Thymic Deletion Research Articles

Cancer Immunotherapy Using AIRE Conditioning of the Tumor Epitopeome.

T cell immune tolerance is established in part through the activity of the Auto-immune Regulator (AIRE) transcription factor in the medullary Thymic Epithelial Cells (mTEC) of the thymus. AIRE induces expression of SELF peripheral tissue-specific antigens for presentation to naïve T cells to promote activation/deletion of potentially autoreactive T cells. We show, for the first time to our knowledge, that tumors mimic the role of AIRE in mTEC to evade immune rejection. Thus, by expressing a broad range of SELF epitopes against which minimal functional T cell reactivities exist because of thymic deletion, AIRE acts as a master controller of SELFNESS, effectively cloaking the tumor from T cell attack. Moreover, we describe a completely novel immunotherapy in which engineered changes in AIRE expression in tumor cells alters their profile of SELFNESS, exposing both AIRE-modified, and parental unmodified, tumor cells to T cell attack. Consistent with our studies, patient RNAseq shows expression of AIRE predicts response to immune therapies with a strong correlation between AIRE expression and markers of TCR signaling. Therefore, by re-setting the immunological SELFNESS of cancer cells, this novel AIRE-mediated immunotherapy 1). converts a highly tolerized T cell compartment into a heteroclitic tumor-reactive T cell population; 2) confers de novo sensitivity to immune checkpoint blockade upon non-immunogenic tumors; 3). completely removes the need to identify potentially immunogenic tumor-associated antigens as targets for generation of de novo CD8 + and helper CD4 + T cell responses; and 4) leads to potent T cell-mediated rejection of aggressive, immunologically cold, non-immunogenic tumors.

Donor derived hematopoietic stem cell niche transplantation facilitates mixed chimerism mediated donor specific tolerance.

Compelling experimental evidence confirms that the robustness and longevity of mixed chimerism (MC) relies on the persistence and availability of donor-derived hematopoietic stem cell (HSC) niches in recipients. Based on our prior work in rodent vascularized composite allotransplantation (VCA) models, we hypothesize that the vascularized bone components in VCA bearing donor HSC niches, thus may provide a unique biologic opportunity to facilitate stable MC and transplant tolerance. In this study, by utilizing a series of rodent VCA models we demonstrated that donor HSC niches in the vascularized bone facilitate persistent multilineage hematopoietic chimerism in transplant recipients and promote donor-specific tolerance without harsh myeloablation. In addition, the transplanted donor HSC niches in VCA facilitated the donor HSC niches seeding to the recipient bone marrow compartment and contributed to the maintenance and homeostasis of stable MC. Moreover, this study provided evidences that chimeric thymus plays a role in MC-mediated transplant tolerance through a mechanism of thymic central deletion. Mechanistic insights from our study could lead to the use of vascularized donor bone with pre-engrafted HSC niches as a safe, complementary strategy to induce robust and stable MC-mediated tolerance in VCA or solid organ transplantation recipients.

Discovering how DO regulates lymphocyte function

HLA-DO or H-2O (DO) in mice is a non-classical, non-polymorphic MHC II molecule primarily expressed in the thymic medulla and B cells. While the common belief is that DO inhibits the activity of DM, controversial data opposes that point of view. Applying physicochemical studies to purified DO, DM, and HLA-DR1, we have demonstrated that DO interacts directly with HLA-DR1 molecules in a peptide-receptive-conformation, augmenting binding of ‘DM-resistant’ peptides, while inhibiting binding of ‘DM-sensitive’ peptides to HLA-DR1. Indeed, we have observed that DO-WT B cells express higher densities of pMHC from naturally processed protein antigens in vivo. Accordingly, DO-KO mice are more susceptible to development of EAE due to presence of larger numbers MOG/IAb-specific CD4 T cells in periphery, likely due to a less effective thymic deletion. Since DO is also expressed in B cells, and B cell antigen presentation is a critical step in differentiation of CD4 memory T cells, we tested the contributions of DO to the development of CD4 memory T cells. To do so, we used DR1+/DO-KO mice, and examined CD4 memory development to DR1 restricted flu specific T cells three months post flu vaccine immunization. We found that consistent with our hypothesis, the DR1+/DO-KO mice had fewer flu specific CD4 memory T cells than DR1+/DO-WT mice. By in vitro and in vivo recall experiments, we also found that the secondary immunological responses in DR1+/DO-KO mice were compromised. In addition, we recovered fewer memory B cells from immunized DR1+/DO-KO mice. These results demonstrate that in the absence of DO, the presentation of cognate foreign antigens in the DO-KO mice is altered and can potentially hamper the proper development of memory cells after the resolution of the infection. These findings support the idea that the expression of DO might ensure the proper development of memory cells.

