Tissue-resident Memory Phenotype Research Articles

Tissue-resident memory T cells invade the brain parenchyma in multiple sclerosis white matter lesions.

Multiple sclerosis is a chronic inflammatory, demyelinating disease, although it has been suggested that in the progressive late phase, inflammatory lesion activity declines. We recently showed in the Netherlands Brain Bank multiple sclerosis-autopsy cohort considerable ongoing inflammatory lesion activity also at the end stage of the disease, based on microglia/macrophage activity. We have now studied the role of T cells in this ongoing inflammatory lesion activity in chronic multiple sclerosis autopsy cases. We quantified T cells and perivascular T-cell cuffing at a standardized location in the medulla oblongata in 146 multiple sclerosis, 20 neurodegenerative control and 20 non-neurological control brain donors. In addition, we quantified CD3+, CD4+, and CD8+ T cells in 140 subcortical white matter lesions. The location of CD8+ T cells in either the perivascular space or the brain parenchyma was determined using CD8/laminin staining and confocal imaging. Finally, we analysed CD8+ T cells, isolated from fresh autopsy tissues from subcortical multiple sclerosis white matter lesions (n = 8), multiple sclerosis normal-appearing white matter (n = 7), and control white matter (n = 10), by flow cytometry. In normal-appearing white matter, the number of T cells was increased compared to control white matter. In active and mixed active/inactive lesions, the number of T cells was further augmented compared to normal-appearing white matter. Active and mixed active/inactive lesions were enriched for both CD4+ and CD8+ T cells, the latter being more abundant in all lesion types. Perivascular clustering of T cells in the medulla oblongata was only found in cases with a progressive disease course and correlated with a higher percentage of mixed active/inactive lesions and a higher lesion load compared to cases without perivascular clusters in the medulla oblongata. In all white matter samples, CD8+ T cells were located mostly in the perivascular space, whereas in mixed active/inactive lesions, 16.3% of the CD8+ T cells were encountered in the brain parenchyma. CD8+ T cells from mixed active/inactive lesions showed a tissue-resident memory phenotype with expression of CD69, CD103, CD44, CD49a, and PD-1 and absence of S1P1. They upregulated markers for homing (CXCR6), reactivation (Ki-67), and cytotoxicity (GPR56), yet lacked the cytolytic enzyme granzyme B. These data show that in chronic progressive multiple sclerosis cases, inflammatory lesion activity and demyelinated lesion load is associated with an increased number of T cells clustering in the perivascular space. Inflammatory active multiple sclerosis lesions are populated by CD8+ tissue-resident memory T cells, which show signs of reactivation and infiltration of the brain parenchyma.

Donor T and NK Cells with a Special Tissue-Resident Memory Phenotype Migrate into the Periphery of Lung Transplant Recipients - A Potential Feature for Tolerance Development

After lung transplantation (LuTx), a transient chimerism of donor cells exists in the blood of recipients due to the migration of lymphocytes from the transplanted lung into the periphery. We aimed to characterize the phenotype of donor CD8+ and CD4+ T and NK cells and to investigate whether they might represent tissue-resident memory (TRM) cells. Lymphocyte dynamics in recipient blood were determined in 97 lung transplant patients directly (T0), 24 hours (T24) and 3 (wks) weeks after LuTx using flow cytometry with lineage-, tissue-, and donor HLA class I allele-specific mAb. The same makers were used to determine the phenotype of lymphocytes present in organ storage solution (perfusate, n=102), recipient explanted lung parenchyma (n=28) and donor trachea (n=17). In peripheral blood of all recipients, donor-derived CD4+ and CD8+ T and CD56dim NK cells were detected at T0, T24 and 3 wks after LuTx and had higher CD69 expression compared to recipient cells (p<0.05) and were mostly CD25- CCR7- memory cells. This phenotype was similar to T and NK cells in corresponding perfusates. In lung parenchyma and trachea, most CD69+ T and NK cells showed coexpression of other tissue residency markers such as CD103, CD49a and PD-1 with significant enrichment in trachea (p<0.05). These markers were not found in circulating donor lymphocytes and perfusates, indicating that they represent distinct memory T and NK subsets. Donor T and NK cells showed higher IFN-γ production with PMA/ionomycin stimulation compared to recipient cells (p<0.05). The presence of these particular TRM cells did not have an impact on the development of PGD 24h after LuTx. However, patients with high frequencies of donor T cells showed a trend towards a CLAD-free survival 2 years post LuTx, although no statistical significance was reached (p=0.15). Our results demonstrate that donor T and NK cells found in the blood of LuTx recipients are a distinct subset from circulating lymphocytes and TRM cells present in lung tissue, since they express CD69 but lack expression of other classical TRM markers. They display a higher functional capacity despite the onset of immunosuppression. Donor T cells might be clinically relevant for tolerance induction and long-term survival after transplantation due to their unique features.

