Thymic Antigen Research Articles

Thymic stromal lymphopoietin-mediated epicutaneous inflammation promotes acute diarrhea and anaphylaxis.

Atopic dermatitis (AD) and food allergy are closely linked; however, the mechanisms that guide the progression of AD to allergic inflammatory responses at other mucosal surfaces, including the gastrointestinal tract, are not well understood. Here, we determined that exposure of mice that have been epicutaneously sensitized with thymic stromal lymphopoietin (TSLP) and antigen to repeated oral doses of the same antigen induced acute diarrhea and anaphylaxis. In this model, loss of TSLP signaling specifically in DCs led to loss of induced allergic diarrhea through lack of sensitization. While TSLP responses were not required during oral allergen challenge, CD4(+) T cells were required and transferred disease when introduced into naive hosts. In addition, oral exposure to the antigen prior to skin sensitization blocked development of allergic disease. Finally, mice lacking the receptor for IL-25 failed to develop acute diarrhea and anaphylaxis, highlighting a role for IL-25 in the initiation of type 2 immunity in the intestine. These results demonstrate a role for TSLP and IL-25 in the atopic march from skin sensitization to food allergic responses and provide a model system for the generation of potential therapeutic interventions.

Clinical Spectrum of SCID: The Key is in the Thymus?

Clinical Spectrum of SCID: The Key is in the Thymus?

From pre-DP, post-DP, SP4, and SP8 Thymocyte Cell Counts to a Dynamical Model of Cortical and Medullary Selection

Cells of the mature αβ T cell repertoire arise from the development in the thymus of bone marrow precursors (thymocytes). αβ T cell maturation is characterized by the expression of thousands of copies of identical αβ T cell receptors and the CD4 and/or CD8 co-receptors on the surface of thymocytes. The maturation stages of a thymocyte are: (1) double negative (DN) (TCR−, CD4− and CD8−), (2) double positive (DP) (TCR+, CD4+ and CD8+), and (3) single positive (SP) (TCR+, CD4+ or CD8+). Thymic antigen presenting cells provide the appropriate micro-architecture for the maturation of thymocytes, which “sense” the signaling environment via their randomly generated TCRs. Thymic development is characterized by (i) an extremely low success rate, and (ii) the selection of a functional and self-tolerant T cell repertoire. In this paper, we combine recent experimental data and mathematical modeling to study the selection events that take place in the thymus after the DN stage. The stable steady state of the model for the pre-DP, post-DP, and SP populations is identified with the experimentally measured cell counts from 5.5- to 17-week-old mice. We make use of residence times in the cortex and the medulla for the different populations, as well as recently reported asymmetric death rates for CD4 and CD8 SP thymocytes. We estimate that 65.8% of pre-DP thymocytes undergo death by neglect. In the post-DP compartment, 91.7% undergo death by negative selection, 4.7% become CD4 SP, and 3.6% become CD8 SP. Death by negative selection in the medulla removes 8.6% of CD4 SP and 32.1% of CD8 SP thymocytes. Approximately 46.3% of CD4 SP and 27% of CD8 SP thymocytes divide before dying or exiting the thymus.

MHC Class II–Alpha Chain Knockout Mice Support Increased Viral Replication That Is Independent of Their Lack of MHC Class II Cell Surface Expression and Associated Immune Function Deficiencies

MHCII molecules are heterodimeric cell surface proteins composed of an α and β chain. These molecules are almost exclusively expressed on thymic epithelium and antigen presenting cells (APCs) and play a central role in the development and function of CD4 T cells. Various MHC-II knockout mice have been generated including MHC-IIAα-/- (I-Aα-/-), MHC-IIAβ-/- (I-β-/-) and the double knockout (I-Aαxβ-/-). Here we report a very striking observation, namely that alphaviruses including the avirulent strain of Semliki Forest virus (aSFV), which causes asymptomatic infection in wild-type C57BL6/J (B6) mice, causes a very acute and lethal infection in I-Aα-/-, but not in I-β-/- or I-Aαxβ-/-, mice. This susceptibility to aSFV is associated with high virus titres in muscle, spleen, liver, and brain compared to B6 mice. In addition, I-Aα-/- mice show intact IFN-I responses in terms of IFN-I serum levels and IFN-I receptor expression and function. Radiation bone marrow chimeras of B6 mice reconstituted with I-Aα-/- bone marrow expressed B6 phenotype, whereas radiation chimeras of I-Aα-/- mice reconstituted with B6 bone marrow expressed the phenotype of high viral susceptibility. Virus replication experiments both in vivo and in vitro showed enhanced virus growth in tissues and cell cultures derived form I-Aα-/- compared to B6 mice. This enhanced virus replication is evident for other alpha-, flavi- and poxviruses and may be of great benefit to producers of viral vaccines. In conclusion, I-Aα-/- mice exhibit a striking susceptibility to virus infections independent of their defective MHC-II expression. Detailed genetic analysis will be carried out to characterise the underlining genetic defects responsible for the observed phenomenon.