Nfkbid Overexpression in Nonobese Diabetic Mice Elicits Complete Type 1 Diabetes Resistance in Part Associated with Enhanced Thymic Deletion of Pathogenic CD8 T Cells and Increased Numbers and Activity of Regulatory T Cells.

Type 1 diabetes (T1D) in both humans and NOD mice is caused by T cell-mediated autoimmune destruction of pancreatic β cells. Increased frequency or activity of autoreactive T cells and failures of regulatory T cells (Tregs) to control these pathogenic effectors have both been implicated in T1D etiology. Due to the expression of MHC class I molecules on β cells, CD8 T cells represent the ultimate effector population mediating T1D. Developing autoreactive CD8 T cells normally undergo extensive thymic negative selection, but this process is impaired in NOD mice and also likely T1D patients. Previous studies identified an allelic variant of Nfkbid, a NF-κB signal modulator, as a gene strongly contributing to defective thymic deletion of autoreactive CD8 T cells in NOD mice. These previous studies found ablation of Nfkbid in NOD mice using the clustered regularly interspaced short palindromic repeats system resulted in greater thymic deletion of pathogenic CD8 AI4 and NY8.3 TCR transgenic T cells but an unexpected acceleration of T1D onset. This acceleration was associated with reductions in the frequency of peripheral Tregs. In this article, we report transgenic overexpression of Nfkbid in NOD mice also paradoxically results in enhanced thymic deletion of autoreactive CD8 AI4 T cells. However, transgenic elevation of Nfkbid expression also increased the frequency and functional capacity of peripheral Tregs, in part contributing to the induction of complete T1D resistance. Thus, future identification of a pharmaceutical means to enhance Nfkbid expression might ultimately provide an effective T1D intervention approach.

Detection of Directly Alloreactive Graft Antigen-Specific CD4+ T Cells in Peripheral Blood

<h3>Purpose</h3> Because the peptide antigens that activate directly alloreactive CD4+ T cells are not known, the phenotype and effects of graft antigen specific CD4+ T cells cannot be established, limiting the ability to accurately diagnose and treat allograft rejection. We hypothesize that a panel of human peptides presented by mismatched human leukocyte antigens (HLA) can be used to detect directly alloreactive CD4+ T cells in peripheral blood. <h3>Methods</h3> We developed a panel of 18 human peptides (18p) presented by HLA-DRB1*04(DR4) in order generate phycoerythrin(PE)- and allophycocyanin(APC)-labelled 18p:DR4 tetramers(18p:DR4^tet). After magnetic bead enrichment of 18p:DR4^tet+ cells, PBMC were stained for non-T lineage-, CD3+, CD8-, CD4+ T cells and quantified using flow cytometry. PBMC were also expanded <i>in vitro</i> for 7 days using 18p with K562 artificial antigen presenting cells (AAPC) with or without DR4 expression before 18p:DR4^tet enrichment and staining as above. <h3>Results</h3> 100-fold more 18p:DR4^tet+ CD4+ T cells were detected directly <i>ex vivo</i> in PBMC from DR4- individuals (Figure 1B, C) than in DR4+ individuals (Figure 1A, C), consistent with thymic deletion of 18p:DR4^tet+ CD4+ T cells in DR4+ individuals and existence of directly alloreactive 18p:DR4^tet+ CD4+ T cells in DR4- individuals. Ten-fold peptide-dependent expansion of 18p:DR4^tet+ CD4+ T cells was seen from PBMC from DR4- but not DR4+ individuals cultured with K562-DR4 expressing AAPC (but not with K562 lacking DR4)(Figure 1D) demonstrating peptide and alloHLA specificity of directly alloreactive CD4+ T cells. <h3>Conclusion</h3> Directly alloreactive CD4+ T cells specific for human peptides presented by alloHLA are detectable directly <i>ex vivo</i> in PBMC of HLA-mismatched individuals. This novel tool will be used to detect and characterize directly alloreactive CD4+ T cells in the PBMC, as well as bronchoalveolar lavage and pleural fluid, from lung transplant recipients in order to better diagnose allograft rejection.

