CD8 T cells were isolated from HLA-mismatched healthy donors and subjected to a stringent stepwise selection procedure to ensure potency and eliminate cross-reactivity. In total, five T cell clones specific for FCRL5 in HLA-A∗01:01, VPREB3 in HLA-A∗24:02, and BOB1 in HLA-B∗35:01 recognized B cell malignancies. For all three specificities, TCR gene transfer into CD8 T cells resulted in cytokine production and efficient killing of multiple B cell malignancies. In conclusion, using this systematic approach we successfully identified three promising TCRs for T cell therapy against B cell malignancies.
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TCR gene therapy is an attractive approach to complement CAR T cell therapies and generate multi-antigen-targeting treatments for B cell malignancies. Following identification of target genes and epitopes Meeuwsen et al. identified three novel TCRs specific for FCRL5, VPREB3, and BOB1. T cells modified with these TCRs specifically lyse patient-derived B cell malignancies.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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