T-cells in human trigeminal ganglia express canonical tissue-resident memory T-cell markers.

BACKGROUND: Trigeminal ganglia (TG) neurons are the main site of lifelong latent herpes simplex virus type 1 (HSV-1) infection. T-cells in ganglia contribute to long-term control of latent HSV-1 infection, but it is unclear whether these cells are bona fide tissue-resident memory T-cells (TRM). We optimized the processing of human post-mortem nervous tissue to accurately phenotype T-cells in human TG ex vivo and in situ. METHODS: Peripheral blood mononuclear cells (PBMC; 5 blood donors) were incubated with several commercial tissue digestion enzyme preparations to determine off-target effect on simultaneous detection of 15 specific T-cell subset markers by flow cytometry. Next, optimized enzymatic digestion was applied to ex vivo phenotype T-cells in paired PBMC, normal appearing white matter (NAWM) and TG of 8 deceased brain donors obtained < 9 h post-mortem by flow cytometry. Finally, the phenotypic and functional markers, and spatial orientation of T-cells in relation to neuronal somata, were determined in TG tissue sections of five HSV-1-latently infected individuals by multiparametric in situ analysis. RESULTS: Collagenase IV digestion of human nervous tissue was most optimal to obtain high numbers of viable T-cells without disrupting marker surface expression. Compared to blood, majority T-cells in paired NAWM and TG were effector memory T-cells expressing the canonical TRM markers CD69, CXCR6 and the immune checkpoint marker PD1, and about half co-expressed CD103. A trend of relatively higher TRM frequencies were detected in TG of latently HSV-1-infected compared to HSV-1 naïve individuals. Subsequent in situ analysis of latently HSV-1-infected TG showed the presence of cytotoxic T-cells (TIA-1+), which occasionally showed features of proliferation (KI-67+) and activation (CD137+), but without signs of degranulation (CD107a+) nor damage (TUNEL+) of TG cells. Whereas majority T-cells expressed PD-1, traits of T-cell senescence (p16INK4a+) were not detected. CONCLUSIONS: The human TG represents an immunocompetent environment in which both CD4 and CD8 TRM are established and retained. Based on our study insights, we advocate for TRM-targeted vaccine strategies to bolster local HSV-1-specific T-cell immunity, not only at the site of recurrent infection but also at the site of HSV-1 latency.

Parallel detection of SARS-CoV-2 epitopes reveals dynamic immunodominance profiles of CD8+ T memory cells in convalescent COVID-19 donors.

Objectives: High-magnitude CD8+ T cell responses are associated with mild COVID-19 disease; however, the underlying characteristics that define CD8+ T cell-mediated protection are not well understood. The antigenic breadth and the immunodominance hierarchies of epitope-specific CD8+ T cells remain largely unexplored and are essential for the development of next-generation broad-protective vaccines. This study identified a broad spectrum of conserved SARS-CoV-2 CD8+ T cell epitopes and defined their respective immunodominance and phenotypic profiles following SARS-CoV-2 infection. Methods: CD8+ T cells from 51 convalescent COVID-19 donors were analysed for their ability to recognise 133 predicted and previously described SARS-CoV-2-derived peptides restricted by 11 common HLA class I allotypes using heterotetramer combinatorial coding, which combined with phenotypic markers allowed in-depth ex vivo profiling of CD8+ T cell responses at quantitative and phenotypic levels. Results: A comprehensive panel of 49 mostly conserved SARS-CoV-2-specific CD8+ T cell epitopes, including five newly identified low-magnitude epitopes, was established. We confirmed the immunodominance of HLA-A*01:01/ORF1ab1637-1646 and B*07:02/N105-113 and identified B*35:01/N325-333 as a third epitope with immunodominant features. The magnitude of subdominant epitope responses, including A*03:01/N361-369 and A*02:01/S269-277, depended on the donors' HLA-I context. All epitopes expressed prevalent memory phenotypes, with the highest memory frequencies in severe COVID-19 donors. Conclusion: SARS-CoV-2 infection induces a predominant CD8+ T memory response directed against a broad spectrum of conserved SARS-CoV-2 epitopes, which likely contributes to long-term protection against severe disease. The observed immunodominance hierarchy emphasises the importance of T cell epitopes derived from nonspike proteins to the overall protective and cross-reactive immune response, which could aid future vaccine strategies.

Organoids as tools for fundamental discovery and translation-a Keystone Symposia report.