Abstract A64: In vitro induction of human tissue resident memory phenotype T-cells for use in adoptive cellular therapy

Abstract Recent reports suggest that tissue resident memory T-cells (TRM) may contribute to antitumor immunity. Specifically, the accumulation of CD103+ tumor-infiltrating lymphocytes (TILRM) in a wide array of solid tumors including breast, lung, ovarian, and melanoma is associated with favorable prognosis. However, relatively little is known regarding TRM differentiation and endogenous TRM are difficult to isolate and culture, impeding their basic study and translational applications such as adoptive cellular therapy (ACT). Thus, a means to generate TRM in vitro would be desirable. TGF-β is known to be critical in establishing TRM populations, and attempts to identify other factors have focused on cytokines. Oxygen tension is another factor that distinguishes the circulation from peripheral tissues; thus, we hypothesized that hypoxia may contribute to a TGF-β1-induced TRM phenotype in human CD8+ T cells. Naive CD8+ T cells from human peripheral blood were activated in normal cell culture conditions (20% O2) or hypoxia (2% O2) and then cultured in the presence of rhTGF-β1. We demonstrate that differentiation of human CD8+ T cells in hypoxia and TGF-β1 in vitro led to the development of a TRM phenotype, characterized by an increase in CD69+CD103+ cells expressing hallmarks of human TRM: CD49a, CD101, and PD-1, as determined by flow cytometric analysis. Hypoxia and TGF-β1 synergized to produce a significantly larger population of TRM phenotype cells than the additive effects of either condition alone. RNA sequencing analysis confirmed that cells differentiated in atmospheric oxygen, hypoxia alone, or hypoxia with TGF-β1 had distinct transcriptional profiles and gene set enrichment analysis (GSEA) showed enrichment for TRM and TILRM gene signatures in CD69+CD103+ cells generated in hypoxia and TGF-β1. We then extended this method to generate MART-1 (melanoma antigen recognized by T-cells 1)-specific in vitro induced TRM by stimulation with autologous dendritic cells in hypoxia and TGF-β1. Hypoxia and TGF-β1 can also be used in a modified rapid expansion protocol (REP) to induce TRM phenotype in antigen-specific T-cells generated via other means, highlighting this method’s potential application to multiple existing ACT modalities. There is great interest in leveraging resident phenotype cells for immunotherapy; however, current approaches focus on using vaccination to induce these cells in vivo as there is no reliable method to generate these cells in vitro. We demonstrate, for the first time, recapitulation of TRM phenotype in vitro from human peripheral blood-derived T cells, enabling a facile means of generating significant numbers of CD8+ TRM phenotype cells for basic research and translational applications such as ACT. Citation Format: Farah Hasan, Yulun Chiu, Rebecca M. Shaw, Junmei Wang, Cassian Yee. In vitro induction of human tissue resident memory phenotype T-cells for use in adoptive cellular therapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A64.