DH consults on setting up list of managers barred from NHS

It was found that donor's serum and serum of patients with rheumatic fever, erysipelas and myasthenia gravis contained autoantibodies to the cytoplasmic antigens and to the antigen of perinuclear zone of differentiated layers cells of human epidermis. Using the serum with different level of autoantibodies to these epidermal antigens it's localization in the human thymus epithelium was determined. It was shown that perinuclear antigen of differentiated epidermis cells is localized in the cytoplasm of cortical and medullar thymus epithelial cells and in the perinuclear zone of some cells in Hassall's corpuscles. The cytoplasmic antigen of differentiated epidermal cells is detected only in the cytoplasm of the Hassall's corpuscle cells. The ability of many tissues (possibly all of them) and in the first turn of epidermis to produce lymphokine along with immunomodulating properties of thymus hetero-organic antigens enables to consider these thymus antigens as a complex of immunomodulating factor proper for other tissues. These factors may provide the competence of certain T-cell subpopulations to different organ's tissues necessary for proceedings them the immunological survey both in normal and pathological conditions.

The Safety Evaluation of NKTT120, an Invariant NKT (iNKT) Cell Depleting Antibody in the Cynomolgus Monkey.

AbstractAbstract 2150Invariant Natural Killer T (iNKT) cells are a small subset of T lymphocytes (ranging from 0.01 – 0.1% of CD3+ T cells). iNKT cells recognize glycolipid antigens presented by the MHC class-I-like protein CD1d rather than peptide antigens. In contrast to most T cell subpopulations, which have diverse sequences for their T Cell Receptors (TCRs), iNKT cells express a uniquely rearranged, highly conserved, semi-invariant TCR-α chain (Vα24-Jα18 in humans), which preferentially pairs with specific TCR-β chains (Vβ11 in humans). iNKT cells are similar to innate cells in their rapid release of cytokines following iTCR antigen binding. They are also adaptive-like, with T cell properties including thymic positive selection and antigen recognition by CD1d presentation. iNKT cells are involved in mediating tissue injury and inflammation in multiple organ systems. Chronic inflammation is associated with the pathophysiology of Sickle Cell Disease (SCD) and our studies and those of others have found an increased ratio of activated iNKT cells in peripheral blood of patients with SCD. The role of iNKT cell activation in the pathology of SCD is supported by studies in a mouse model of SCD (Wallace et al. Blood 114:667, 2009). These data suggest that iNKT cell reduction and/or depletion would be effective in reducing the inflammatory state in SCD. To this end, we have developed a humanized monoclonal antibody (NKTT120) that exclusively binds to the CDR3 loop of the human and non-human primate (NHP) invariant T cell receptor and depletes iNKT cells. This antibody could provide an effective therapeutic intervention to modulate iNKT cell numbers, and thus their ability to mediate inflammation in SCD. The current study was designed to assess the overall safety of NKTT120 in the cynomolgus monkey (Macaca fascicularis). The cynomolgus monkey was selected for testing because NKTT120 is only active in human and old world NHP species. Thirty-two cynomolgus monkeys (3–5 kg) equally divided between males and females were studied. The groups consisted of a vehicle control and treatment groups that received 0.3 mg/kg, 3 mg/kg, and 10 mg/kg NKTT120 IV weekly for a total of 5 doses. Two animals per sex in the vehicle and the 10 mg/kg groups were recovered for an additional 2 months following the last dose. Animals were evaluated for food intake, body weight and general health. Standard hematology, coagulation testing and clinical chemistry testing were also performed. In addition, iNKT cell number and other lymphocytes