Complex three-dimensional in vitro organ-like models, or organoids, offer a unique biological tool with distinct advantages over two-dimensional cell culture systems, which can be too simplistic, and animal models, which can be too complex and may fail to recapitulate human physiology and pathology. Significant progress has been made in driving stem cells to differentiate into different organoid types, though several challenges remain. For example, many organoid models suffer from high heterogeneity, and it can be difficult to fully incorporate the complexity of in vivo tissue and organ development to faithfully reproduce human biology. Successfully addressing such limitations would increase the viability of organoids as models for drug development and preclinical testing. On April 3-6, 2022, experts in organoid development and biology convened at the Keystone Symposium "Organoids as Tools for Fundamental Discovery and Translation" to discuss recent advances and insights from this relatively new model system into human development and disease.

Two sides of the same coin: Protective versus pathogenic CD4+ resident memory T cells.

The ability of the adaptive immune system to form memory is key to providing protection against secondary infections. Resident memory T cells (TRM) are specialized T cell populations that reside within tissue sites where they await reencounter with their cognate antigen. TRM are distinct from circulating memory cells, including central and effector memory T cells, both functionally and transcriptionally. Since the discovery of TRM, most research has focused on CD8+ TRM, despite that CD4+ TRM are also abundant in most tissues. In the past few years, more evidence has emerged that CD4+ TRM can contribute both protective and pathogenic roles in disease. A complexity inherent to the CD4+ TRM field is the ability of CD4+ T cells to polarize into a multitude of distinct subsets and recognize not only viruses and intracellular bacteria but also extracellular bacteria, fungi, and parasites. In this review, we outline the key features of CD4+ TRM in health and disease, including their contributions to protection against SARS-CoV-2 and potential contributions to immunopathology associated with COVID-19.

Allo-reactive tissue-resident T cells causing damage: An inside job.

Tissue-resident memory T cells (TRM cells) reside in the epithelium and contribute to the first line defense against invading pathogens. Snyder et al. (2022. J. Exp. Med.https://doi.org/10.1084/jem.20212059) now report that clonally expanded, recipient T cells persist as TRM cells in human lung allografts despite intensive immunosuppression. Their persistence may contribute to chronic allograft dysfunction.

The endothelial diapedesis synapse regulates transcellular migration of human T lymphocytes in a CX3CL1- and SNAP23-dependent manner.

Understanding how cytotoxic T lymphocytes (CTLs) efficiently leave the circulation to target cancer cells or contribute to inflammation is of high medical interest. Here, we demonstrate that human central memory CTLs cross the endothelium in a predominantly paracellular fashion, whereas effector and effector memory CTLs cross the endothelium preferably in a transcellular fashion. We find that effector CTLs show a round morphology upon adhesion and induce a synapse-like interaction with the endothelium where ICAM-1 is distributed at the periphery. Moreover, the interaction of ICAM-1:ß2integrin and endothelial-derived CX3CL1:CX3CR1 enables transcellular migration. Mechanistically, we find that ICAM-1 clustering recruits the SNARE-family protein SNAP23, as well as syntaxin-3 and -4, for the local release of endothelial-derived chemokines like CXCL1/8/10. In line, silencing of endothelial SNAP23 drives CTLs across the endothelium in a paracellular fashion. In conclusion, our data suggest that CTLs trigger local chemokine release from the endothelium through ICAM-1-driven signals driving transcellular migration.

How to Reliably Define Human CD8+ T-Cell Subsets: Markers Playing Tricks.

In recent years, our understanding about the functional complexity of CD8+ T-cell populations has increased tremendously. The immunology field is now facing challenges to translate these insights into phenotypic definitions that correlate reliably with distinct functional traits. This is key to adequately monitor and understand compound immune responses including vaccination and immunotherapy regimens. Here we will summarize our understanding of the current state in the human CD8+ T-cell subset characterization field. We will address how reliably the currently used cell surface markers are connected to differentiation status and function of particular subsets. By restricting ourselves to CD8+ αß T cells, we will focus mostly on major histocompatibility complex (MHC) class I-restricted virus- and tumor-specific T cells. This comes with a major advantage as fluorescently labeled peptide-loaded MHC class I multimers have been widely used to identify and characterize these cells.

Divergent SARS-CoV-2-specific T- and B-cell responses in severe but not mild COVID-19 patients.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the current coronavirus disease 2019 (COVID-19) pandemic. Understanding the immune response that provides specific immunity but may also lead to immunopathology is crucial for the design of potential preventive and therapeutic strategies. Here, we characterized and quantified SARS-CoV-2-specific immune responses in patients with different clinical courses. Compared to individuals with a mild clinical presentation, CD4+ T-cell responses were qualitatively impaired in critically ill patients. Strikingly, however, in these patients the specific IgG antibody response was remarkably strong. Furthermore, in these critically ill patients, a massive influx of circulating T cells into the lungs was observed, overwhelming the local T-cell compartment, and indicative of vascular leakage. The observed disparate T- and B-cell responses could be indicative of a deregulated immune response in critically ill COVID-19 patients.