Donor T and NK Cells are Derived from the Donor Lung Parenchyme and Represent Tissue-Resident Memory Cells - An Important Feature for Tolerance Development

Purpose In previous studies, we identified donor lymphocytes in peripheral blood of recipients directly after transplantation and persisting at least three weeks that were characterized by the tissue retention marker CD69. In order to determine their origin, we hypothesized that donor T and NK cells have a peculiar tissue-resident memory phenotype that is shared between cells derived from lung perfusates, trachea parenchyma and lymph nodes. Methods Donor lymphocytes in recipient blood were determined in 27 lung transplant patients at T0, T24, 3 weeks by staining of donor HLA class I molecules in combination with lineage- and tissue-specific markers using flow cytometry. The phenotype of T and NK cells in perfusates (n=30), donor trachea (n=5), and lymph node (n=10), recipient explanted parenchyma (n=15) was compared to circulating cells using the same markers. Results In peripheral blood of all lung transplant recipients, donor derived T and NK cells were detected at T0, T24 and 3 weeks and had higher CD69 expression compared to recipient cells (p=0.001 to 0.03) and were mostly CD25−. This phenotype was similar to T and NK cells in corresponding perfusates, with significantly increased CD69 expression compared to circulating PBMCs (all p Conclusion Our results suggest that donor T and NK cells found in the periphery of lung transplant recipients are derived from lung parenchyma and represent tissue-resident memory cells. This transient chimerism in recipient blood might be clinically relevant for tolerance induction after transplantation due to unique features of TRM T and NK cells.

Tissue-Resident Memory CD8+ T Cells Acting as Mediatorsof Salivary Gland Damage in a Murine Model of Sjögren's Syndrome.

Although a role for CD4+ T cells in the pathogenesis of Sjögren's syndrome (SS) has been documented, the pathogenic significance of CD8+ T cells is unclear. The aim of this study was to investigate the role of CD8+ T cells in the development of SS. Flow cytometry and immunofluorescence analyses were utilized to detect T cell infiltration within the labial salivary glands of patients with primary SS. In parallel, p40-/- CD25-/- mice were used as a murine model of SS. In addition, mice with genetic knockout of CD4, CD8a, or interferon-γ (IFNγ) were crossed with p40-/- CD25-/- mice to study the pathogenic significance of specific lineage subpopulations, including functional salivary gland tests as well as histopathologic and serologic data. A CD8+ T cell-specific depletion antibody was used in this murine SS model to evaluate its potential as a therapeutic strategy. CD8+ T cells with a tissue-resident memory phenotype outnumbered CD4+ T cells in the labial salivary glands of patients with SS, and were primarily colocalized with salivary duct epithelial cells and acinar cells. Furthermore, infiltrating CD8+ T cells with a CD69+CD103+/- tissue-resident phenotype and with a significant elevation of IFNγ production were dominant in the submandibular glands of mice in this murine SS model. CD8a knockout abrogated the development of SS in these mice. Knockout of IFNγ decreased CD8+ T cell infiltration and gland destruction. More importantly, depletion of CD8+ T cells fully protected mice against the pathologic manifestations of SS, even after the onset of disease. These data reveal the pathogenic significance of CD8+ T cells in the development and progression of SS in the salivary glands. Treatment directed against CD8+ T cells may be a rational therapy for the management of SS in human subjects.

Co-expression of CD39 and CD103 identifies tumor-reactive CD8 T cells in human solid tumors

Identifying tumor antigen-specific T cells from cancer patients has important implications for immunotherapy diagnostics and therapeutics. Here, we show that CD103+CD39+ tumor-infiltrating CD8 T cells (CD8 TIL) are enriched for tumor-reactive cells both in primary and metastatic tumors. This CD8 TIL subset is found across six different malignancies and displays an exhausted tissue-resident memory phenotype. CD103+CD39+ CD8 TILs have a distinct T-cell receptor (TCR) repertoire, with T-cell clones expanded in the tumor but present at low frequencies in the periphery. CD103+CD39+ CD8 TILs also efficiently kill autologous tumor cells in a MHC-class I-dependent manner. Finally, higher frequencies of CD103+CD39+ CD8 TILs in patients with head and neck cancer are associated with better overall survival. Our data thus describe an approach for detecting tumor-reactive CD8 TILs that will help define mechanisms of existing immunotherapy treatments, and may lead to future adoptive T-cell cancer therapies.