Candidiasis in patients with APS-1: low IL-17, high IFN-γ, or both?

Chronic mucocutaneous candidiasis (CMC) is one of the earliest and most frequent clinical manifestations of autosomal recessive autoimmune polyendocrine syndrome type 1 (APS-1), a monogenic inborn error of immunity caused by deleterious variants of the autoimmune regulator (AIRE) gene. APS-1 patients suffer from various autoimmune diseases, due to the defective thymic deletion of autoreactive T cells, and the development of a large range of autoantibodies (auto-Abs) against various tissue antigens, and some cytokines. The mechanisms underlying CMC remained elusive for many years, until the description in 2010 of high serum titers of neutralizing auto-Abs against IL-17A, IL-17F, and/or IL-22, which are present in almost all APS-1 patients. Excessively high mucosal concentrations of IFN-γ were recently proposed as an alternative mechanism for CMC in APS-1.

B7-CD28 co-stimulation enforces central tolerance by thymic clonal deletion and Treg cell generation through distinct mechanisms

Abstract To establish central T cell tolerance in the thymus, thymocytes expressing TCR strongly reactive to self-Ag-MHC complex are eliminated from the conventional T cell (Tconv) repertoire by two distinct processes, clonal deletion or clonal diversion to non-conventional T cell lineages such as Foxp3+ regulatory T (Treg) cells. We analyzed the requirements for B7-CD28 co-stimulation for thymic clonal deletion and Treg cell generation of endogenous tissue-restricted-antigen (TRA)-specific thymocytes. We found that: 1) both clonal deletion and Treg cell generation of TRA-specific thymocytes were B7-CD28-dependent, and that clonal deletion had a greater effect than Treg diversion in eliminating TRA-specific T cells from the Tconv repertoire; 2) B7-expressing APC requirements differed for clonal deletion and Treg generation; and 3) although B7-CD28 mediated both clonal deletion and Treg cell generation, CD28 cytoplasmic domain requirements for these two processes were different. These results indicate that qualitatively distinct CD28 signaling determines fates of strongly self-reactive thymocytes. To further characterize the CD28 signaling pathways involved in clonal deletion and Treg cell generation, single cell transcriptomic analyses of TRA-specific thymocyte are underway.

Loss of the Class II Accessory Molecule HLA-DO leads to altered TCR avidity

Abstract HLA-DO (DO in human; H2-O in mice) is a highly conserved non-classical MHC class II accessory molecule. Unlike the main Class II peptide editor HLA-DM, expression of DO is restricted to the thymic medulla, B cells and certain dendritic cell subsets. Although DO forms a stable complex with HLA-DM, its biological functions remain poorly understood. Previously, our lab provided biochemical evidence in support of a model where DO enhances the binding of DM-resistant peptides, and reduces the binding of DM-sensitive peptides to the HLA-DR1 (DR1) molecules in vitro. Changes in the quantities of either DM-resistant of DM-sensitive peptides could therefore lead to alterations in both thymic deletion, and peripheral activation of CD4 T cells. Here, using both in vivo antigen presentation as well as a novel avidity measurement tool, z-Movi™ (LUMICKS), we found that: (1) brain recovered APCs from diseased DO-KO mice present less of the MOG(35–55) epitope, (2) naïve DO-KO CD4 T cells have a more avid interaction with their own APCs, and (3) that hemagglutinin specific, DR1 restricted CD4 T cells had a less avid interaction with B cells from flu vaccinated DR1+DO-KO mice as compared to B cells from vaccinated DR1+DO-WT mice. These avidity changes could explain two things: (1) why we find that despite DR1+DO-KO mice have similar precursor levels of collagen specific CD4 T cells as DR1+DO-WT mice, they are more susceptible to the development of Collagen Induced Arthritis, even though we previously showed that the immunodominant collagen epitope is DM-sensitive. And, (2) why we find increased levels of regulatory T cells in the absence of DO.