were monitored during the study by FACS analysis. The repeat dose injections were well tolerated by all dose groups. No deaths or serious adverse events were reported during dosing or in the recovery period. Body weight, food intake and clinical evaluation, hematology, coagulation assays, and clinical chemistry were similar for vehicle control and all dosing groups. As expected, iNKT cells were depleted within 24 hours to below level of detection and remained depleted throughout the dosing period at all doses tested. The iNKT cell numbers of the 10 mg/kg recovery animals remained depleted throughout the 2 month recovery. However, there was no change in other cells of the lymphocytic series at any dose or at any time point evaluated. Overall our study showed that we can safely and specifically deplete iNKT cells in non-human primates following administration of NKTT120. These data support the use of NKTT120 as a therapeutic intervention in conditions such as sickle cell disease where iNKT modulation could be beneficial.Percent of Gated Cells (Mean ± SD) Estimated by FACS Analysis Before and Following NKTT120 IV Administration to Cynomolgus Macaques Shows Specific iNKT Cell Depletion at all Doses and Time pointsLymphocyte ProfilesPost Dose 1Post Dose 52 Months Post Dose 5Vehicle0.3 mg/kg3 mg/kg10 mg/kgVehicle0.3 mg/kg3 mg/kg10 mg/kgVehicle10 mg/kgiNKT Cells*0.05±.030.000.000.000.06±0.050.000.000.000.06±.010.00NK Cells**11±610±311±512±710±59±56±47±67±35±4Mature T cells**69±761±763±657±764±861±1059±1159±878±670±7CD8 T Cells**26±425±623±319±622±422±820±719±528±220±2CD4 T Cells**40±635±637±635±737±635±735±537±846±447±5B Cells**5±38±27±49±35±35±22±26±36±38±3*%CD3 Cells.**% Lymphocytes. Disclosures:Macdonald:NKT Therapeutics: Employment, Equity Ownership. Scheuplein:NKT Therapeutics: Employment, Equity Ownership. Thariath:NKT Therapeutics: Employment, Equity Ownership. LeBel:MPI Research: Employment; NKT Therapeutics: Research Funding. Zeigler:MPI Research: Employment; NKT Therapeutics: Research Funding. Truneh:NKT Therapeutics: Consultancy, Equity Ownership. Mashal:NKT Therapeutics: Employment, Equity Ownership. Schaub:NKT Therapeutics: Employment, Equity Ownership.

Cathepsin S dominates autoantigen processing in human thymic dendritic cells

The interaction of developing thymocytes with peptide-MHC complexes on thymic antigen presenting cells (APC) is crucial for T cell development, both for positive selection of “useful” thymocytes as well as negative selection of autoreactive thymocytes to prevent autoimmunity. The peptides presented on MHC II molecules are generated by lysosomal proteases such as the cathepsins. At the same time, lysosomal proteases will also destroy other potential T cell epitopes from self-antigens. This will lead to a lack of presentation on negatively selecting thymic antigen presenting cells and consequently, escape of autoreactive T cells recognizing these epitopes. In order to understand the processes that govern generation or destruction of self-epitopes in thymic APC, we studied the antigen processing machinery and epitope processing in the human thymus. We find that each type of thymic APC expresses a different signature of lysosomal proteases, providing indirect evidence that positive and negative selection of CD4+ T cells might occur on different sets of peptides, in analogy to what has been proposed for CD8+ T cells. We also find that myeloid dendritic cells (DC) are more efficient in processing autoantigen than plasmacytoid DC. In addition, we observed that cathepsin S plays a central role in processing of the autoantigens myelin basic protein and proinsulin in thymic dendritic cells. Cathepsin S destroyed a number of known T cell epitopes, which would be expected to result in lack of presentation and consequently, escape of autoreactive T cells. Cathepsin S therefore appears to be an important factor that influences selection of autoreactive T cells.