Role of Graft-derived Graft-versus-Host T cells in Facilitating Multilineage Blood Chimerism after Human Intestinal Transplantation

Long-term multilineage chimerism often appears in blood following human intestinal transplantation (ITx), especially in multivisceral transplant (MVTx) recipients. Patients with donor T cell macrochimerism (>4%) in blood, frequently without graft-versus-host disease (GVHD), displayed less graft rejection and donor-specific antibody (DSA) production and slower graft T cell replacement by the recipient. To address the hypothesis that the balance of graft-versus-host (GvH) and host-versus-graft (HvG) alloreactivity might underly these relationships, we analyzed the phenotype, repertoire and alloreactivity of donor- and recipient-derived T cells in the intestinal allograft, peripheral blood and bone marrow (BM) of ITx recipients. We identified GvH and HvG clones on the basis of high throughput TCRβ CDR3 sequencing from pre-Tx donor and recipient mixed lymphocyte reactions as we have described1. Enrichment of GvH compared to HvG clones in early intestinal biopsies and absence of Class I DSA were associated with donor T cell macrochimerism. Local expansion of GvH clones in the graft was likely driven by uniformly rapid replacement of recipient antigen presenting cells (APCs) within allografts early post-Tx. Cumulative frequency of GvH clones in blood early post-Tx was positively correlated with the peak level of donor T cell chimerism (Fig.1).Unlike donor ileal lymphocytes, which display a tissue-resident memory phenotype, long-term circulating donor T cells were markedly enriched for the recent thymic emigrant phenotype compared with recipient cells, and GvH clones were undetectable among these late circulating naïve donor-derived T cells. Enrichment of donor circulating naïve B cells and T cells and recurrent myeloid chimerism late post-Tx are consistent with our observation that donor-derived hematopoietic stem and progenitor cells are present in intestinal allografts and are detectable in the circulation of patients with macrochimerism. Multilineage chimerism might reflect a lymphohematopoietic GvH response (LGVHR) migrating from recipient circulation to the BM, making “space” for engraftment of progenitor cells from the graft. Indeed, 0.0356% of CD45+ cells in the BM of an MVTx patient on day 105 post-Tx were donor-derived, among which 72.9% were T cells and 1.37% were CD34+ progenitors. TCR sequencing of sorted donor cells in recipient BM identified donor-mappable clones, among CD4s of which 60% by frequency were GvH-reactive. GvH CD4 cells did not show marked enrichment in donor-mappable PBMCs on the same day. (Fig.2)Together, these data suggest that donor GvH-reactive T cells expanding within the graft in response to recipient APCs entering the graft migrate into the recipient circulation and BM, playing a key role in promoting and maintaining mixed chimerism. LGVHR without GVHD in ITx recipients suggests a novel approach to achieving intestinal allograft tolerance. 1. Zuber et al., 2016 Science Immunology. The study was funded by the National Institute of Allergy and Infectious Diseases grant P01 AI106697. Research reported in this study was performed in the CCTI Flow Cytometry Core, supported in part by the Office of the Director, National Institutes of Health under awards S10RR027050 and S10OD020056.

SOX2 as a target for immunotherapy of pediatric gliomas.