Failure of thymic deletion and instability of autoreactive Tregs drive autoimmunity in immune-privileged liver.

The liver is an immune-privileged organ that can deactivate autoreactive T cells. Yet in autoimmune hepatitis (AIH), autoreactive T cells can defy hepatic control and attack the liver. To elucidate how tolerance to self-antigens is lost during AIH pathogenesis, we generated a spontaneous mouse model of AIH, based on recognition of an MHC class II–restricted model peptide in hepatocytes by autoreactive CD4+ T cells. We found that the hepatic peptide was not expressed in the thymus, leading to deficient thymic deletion and resulting in peripheral abundance of autoreactive CD4+ T cells. In the liver, autoreactive CD4+ effector T cells accumulated within portal ectopic lymphoid structures and maturated toward pathogenic IFN-γ and TNF coproducing cells. Expansion and pathogenic maturation of autoreactive effector T cells was enabled by a selective increase of plasticity and instability of autoantigen-specific Tregs but not of nonspecific Tregs. Indeed, antigen-specific Tregs were reduced in frequency and manifested increased IL-17 production, reduced epigenetic demethylation, and reduced expression of Foxp3. As a consequence, autoantigen-specific Tregs had a reduced suppressive capacity, as compared with that of nonspecific Tregs. In conclusion, loss of tolerance and the pathogenesis of AIH were enabled by combined failure of thymic deletion and peripheral regulation.

DOCK8 deficiency diminishes thymic T-regulatory cell development but not thymic deletion.

ObjectiveTo define the effect of DOCK8 deficiency on thymic tolerance in mice.MethodsThymocytes from wild‐type (Dock8+/+) and DOCK8‐deficient (Dock8pri/pri) mice were examined by flow cytometry. Some mice had transgenic expression of the BCL2 anti‐apoptotic protein in haemopoietic cells. Some mice expressed the transgenic 3A9 T‐cell receptor (TCR), which triggers thymocyte deletion in mice also expressing hen egg lysozyme under the insulin promoter.ResultsIn Dock8pr/pri mice, the proportion of thymocytes induced to acquire tolerance at the immature CCR7− stage was normal. Deletion of strongly self‐reactive CD4+ thymocytes occurred efficiently in Dock8pri/pri mice in a TCR‐transgenic model that requires self‐antigen transfer from epithelial cells to bone marrow (BM)‐derived antigen‐presenting cells. Thymic Foxp3+ T‐regulatory cells (TREG) and Helios+ Foxp3− TREG precursors were decreased in Dock8pri/pri mice, including when apoptosis was inhibited by BCL2 transgene expression. Dock8pri/pri thymic TREG expressed CD25 and CTLA‐4 at normal levels. The results suggest that DOCK8 deficiency does not affect the function of BM‐derived antigen‐presenting cells in the thymus, the TCR self‐reactivity threshold that activates tolerance mechanisms in thymocytes or the apoptotic deletion of these thymocytes. However, DOCK8 is required to prevent a subset of developing TREG cells from undergoing cell death via a mechanism that is distinct from apoptosis.ConclusionDOCK8 deficiency diminishes TREG development in the thymus without compromising thymocyte deletion.

RANK links thymic regulatory T cells to fetal loss and gestational diabetes in pregnancy.

Successful pregnancies rely on adaptations within the mother1, including marked changes within the immune system2. It has long been known that the thymus, the central lymphoid organ, changes markedly during pregnancy3. However, the molecular basis and importance of this process remain largely obscure. Here we show that the osteoclast differentiation receptor RANK4,5 couples female sex hormones to the rewiring of the thymus during pregnancy. Genetic deletion of Rank (also known as Tnfrsf11a) in thymic epithelial cells results in impaired thymic involution and blunted expansion of natural regulatory T (Treg) cells in pregnant female mice. Sex hormones, in particular progesterone, drive the development of thymic Treg cells through RANK in a manner that depends on AIRE+ medullary thymic epithelial cells. The depletion of Rank in the mouse thymic epithelium results in reduced accumulation of natural Treg cells in the placenta, and an increase in the number of miscarriages. Thymic deletion of Rank also results in impaired accumulation of Treg cells in visceral adipose tissue, and is associated with enlarged adipocyte size, tissue inflammation, enhanced maternal glucose intolerance, fetal macrosomia, and a long-lasting transgenerational alteration in glucose homeostasis, which are all key hallmarks of gestational diabetes. Transplantation of Treg cells rescued fetal loss, maternal glucose intolerance and fetal macrosomia. In human pregnancies, we found that gestational diabetes also correlates with a reduced number of Treg cells in the placenta. Our findings show that RANK promotes the hormone-mediated development of thymic Treg cells during pregnancy, and expand the functional role of maternal Treg cells to the development of gestational diabetes and the transgenerational metabolic rewiring of glucose homeostasis.