Complete Diabetes Protection Despite Delayed Thymic Tolerance in NOD8.3 TCR Transgenic Mice Due to Antigen-Induced Extrathymic Deletion of T Cells

Prevention of autoimmunity requires the elimination of self-reactive T cells during their development in the thymus and maturation in the periphery. Transgenic NOD mice that overexpress islet-specific glucose 6 phosphatase catalytic subunit–related protein (IGRP) in antigen-presenting cells (NOD-IGRP mice) have no IGRP-specific T cells. To study the relative contribution of central and peripheral tolerance mechanisms to deletion of antigen-specific T cells, we crossed NOD-IGRP mice to highly diabetogenic IGRP206–214 T-cell receptor transgenic mice (NOD8.3 mice) and studied the frequency and function of IGRP-specific T cells in the thymus and periphery. Peripheral tolerance was extremely efficient and completely protected NOD-IGRP/NOD8.3 mice from diabetes. Peripheral tolerance was characterized by activation of T cells in peripheral lymphoid tissue where IGRP was expressed followed by activation-induced cell death. Thymectomy showed that thymic output of IGRP-specific transgenic T cells compensated for peripheral deletion to maintain peripheral T-cell numbers. Central tolerance was undetectable until 10 weeks and complete by 15 weeks. These in vivo data indicate that peripheral tolerance alone can protect NOD8.3 mice from autoimmune diabetes and that profound changes in T-cell repertoire can follow subtle changes in thymic antigen presentation.

The AIRE gene and susceptibility to rheumatoid arthritis in the Spanish population

AIRE is a transcriptional regulator that plays a functional role in thymocyte education and negative selection by controlling the expression of peripheral antigens in thymus. This gene has been associated with the susceptibility to rheumatoid arthritis (RA) in a recent GWAS carried out in the Japanese population; nevertheless, no association has been described in Caucasian populations.

Autoimmune regulator (AIRE) contributes to Dectin-1–induced TNF-α production and complexes with caspase recruitment domain–containing protein 9 (CARD9), spleen tyrosine kinase (Syk), and Dectin-1

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) syndrome is a complex immunologic disease caused by mutation of the autoimmune regulator (AIRE) gene. Autoimmunity in patients with APECED syndrome has been shown to result from deficiency of AIRE function in transcriptional regulation of thymic peripheral tissue antigens, which leads to defective T-cell negative selection. Candidal susceptibility in patients with APECED syndrome is thought to result from aberrant adaptive immunity. To determine whether AIRE could function in anticandidal innate immune signaling, we investigated an extrathymic role for AIRE in the immune recognition of β-glucan through the Dectin-1 pathway, which is required for defense against Candida species. Innate immune signaling through the Dectin-1 pathway was assessed in both PBMCs from patients with APECED syndrome and a monocytic cell line. Subcellular localization of AIRE was assessed by using confocal microscopy. PBMCs from patients with APECED syndrome had reduced TNF-α responses after Dectin-1 ligation but in part used a Raf-1-mediated pathway to preserve function. In the THP-1 human monocytic cell line, reducing AIRE expression resulted in significantly decreased TNF-α release after Dectin-1 ligation. AIRE formed a transient complex with the known Dectin-1 pathway components phosphorylated spleen tyrosine kinase and caspase recruitment domain-containing protein 9after receptor ligation and localized with Dectin-1 at the cell membrane. AIRE can participate in the Dectin-1 signaling pathway, indicating a novel extrathymic role for AIRE and a defect that likely contributes to fungal susceptibility in patients with APECED syndrome.

Systematic Variations in Immune Response-Related Gene Transcript Abundance Suggest New Questions about Environmental Influences on Lacrimal Gland Immunoregulation

Retrospective analyses were undertaken to assess the hypothesis that environmental variables influenced immunophysiological status of lacrimal glands from untreated female rabbits that had been housed out-of-doors until they were acquired for use as controls for experimental studies. Rabbits were euthanized within 5 days of arrival at University Vivaria. Glands were divided for histology and RNA extraction. Transcript abundances were determined with real time RT-PCR. Sections were stained for CD18 and rabbit thymic lymphocyte antigen. Environmental variables assessed were mean daily high temperature, low humidity, high temperature/low humidity ratio, and days with above average temperature/humidity ratio ("adverse days") during the prior 30 days. Spearman's analyses revealed numerous significant correlations. Numbers of T cells and abundances of mRNAs for CD8; CCL2, and CCL4; IL-1α and IL-1β; the T(H)1 cytokine, IL-2; and the T(H)2- and B cell cytokines, IL-4, IL-6, IL-10, APRIL, and BAFF, all increased with adverse days, while IFN-γ mRNA abundance decreased. Glands from the group exposed to the most adverse days remained free of immunopathological lesions. Glands from the group exposed to the highest temperatures fell above the regression curves for IL-4, APRIL, and BAFF calculated for the other groups and had significantly higher abundances of mRNAs for prolactin, IL-18, CCL21, CCL28, CXCL8, and CXCL13. One of six glands from this group contained small immune cell aggregates; the others appeared normal. The only gland that presented with frank histopathology was from a group that had experienced benign conditions. Increasing adverse days correlated with increasing abundances of transcripts, including mRNAs for IL-2, IL-10, and CD8, outside the T(H)1/T(H)2 paradigm. The findings raise intriguing questions as to whether and how such changes might be associated with homeostatic phenomena.