e22012 Background: New treatments are needed to improve outcomes for pediatric gliomas. Immune checkpoint inhibitors are effective therapies in tumors with a high mutation burden that express multiple neo-antigens. However, for pediatric tumors that carry few mutations, there is a need to identify new antigenic targets of anti-tumor immunity. SOX2 is an embryonal stem cell antigen implicated in the biology of glioma initiating cells. Expression of SOX2 by pediatric glial tumors, and the capacity of the immune system in these patients to recognize SOX2, has not been studied. Methods: We examined the expression of SOX2 on paraffin-embedded tissue from pediatric glial tumors (n = 30). The presence of T cell immunity to SOX2 was examined in both blood and tumor-infiltrating T cells using antigen-dependent cytokine and T cell proliferation assays (n = 15). The nature of tumor-infiltrating immune cells in glial tumors (n = 4) was analyzed using single cell mass cytometry. Results: SOX2 is expressed by tumor cells but not surrounding normal tissue in all low grade gliomas (n = 15), high grade gliomas (n = 7), ependymomas (n = 3) and in 60% of oligodendrogliomas (n = 5). T cells against SOX2 can be detected in blood and tumor tissue in 33% of patients. CD4 and CD8 tumor infiltrating T-cells display a higher proportion of PD-1 expression compared to circulating T cells (p &lt; 0.05). Glial CD4 and CD8 T cells are enriched for tissue resident memory phenotype (TRM; CD45RO+, CD69+, CCR7-) and the expression of PD-1 is primarily on these TRM cells (p &lt; 0.05). A subset of CD4 and CD8 TRM cells also co-express multiple inhibitory checkpoints including PD-L1 and TIGIT. Glial tumors also contain NK cells with reduced expression of lytic granzyme (p &lt; 0.05). Conclusions: Our data demonstrate in vivo immunogenicity of SOX2, which is specifically overexpressed on pediatric glial tumor cells. Our data also suggest that the TRM subset of tumor-infiltrating T cells may be key targets for immune checkpoint blockade, and harnessing tumor immunity will likely require the combined targeting of multiple inhibitory checkpoints. Future efforts to target SOX2 with dendritic cell vaccines combined with immune checkpoint blockade could provide effective tumor immunity and improve outcomes in pediatric brain tumors.

Distinct dominant T-cell receptors with a tissue resident memory phenotype in individual melanoma metastases.

3 Background: Expression of inhibitory immune checkpoints (ICPs) within tumors has emerged as an important barrier for effective anti-tumor immunity. Antibody-mediated blockade of ICPs can lead to durable responses in patients. Interestingly, only a small subset of tumor infiltrating lymphocytes (TILs) express these checkpoints and there is a need to better understand the characteristics of this subset. We undertook this study to understand characteristics of TILs within melanoma. Methods: We used single cell mass cytometry, gene expression profiling of purified T cell subsets, T cell receptor (TCR) sequencing as well as functional studies to understand the characteristics of TILs in melanoma patients (n=50). We also performed exome sequencing of tumor cells in some patients. Results: We find that TILs are functionally and phenotypically distinct from circulating T cells. They express higher levels of inhibitory ICPs (PD-1, TIM-3) and secrete less IL2, IFNg and TNFa than T cells in circulation. Expression of vascular endothelial growth factor within tumors correlated with reduced T cell infiltration. Expression of ICPs (PD-1, TIM-3, PD-L1) were enriched in T cells with a phenotype and expression profile of tissue resident memory T (TRM) cells with most cells expressing multiple checkpoints. Within the myeloid compartment, ICPs were predominantly expressed on CD14+CD16+ subset. TCR sequencing revealed that individual melanoma metastases revealed that the top clones within each of the lesions have distinct TCRs. Concurrent TCR and tumor exome sequencing of individual metastases in the same patient revealed that inter-lesional diversity of TCRs exceeded differences in mutation/neoantigen load in tumor cells. Conclusions: Our findings suggest that TRM cells and CD16+ myeloid cells may be the major target of ICP blockade within tumors. The ability to activate, and retain TRM cells may be an important determinant of the T cell content of the tumor microenvironment and should be a goal for future vaccines. Importantly, our study illustrates inter-lesional diversity of TCRs within individual metastases which may differentially impact the outcome of immune therapy at each site.

A context-dependent role for IL-21 in modulating the differentiation, distribution, and abundance of effector and memory CD8 T cell subsets