B7-CD28 co-stimulation modulates central tolerance via thymic clonal deletion and Treg generation through distinct mechanisms

The molecular and cellular mechanisms mediating thymic central tolerance and prevention of autoimmunity are not fully understood. Here we show that B7-CD28 co-stimulation and B7 expression by specific antigen-presenting cell (APC) types are required for clonal deletion and for regulatory T (Treg) cell generation from endogenous tissue-restricted antigen (TRA)-specific thymocytes. While B7-CD28 interaction is required for both clonal deletion and Treg induction, these two processes differ in their CD28 signaling requirements and in their dependence on B7-expressing dendritic cells, B cells, and thymic epithelial cells. Meanwhile, defective thymic clonal deletion due to altered B7-CD28 signaling results in the accumulation of mature, peripheral TRA-specific T cells capable of mediating destructive autoimmunity. Our findings thus reveal a function of B7-CD28 co-stimulation in shaping the T cell repertoire and limiting autoimmunity through both thymic clonal deletion and Treg cell generation.

Could cross-reactivity rescue Foxp3+ regulatory T cell precursors from thymic deletion?

Thymocytes that bind with high affinity to peptides displayed by MHC class II (pMHC-II) are deleted while low-affinity binders differentiate into naive CD4+ T cells. However, Foxp3+ regulatory T cells (Tregs) seem to defy this binary choice as their precursors require high-affinity interaction with pMHC-II for maturation in the thymus. Here, we rely on the antigen-specific interpretive framework, SPIRAL (Specific ImmunoRegulatory Algorithm), to propose that Tregs escape thymic deletion by forming dyads with IL-2-producing T cells via antigen cross-reactivity. This interpretation reconciles contradictions related to Treg ontogeny in the thymus and their role in modulating antigen-specific immune responses.

COVID‐19 in bone marrow transplant recipients: reflecting on a single centre experience

Covid-19 disease is caused by a novel SARS-CoV-2 virus and has been declared a pandemic on the 9th of March by WHO. Hallmark of COVID-19 management is supportive care and there is still no convincing evidence on a treatment which will reduce mortality. Severe COVID-19 associated sepsis characterized by acute respiratory distress syndrome (ARDS), secondary bacterial pneumonias thrombotic complications, myocarditis, and gastrointestinal involvement are more prevalent in those with comorbidities such as hypertension, diabetes, cardiac disease, cancer and age>70 years (Liang et al; 2020, Guan et al; 2019).

Loss of the Class II Accessory Molecule HLA-DO leads to presentation of lower abundance of DM-resistant epitopes

Abstract HLA-DO (DO in human; H2-O in mice) is a highly conserved non-classical MHC class II accessory molecule. Unlike the main Class II peptide editor HLA-DM, expression of DO is restricted to the thymic medulla, B cells and certain dendritic cell subsets. Although DO forms a stable complex with HLA-DM, its biological function remains poorly understood. Previously, our lab provided biochemical evidence in support of a model where DO enhances the binding of DM-resistant peptides, and reduces the binding of DM-sensitive peptides to the HLA-DR1 (DR1) molecules in vitro. Changes in the quantities of either DM-resistant of DM-sensitive peptides could therefore lead to alterations in both the thymic deletion, and peripheral activation of CD4 T cells. Here, using in vivo antigen presentation in an autoimmune as well as infectious model, we found that DO-KO mice present a lower abundance of MOG(35-55)/I-Ab and H5N1-HA(259-274)/DR1 complexes by B cells. Using 2D2 Tg T cells specific for MOG(35-55)/I-Ab as a readout system, we observed a less effective T cell activation upon co-culture of brain recovered APCs from diseased DO-KO mice. When we vaccinated DR1+DO-KO and DR1+DO-WT mice with flu vaccine, we found that HA(259-274)/DR1 specific CD4 T cells had a less avid interaction with B cells presenting HA(259-274)/DR1 from DR1+DO-KO as compared to B cells from vaccinated DR1+DO-WT mice as measured by a novel avidity measurement tool, z-MoviTM.