Ontogenesis of rat immune system: Proteasome expression in different cell populations of the developing thymus

Immune proteasomes in thymus are involved in processing of self-antigens, which are presented by MHC class I molecules for rejection of autoreactive thymocytes in adults and probably in perinatal rats. The distribution of immune proteasome subunits LMP7 and LMP2 in thymic cells have been investigated during rat perinatal ontogenesis. Double immunofluorescent labeling revealed LMP7 and LMP2 in thymic epithelial and dendritic cells, as well as in CD68 positive cells – macrophages, monocytes – at all developmental stages. LMP2 and LMP7 were also detected by flow cytometry in almost all thymic CD90 lymphocytes through pre- and postnatal ontogenesis. Our results demonstrate that the immune proteasomes are expressed in all types of thymic antigen presenting cells during perinatal ontogenesis, suggesting the establishment of the negative selection in the thymus at the end of fetal life. The observation of the immune proteasome expression in T lymphocytes suggests their role in thymocyte differentiation besides antigen processing in thymus.

Cutting Edge: γδ Intraepithelial Lymphocytes of the Small Intestine Are Not Biased toward Thymic Antigens

gammadelta Tau cells, together with alphabeta Tau cells, are abundantly present in the epithelial layer of the small intestine (IEL) and are essential for the host's first line of defense. Whether or not gammadelta IELs, like alphabeta IELs, are derived from thymocytes that encounter self-Ags in the thymus is unclear. In this study, we report that a natural population of gammadelta T cells that are specific for the nonclassical MHC class I molecules T10 and T22 are present in the IEL compartment of mice that do not express T10/T22. Furthermore, the small intestinal homing receptor CCR9 is preferentially expressed on gammadelta thymocytes that have yet to encounter a ligand, and gammadelta thymocytes with high affinity for self-ligand are CCR9(low). These observations suggest that the Ag-specific repertoire of gammadelta IELs is not biased toward thymic Ags. Instead, gammadelta IELs appear suited to respond to novel Ags revealed in pathological settings.

Induction of Human T-Cell Tolerance to Pig Xenoantigens via Thymus Transplantation in Mice with an Established Human Immune System

Thymus xenotransplantation has been shown to induce tolerance to porcine xenografts in mice and to permit survival of alpha1,3Gal-transferase knockout porcine kidney xenografts for months in nonhuman primates. We evaluated the ability of porcine thymus xenotransplantation to induce human T-cell tolerance using a humanized mouse (hu-mouse) model, where a human immune system is preestablished by implantation of fetal human thymus tissue under the kidney capsule and intravenous injection of CD34(+) hematopoietic stem/progenitor cells. Human T-cell depletion with an anti-CD2 mAb following surgical removal of human thymic grafts prevented the initial rejection of porcine thymic xenografts in hu-mice. In these hu-mice, porcine thymic grafts were capable of supporting human thymopoiesis and T-cell development, and inducing human T-cell tolerance to porcine xenoantigens. Human T cells from these mice responded strongly to third-party pig, but not to the thymic donor swine leukocyte antigen (SLA)-matched pig stimulators in a mixed lymphocyte reaction (MLR) assay. Anti-pig xenoreactive antibodies declined in these hu-mice, whereas antibody levels increased in nontolerant animals that rejected porcine thymus grafts. These data show that porcine thymic xenotransplantation can induce donor-specific tolerance in immunocompetent hu-mice, supporting this approach for tolerance induction in clinical xenotransplantation.

IgM, IgG, and IgA anti-DNA topoisomerase I antibodies in systemic sclerosis.