Abstract The activation of naïve CD8 T cells typically results in the formation of effector cells (TE) as well as phenotypically distinct memory cells that are retained over time. Memory CD8 T cells can be further subdivided into central memory (TCM), effector memory (TEM) and tissue-resident memory (TRM) subsets, which cooperate to confer immunological protection. Using mixed bone marrow chimeras and adoptive transfer studies in which CD8 T cells either do or do not express the IL-21 receptor (IL-21R), we discovered that under homeostatic or lymphopenic conditions IL-21 acts directly on CD8 T cells to favor the accumulation of TE/TEM populations. The inability to perceive IL-21 signals under competitive conditions also resulted in lower levels of TRM phenotype cells and reduced expression of granzyme B in the small intestine. Furthermore, IL-21 differentially promoted the expression of the chemokine receptor CX3CR1 and the integrin α4β7 on circulating CD8 T cells in the mixed bone marrow chimeras and on CD8 T cells primed in vitro. Thus, IL-21 may influence CD8 T cell migration by modulating the expression of CX3CR1 and α4β7. The requirement for IL-21 to establish CD8 TE/TEM and TRM subsets was overcome by acute lymphocytic choriomeningitis virus infection. Nevertheless, memory virus-specific CD8 T cells remained dependent on IL-21 for optimal accumulation in tissues under lymphopenic conditions. Overall, this study reveals a context-dependent role for IL-21 in sustaining effector-phenotype CD8 T cells and influencing their migratory properties, accumulation, and functions.

A Context-Dependent Role for IL-21 in Modulating the Differentiation, Distribution, and Abundance of Effector and Memory CD8 T Cell Subsets.

The activation of naive CD8 T cells typically results in the formation of effector cells (TE) as well as phenotypically distinct memory cells that are retained over time. Memory CD8 T cells can be further subdivided into central memory, effector memory (TEM), and tissue-resident memory (TRM) subsets, which cooperate to confer immunological protection. Using mixed bone marrow chimeras and adoptive transfer studies in which CD8 T cells either do or do not express IL-21R, we discovered that under homeostatic or lymphopenic conditions IL-21 acts directly on CD8 T cells to favor the accumulation of TE/TEM populations. The inability to perceive IL-21 signals under competitive conditions also resulted in lower levels of TRM phenotype cells and reduced expression of granzyme B in the small intestine. IL-21 differentially promoted the expression of the chemokine receptor CX3CR1 and the integrin α4β7 on CD8 T cells primed in vitro and on circulating CD8 T cells in the mixed bone marrow chimeras. The requirement for IL-21 to establish CD8 TE/TEM and TRM subsets was overcome by acute lymphocytic choriomeningitis virus infection; nevertheless, memory virus-specific CD8 T cells remained dependent on IL-21 for optimal accumulation in lymphopenic environments. Overall, this study reveals a context-dependent role for IL-21 in sustaining effector phenotype CD8 T cells and influencing their migratory properties, accumulation, and functions.

Characterisation of the lymphoid stress surveillance response in human intestine

BackgroundThe human intestine harbours an immense repertoire of T cells, many having proved difficult to isolate and characterise. The lymphoid stress surveillance response (LSSR), whereby unconventional T cells respond to epithelial dysregulation in synchrony with innate myeloid cells, was first described in mice. We investigated the potential for LSSR in human intestine, and whether this response could contribute to gut inflammation. MethodsWe obtained colonic biopsy samples at endoscopy from healthy donors. We applied a novel explant culture system (from one described in human skin) that permits outgrowth of large numbers of highly viable T cells, to complete flow-cytometric phenotyping and functional studies. To validate our method we compared T cell isolation methods. FindingsValidation of our method demonstrated that cell yield was twice as high as with conventionally isolated cells. γδ T cells comprised a substantial, underappreciated proportion of total intestinal lymphocytes (mean 9·5%, SD 9). Gut γδ T cells expressed a tissue-resident memory phenotype, including the activating receptor NKG2D. As previously demonstrated by us in human skin, specific cytokines licensed such cells to make purely innate-like cytotoxic responses, both to recombinant NKG2D ligands and to colonic tumour cell lines. Responses to colonic tumour cell lines were abrogated by antibodies that block NKG2D, demonstrating common functional potentials at distinct epithelial surfaces. InterpretationThe study shows that the human intestine harbours a large number of tissue-resident γδ T cells that can make rapid, innate-like responses to epithelial cell dysregulation, akin to those shown in our laboratory both in vivo for their mouse counterparts, and in skin. We can now investigate the contribution of LSSR to inflammatory gastrointestinal diseases characterised by tissue dysregulation and the upregulation of stress ligands. FundingFrancis-Crick Institute - National Institute for Health Research Biomedical Research Centre (Clinical Research Training Fellowship), Wellcome Trust.