Measuring Thymic Clonal Deletion at the Population Level.

Clonal deletion of T cells specific for self-antigens in the thymus has been widely studied, primarily by approaches that focus on a single receptor (using TCR transgenes) or a single specificity (using peptide-MHC tetramers). However, less is known about clonal deletion at the population level. In this article, we report an assay that measures cleaved caspase 3 to define clonal deletion at the population level. This assay distinguishes clonal deletion from apoptotic events caused by neglect and approximates the anatomic site of deletion using CCR7. This approach showed that 78% of clonal deletion events occur in the cortex in mice. Medullary deletion events were detected at both the semimature and mature stages, although mature events were associated with failed regulatory T cell induction. Using this assay, we showed that bone marrow-derived APC drive approximately half of deletion events at both stages. We also found that both cortical and medullary deletion rely heavily on CD28 costimulation. These findings demonstrate a useful strategy for studying clonal deletion within the polyclonal repertoire.

Mechanism of action of posttransplantation cyclophosphamide: more than meets the eye.

For high-risk and refractory hematological malignancies, allogeneic hematopoietic stem cell transplantation (alloHSCT) is the only available curative therapy, with benefits derived from the antigenic disparity between recipient cancer and the incoming immune system. This immunologic mismatch can also lead to lethal graft-versus-host disease (GVHD), and immunosuppression strategies, including high-dose posttransplantation cyclophosphamide (PTCy), have been developed to allow for safe alloHSCT delivery. In this issue of JCI, Wachsmuth et al. present the results of preclinical studies designed to evaluate the mechanisms that underlie efficacy of PTCy after alloHSCT. The results of this study challenge previous reports indicating that alloreactive T cell elimination and thymic clonal deletion are primary mediators of PTCy efficacy and provide strong evidence to support FoxP3+CD4+ Tregs as important effectors of PTCy benefits.

Nfkbid overexpression in NOD mice elicits complete type 1 diabetes resistance

Abstract Type 1 diabetes (T1D) in both humans and the NOD mouse model is caused by the autoimmune mediated killing of pancreatic β-cells. Increased frequency or activity of autoreactive T cells and failures of regulatory T cells (Tregs) to control autoreactive T cells have both been implicated in T1D etiology. Due to the expression of MHC-I molecules on β-cells, autoreactive CD8+ T cells are likely the ultimate mediators of this destruction. Developing CD8+ T cells undergo selection processes in the thymus whereby most autoreactive clonotypes are eliminated during their development. Fine mapping of a genetic locus associated with defective thymic deletion of autoreactive CD8+ AI4 TCR transgenic (AI4) T cells in NOD mice led to the identification of Nfkbid, a NFκB signal modulator, as contributing to this phenotype. Ablation of Nfkbid in NOD mice by CRISPR/Cas9 results in a decreased frequency of autoreactive CD8+ AI4 T cells and an unexpected acceleration of T1D incidence, associated with reductions in the frequency and functional capacity of peripheral Tregs. Transgenic overexpression of Nfkbid in NOD mice paradoxically results in further decreased levels of circulating autoreactive CD8+ AI4 T cells. However, transgenic elevation of Nfkbid expression also increased the frequency and functional capacity of peripheral Tregs with a striking complete absence of T1D development in these mice. Studies are ongoing to define the mechanisms of T1D resistance mediated by transgenic overexpression of the transcriptional co-factor Nfkbid.

A Hypermorphic Nfkbid Allele Contributes to Impaired Thymic Deletion of Autoreactive Diabetogenic CD8+ T Cells in NOD Mice.