Anti-DNA topoisomerase I (anti-topo I) antibodies have been broadly studied in systemic sclerosis (SSc). The use of different native and molecularly cloned antigens has shown a predominant IgG response, and a variable frequency of positive IgM and IgA tests. We report herein the serological findings of SSc using a recombinant topo I obtained through a standard bacterial system. Anti-topo I antibodies were studied in 45 SSc patients and 85 healthy controls through ELISA and western blot. Escherichia coli XL1-blue strain and pT7-7 vector were used to amplify a DNA topo I cDNA clone, and to obtain the recombinant polypeptide. The latter was purified by affinity chromatography, and the enzymatic and antigenic properties were evaluated through specific tests. A native antigen was included for comparison. The SSc group disclosed positive IgM (20%), IgG (86.6%), and IgA (26.6%) anti-topo I tests with the recombinant polypeptide, and a purified calf thymus antigen yielded similar results. IgG autoantibodies were frequently associated with skin involvement, esophageal dysfunction, and restrictive lung disease. The recombinant protein showed a molecular weight of 86.6 kDa, a positive topo I activity using a supercoiled pBR322 DNA relaxation test, and its carboxyl terminus region was recognized by specific antibodies. This report confirms that different immunoglobulin classes with anti-topo I activity may occur in SSc. IgG was the predominant serological feature with both, the recombinant and native antigens. The study also demonstrates the association between high levels of these autoantibodies and some clinical manifestations of SSc.

Mechanisms of an autoimmunity syndrome in mice caused by a dominant mutation in Aire

Homozygous loss-of-function mutations in AIRE cause autoimmune polyglandular syndrome type 1 (APS 1), which manifests in a classic triad of hypoparathyroidism, adrenal insufficiency, and candidiasis. Interestingly, a kindred with a specific G228W AIRE variant presented with an autosomal dominant autoimmune phenotype distinct from APS 1. We utilized a novel G228W-knockin mouse model to show that this variant acted in a dominant-negative manner to cause a unique autoimmunity syndrome. In addition, the expression of a large number of Aire-regulated thymic antigens was partially inhibited in these animals, demonstrating the importance of quantitative changes in thymic antigen expression in determining organ-specific autoimmunity. Furthermore, the dominant-negative effect of the G228W variant was exerted through recruitment of WT Aire away from active sites of transcription in the nucleus of medullary thymic epithelial cells in vivo. Together, these results may demonstrate a mechanism by which autoimmune predisposition to phenotypes distinct from APS 1 can be mediated in a dominant-negative fashion by Aire.

Lesions in the thymus and bone marrow in chicks with experimentally induced chicken infectious anemia disease

One-day-old SPF chicks were inoculated with the Cux-l strain of chicken infectious anemia virus (CIAV), and the clinical development of disease and its macroscopic and microscopic alterations in the thymus and bone marrow, were observed. Tissue sections of thymus and bone marrow were stained using the streptavidin-biotin peroxidase method and examined under light microscope for evaluation of antigenic intensities in tissues. Those findings were then compared with blood parameters and ELISA results obtained through collected sera during sacrifice procedures. We sought to determine: the localization of viral antigens in thymus and bone marrow tissues after inoculation, the correlation between antigen intensities and hematologic, serologic and histopathologic findings, definitive diagnostic criteria using histopathologic and immunoperoxidase methods, and the reliability of these methods in the diagnosis of CIAV infection. For this purpose, 83, one-day-old SPF chicks were used. The birds were divided into experimental (n = 52) and control (n = 26) groups. A virus dose of TCID50 of 100,000/ml was administered intramuscularly to every bird in the experimental group. Based on the results of this study, we have suggested that clinical examination, along with macroscopic and microscopic evaluation of the thymus and bone marrow, maybe undertaken starting from day 7 post-inoculation (PI). ELISA, might be of value, as it might give consistent results starting from day 14 PI. However, the most reliable results were obtained through examination of thymus and bone marrow sections from infected birds stained by immunoperoxidase technique, as early as day 4 PI.