Quantitative and Qualitative Analysis of Bone Marrow CD8(+) T Cells from Different Bones Uncovers a Major Contribution of the Bone Marrow in the Vertebrae.

Bone marrow (BM) plays an important role in the long-term maintenance of memory T cells. Yet, BM is found in numerous bones throughout the body, which are not equal in structure, as they differ in their ratio of cortical and trabecular bone. This implies that BM cells within different bones are subjected to different microenvironments, possibly leading to differences in their frequencies and function. To address this, we examined BM from murine tibia, femur, pelvis, sternum, radius, humerus, calvarium, and the vertebrae and analyzed the presence of effector memory (TEM), central memory (TCM), and naïve (TNV) CD8+ T cells. During steady-state conditions, the frequency of the total CD8+ T cell population was comparable between all bones. Interestingly, most CD8+ T cells were located in the vertebrae, as it contained the highest amount of BM cells. Furthermore, the frequencies of TEM, TCM, and TNV cells were similar between all bones, with a majority of TNV cells. Additionally, CD8+ T cells collected from different bones similarly expressed the key survival receptors IL-7Rα and IL-15Rβ. We also examined BM for memory CD8+ T cells with a tissue-resident memory phenotype and observed that approximately half of all TEM cells expressed the retention marker CD69. Remarkably, in the memory phase of acute infection with the lymphocytic choriomeningitis virus (LCMV), we found a massive compositional change in the BM CD8+ T cell population, as the TEM cells became the dominant subset at the cost of TNV cells. Analysis of Ki-67 expression established that these TEM cells were in a quiescent state. Finally, we detected higher frequencies of LCMV-specific CD8+ T cells in BM compared to spleen and found that BM in its entirety contained fivefold more LCMV-specific CD8+ T cells. In conclusion, although infection with LCMV caused a dramatic change in the BM CD8+ T cell population, this did not result in noticeable differences between BM collected from different bones. Our findings suggest that in respect to CD8+ T cells, BM harvested from a single bone is a fair reflection of the rest of the BM present in the murine body.

CCL21 dependent CD4+ T cell migration plays a role in protective immune responses against Toxoplasma infection in the brain. (99.22)

Abstract The chemokine receptor, CCR7 and interactions with its ligands CCL19 and CCL21 are well known to facilitate priming of T cells in lymphoid tissue. Our data demonstrate upregulation of CCR7, CCL19 and CCL21 in the brain during Toxoplasma gondii infection at a time point coincident with the peak of T cell infiltration in the CNS. CNS resident astrocytes significantly upregulate CCL21 following direct infection with Toxoplasma tachyzoites and in the absence of CCL21, plt/plt mice were less efficient in controlling parasite replication during chronic infection. Although T cells enter the infected brain in a CCR7 independent manner, migration of extravasated CD4+T cells from the perivascular area into the CNS parenchyma is CCL21-dependent. Our data also demonstrate accumulation of CD4+CCR7 expressing T cells in the infected brain. A significant proportion of these CCR7+T cells express memory T cell markers including IL-7R (Interleukin 7 Receptor) and CD103 (tissue resident memory phenotype). These data indicate CCR7-CCL21 interactions play a role in guiding CD4+CCR7+ memory T cell migration in the brain to prevent parasite reactivation and disease. Analysis of the behavior and phenotype of this memory T cell population in the absence of CCR7 or CCL21 will be presented. These data address the potential contribution of CCR7/CCL21 mediated interactions to maximize memory responses in the brain during Toxoplasma infection.