In both NOD mice and humans, the development of type 1 diabetes (T1D) is dependent in part on autoreactive CD8+ T cells recognizing pancreatic β cell peptides presented by often quite common MHC class I variants. Studies in NOD mice previously revealed that the common H2-Kd and/or H2-Db class I molecules expressed by this strain aberrantly lose the ability to mediate the thymic deletion of pathogenic CD8+ T cell responses through interactions with T1D susceptibility genes outside the MHC. A gene(s) mapping to proximal chromosome 7 was previously shown to be an important contributor to the failure of the common class I molecules expressed by NOD mice to mediate the normal thymic negative selection of diabetogenic CD8+ T cells. Using an inducible model of thymic negative selection and mRNA transcript analyses, we initially identified an elevated Nfkbid expression variant as a likely NOD-proximal chromosome 7 region gene contributing to impaired thymic deletion of diabetogenic CD8+ T cells. CRISPR/Cas9-mediated genetic attenuation of Nfkbid expression in NOD mice resulted in improved negative selection of autoreactive diabetogenic AI4 and NY8.3 CD8+ T cells. These results indicated that allelic variants of Nfkbid contribute to the efficiency of intrathymic deletion of diabetogenic CD8+ T cells. However, although enhancing thymic deletion of pathogenic CD8+ T cells, ablating Nfkbid expression surprisingly accelerated T1D onset that was associated with numeric decreases in both regulatory T and B lymphocytes in NOD mice.

Bruno Kyewski 1950‐2018

Bruno Kyewski, a dear friend and colleague, passed away in March 2018 after a long battle against early metastasizing prostate cancer. With him we have lost a remarkable and visionary scientist who more than 40 years ago set out to unravel the immunological secrets of the thymus and its role in self-tolerance. Bruno was born in the idyllic village of Bad Peterstal in the Black Forest and studied medicine in Bonn and Zürich. He earned his MD in 1978 with Hartmut Wekerle at the Max-Planck-Institute for Immunobiology in Freiburg, in those days one of the cradles of immunologists in Germany. Inspired by Wekerle´s thymic nurse cells Bruno developed his interest and passion for the role of the thymus in shaping the T cell repertoire and self-tolerance. He continued to work on the thymus as a postdoc in Henry Kaplan's laboratory at Stanford University Medical School, where he discovered that circulating protein antigens were efficiently presented in the thymus by MHC class II-positive dendritic cells, whereas cortical epithelial cells failed to do so, despite their high MHC class II expression. Thereafter, he joined the German Cancer Research Center in Heidelberg as a principal investigator, where he became Head of the Division of Developmental Immunology in 2004. For a long time it was assumed, and then shown with T cell receptor transgenic mice, that autoreactive T cells could be deleted in the thymus. It was believed that at least some peripheral tissue-restricted antigens could be picked up by dendritic cells and presented in the thymus, resulting in central tolerance. For those organ-specific antigens that would not find their way into the thymus, it was assumed that peripheral tolerance mechanisms would take care of auto-reactivity. This hypothesis was plausible and made perfect sense. No one could imagine the existence of a particular cell type which would express all or almost all organ-specific proteins, but Bruno did. In a brilliant set of experiments he discovered that thymic medullary epithelial cells expressed a large number of antigens that had been believed to be restricted to peripheral tissues, thereby resulting in thymic deletion of T cells specific for peripheral organs. With these stunning findings Bruno provided a crucial part of the mechanistic basis for the groundbreaking observation made by the Nobel laureate Sir Peter Medawar half a century earlier in the 1950s that immunological tolerance to the body's own tissues is not inborn but rather is acquired by the developing immune system. Most importantly, Bruno's elegant studies stimulated new directions of research, such as the genetic mechanisms for promiscuous gene expression and the consequences for autoimmune diseases and cancer vaccination. In addition to that work, Bruno served as associate editor at the International Journal of Cancer for over 15 years. Here, too, he made a significant contribution to the journal's development with his expertise. Bruno was a unique friend and colleague, warmhearted and very modest, almost too modest for this world. He would listen carefully and help to solve scientific problems, but his curiosity and interests were not limited to science. In discussions and conversations he would often drift off to literature, philosophy, art, and music, especially Schubert's music, which deeply moved him. He loved to spend vacations in France together with his family, where he enjoyed French cuisine and savoir vivre. His thoughts and comments were always enlightening and never superficial. Could I choose a piece of art for him it would be Rodin's sculpture The Thinker. Bruno would have loved to continue his research and life with his family, but fate wouldn't let him. We already miss him very much. Günter J. Hämmerling Tumor Immunology Program German Cancer Research Center