Haematopoietic antigen‐presenting cells in the human thymic cortex: evidence for a role in selection and removal of apoptotic thymocytes

Only a small proportion of thymocytes survive T cell selection in the thymus and leave the thymus as mature T cells. The vast majority of thymocytes undergo cell death during selection, either due to failure to undergo positive selection on self peptide-MHC presented by thymic antigen presenting cells (APC) or due to negative selection. In the murine thymus it has been shown that most thymocytes that fail selection undergo apoptosis in the thymic cortex and are removed by cortical macrophages. However, it is unknown how apoptotic thymocytes are cleared from the cortex of the human thymus. Here we report the identification of antigen-presenting cells of haematopoietic origin (hAPCs) by expression of dendritic cell (DC) specific C-type lectin DC-SIGN (CD209) in the cortex of the human thymus, and show that these cells exhibit features of both immature DCs and macrophages. The analysis of cellular markers, in particular the expression of the molecular chaperone HLA-DM, on cortical hAPCs further suggests that these hAPCs may participate in selection of thymocytes in the cortex. Using in situ terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL), we demonstrated that these cortical hAPCs are surrounded by apoptotic, TUNEL(+) thymocytes in situ. Futhermore, in situ immuno-cryo-electron microscopy suggests that cortical hAPCs take up and remove apoptotic thymocytes. Thus, DC-SIGN(+) hAPCs in the human thymic cortex appear to function in thymocyte selection and removal of apoptotic thymocytes from the thymic cortex.

CD8 Raft Localization Is Induced by Its Assembly into CD8αβ Heterodimers, Not CD8αα Homodimers

The coreceptor CD8 is expressed as a CD8alphabeta heterodimer on major histocompatibility complex class I-restricted TCRalphabeta T cells, and as a CD8alphaalpha homodimer on subsets of memory T cells, intraepithelial lymphocytes, natural killer cells, and dendritic cells. Although the role of CD8alphaalpha is not well understood, it is increasingly clear that this protein is not a functional homologue of CD8alphabeta. On major histocompatibility complex class I-restricted T cells, CD8alphabeta is a more efficient TCR coreceptor than CD8alphaalpha. This property has for the mouse protein been attributed to the recruitment of CD8alphabeta into lipid rafts, which is dependent on CD8beta palmitoylation. Here, these divergent distributions of CD8alphabeta and CD8alphaalpha are demonstrated for the human CD8 proteins as well. However, although palmitoylation of both CD8alpha and CD8beta chains was detected, this modification did not contribute to raft localization. In contrast, arginines in the cytoplasmic domain are crucial for raft localization of CD8betabeta. Most strikingly, the assembly of a non-raft localized CD8beta chain with a non-raft localized CD8alpha chain resulted in raft-localized CD8alphabeta heterodimers. Using chimeric CD8 proteins, this property of the heterodimer was found to be determined by the assembly of CD8alpha and CD8beta extracellular regions. The presence of two CD8alpha extracellular regions, on the other hand, appears to preclude raft localization. Thus, heterodimer formation and raft association are intimately linked for CD8alphabeta. These results emphasize that lipid raft localization is a key feature of human CD8alphabeta that clearly distinguishes it from CD8alphaalpha.

Crystal Structure of the TCR Co-receptor CD8αα in Complex with Monoclonal Antibody YTS 105.18 Fab Fragment at 2.88 Å Resolution

The CD8 glycoprotein functions as an essential element in the control of T-cell selection, maturation and the TCR-mediated response to peptide antigen. CD8 is expressed as both heterodimeric CD8alphabeta and homodimeric CD8alphaalpha isoforms, which have distinct physiological roles and exhibit tissue-specific expression patterns. CD8alphaalpha has previously been crystallized in complex with class I pMHC and, more recently, with the mouse class Ib thymic leukemia antigen (TL). Here, we present the crystal structure of a soluble form of mouse CD8alphaalpha in complex with rat monoclonal antibody YTS 105.18 Fab fragment at 2.88 A resolution. YTS 105.18, which is commonly used in the blockade of CD8+ T-cell activation in response to peptide antigen, is specific for mouse CD8alpha. The YTS 105.18 Fab is one of only five rat IgG Fab structures to have been reported to date. Analysis of the YTS 105.18 Fab epitope on CD8alpha reveals that this antibody blocks CD8 activity by hydrogen bonding to residues that are critical for interaction with both class I pMHC and TL. Structural comparison of the liganded and unliganded forms of soluble CD8alphaalpha indicates that the mouse CD8alphaalpha immunoglobulin-domain dimer does not undergo significant structural alteration upon interaction either with class I pMHC or TL.