The Historical Background of Convulsions in Childhood

<h3>Background</h3> The pathogenesis of Spondyloarthritis (SpA) remains unknown but its strong association with some alleles of HLA-B*27 is peculiar. The arthritogenic antigen hypothesis assumes the existence of specific peptides presented by risk-conferring HLA-B*27 alleles to antigen-specific CD8+ T cells, which then initiate or sustain autoimmune reactions. Several studies analyzing T cell receptor (TCR) repertoire found preferred Variable TCR chains and motifs in the hypervariable complementary determining region (CDR) 3, but analyzed only TCR β-chains in bulk analyses^1,2. <h3>Objectives</h3> To analyze full sequence information of TCR including matching α- and β-chains from single CD8+ T cells and characterize the transcriptomes of expanded and non-expanded clonotypes in synovial fluid (SF) of SpA patients. <h3>Methods</h3> We included 17 patients with active gonarthritis: 10 patients with HLA-B27 positive (B27pos) SpA, 4 with HLA-B27 negative (B27neg) SpA and 3 rheumatoid arthritis (RA) patients. Antigen-experienced CD8+ T cells were sorted out of SF by flow cytometry. Single cell sequencing was performed for all patients to analyze matching TCR α- and β-chains. For 7 patients (3 B27pos SpA, 2 B27neg SpA, 2 RA), additionally whole transcriptome analyses were performed. <h3>Results</h3> We found strong biases when analyzing α and β chains of TCR Variable regions and CDR1 and CDR2 sequences (Figure 1 a,b): AV21, AV12-2, and AV17 were highly enriched in B27pos SpA as compared to B27neg subjects. Amongst the highest expressed clones, we could confirm enrichment for previously described TRBV genes as BV19, BV5-1 and BV6-2. We examined TCR α/β combinations and focused on those detected in at least three different B27pos SpA but not in any of the B27neg patients (Figure 1 c-f). The combinations TRBV19/TRAV21 and TRBV6-2/TRAV21 were most likely specific for B27pos SpA and might reflect interaction of these TCR chains with HLA-B*27. Sequences of CDR3 loops, which predominantly interact with HLA-bound antigenic peptides, revealed striking common structural motifs in α- and β-chains. Focusing on the most prominent TRAV21 chains pairing with TRBV19, 5-1 and 6-2 chains, revealed identical sequences in different patients and striking common structural motifs in α- and β-CDR3 sequences in other patients. Such marked similarities in the antigen-recognition loops of the β-chains associated with TRAV21 suggest common or highly similar antigens. Gene expression levels provided evidence that expanded cell populations had tissue resident memory (TRM) phenotypes (elevated expression of activation, migration and tissue retention markers, downregulated genes characteristic for T cell egress), while this phenotype was not very pronounced in non-expanded cells. Furthermore, markers for T cell exhaustion and apoptosis were elevated in expanded cells of B27pos SpA patients. <h3>Conclusion</h3> Analysis of single antigen experienced CD8+ T cells from SF of B27pos SpA patients revealed significant clonal expansions and common motifs in the CDR loops. Two of the four CDR1 and CDR2 loops were highly homologous suggesting that these loops interact with α-helices of HLA-B*27. Common motifs in CDR3 loops of expanded clonotypes suggest recognition of a limited set of antigenic peptides presented by HLA-B*27. Many of the expanded clonotypes showed a TRM phenotype, were exhausted and on the way to become apoptotic, which suggests that these clones had sustained contact to specific antigens. <h3>References</h3> [1]Komech, et al. Rheumatology 2018 [2]Hanson, et al. A&amp;R 2020 <h3>Acknowledgements</h3> We thank all patients included in this study for their participation. This work was funded by the German Research Foundation (DFG) through grants DO 420/4 to KDo, PO 2124/2-1 to DP, and SyNergy (EXC 2145 SyNergy – ID 390857198) to KDo. Judith Rademacher and Katharina Deschler contributed equally. JR is participant in the BIH-Charité Clinician Scientist Program funded by the Charité –Universitätsmedizin Berlin and the Berlin Institute of Health. The authors would like to thank Martina Seipel for excellent technical assistance, Sabrina Sron for patient recruitment and study coordination, and Hildrun Haibel, Mikhail Protopopov, Fabian Proft, Valeria Rios Rodriguez and Laura Spiller for recruiting patients for this study. <h3>Disclosure of Interests</h3> None declared