Blimp-1 Rather Than Hobit Drives the Formation of Tissue-Resident Memory CD8+ T Cells in the Lungs.

Tissue-resident memory CD8+ T (TRM) cells that develop in the epithelia at portals of pathogen entry are important for improved protection against re-infection. CD8+ TRM cells within the skin and the small intestine are long-lived and maintained independently of circulating memory CD8+ T cells. In contrast to CD8+ TRM cells at these sites, CD8+ TRM cells that arise after influenza virus infection within the lungs display high turnover and require constant recruitment from the circulating memory pool for long-term persistence. The distinct characteristics of CD8+ TRM cell maintenance within the lungs may suggest a unique program of transcriptional regulation of influenza-specific CD8+ TRM cells. We have previously demonstrated that the transcription factors Hobit and Blimp-1 are essential for the formation of CD8+ TRM cells across several tissues, including skin, liver, kidneys, and the small intestine. Here, we addressed the roles of Hobit and Blimp-1 in CD8+ TRM cell differentiation in the lungs after influenza infection using mice deficient for these transcription factors. Hobit was not required for the formation of influenza-specific CD8+ TRM cells in the lungs. In contrast, Blimp-1 was essential for the differentiation of lung CD8+ TRM cells and inhibited the differentiation of central memory CD8+ T (TCM) cells. We conclude that Blimp-1 rather than Hobit mediates the formation of CD8+ TRM cells in the lungs, potentially through control of the lineage choice between TCM and TRM cells during the differentiation of influenza-specific CD8+ T cells.

Expression of IL-7Rα and KLRG1 defines functionally distinct CD8+ T-cell populations in humans.

During acute viral infections in mice, IL-7Rα and KLRG1 together are used to distinguish the short-lived effector cells (SLEC; IL-7Rαlo KLRGhi ) from the precursors of persisting memory cells (MPEC; IL-7Rαhi KLRG1lo ). We here show that these markers can be used to define distinct subsets in the circulation and lymph nodes during the acute phase and in "steady state" in humans. In contrast to the T cells in the circulation, T cells derived from lymph nodes hardly contain any KLRG1-expressing cells. The four populations defined by IL-7Rα and KLRG1 differ markedly in transcription factor, granzyme and chemokine receptor expression. When studying renal transplant recipients experiencing a primary hCMV and EBV infection, we also found that after viral control, during latency, Ki-67-negative SLEC can be found in the peripheral blood in considerable numbers. Thus, combined analyses of IL-7Rα and KLRG1 expression on human herpes virus-specific CD8+ T cells can be used to separate functionally distinct subsets in humans. As a noncycling IL-7Rαlo KLRG1hi population is abundant in healthy humans, we conclude that this combination of markers not only defines short-lived effector cells during the acute response but also stable effector cells that are formed and remain present during latent herpes infections.

Peripheral and systemic antigens elicit an expandable pool of resident memory CD8+ T cells in the bone marrow.

BM has been put forward as a major reservoir for memory CD8+  T cells. In order to fulfill that function, BM should "store" memory CD8+ T cells, which in biological terms would require these "stored" memory cells to be in disequilibrium with the circulatory pool. This issue is a matter of ongoing debate. Here, we unequivocally demonstrate that murine and human BM harbors a population of tissue-resident memory CD8+ T (TRM ) cells. These cells develop against various pathogens, independently of BM infection or local antigen recognition. BM CD8+ TRM cells share a transcriptional program with resident lymphoid cells in other tissues; they are polyfunctional cytokine producers and dependent on IL-15, Blimp-1, and Hobit. CD8+ TRM cells reside in the BM parenchyma, but are in close contact with the circulation. Moreover, this pool of resident T cells is not size-restricted and expands upon peripheral antigenic re-challenge. This works extends the role of the BM in the maintenance of CD8+ T cell memory to include the preservation of an expandable reservoir of functional, non-recirculating memory CD8+ T cells, which develop in response to a large variety of peripheral antigens.

Functional Heterogeneity of CD4+ Tumor-Infiltrating Lymphocytes With a Resident Memory Phenotype in NSCLC.

Resident memory T cells (TRM) inhabit peripheral tissues and are critical for protection against localized infections. Recently, it has become evident that CD103+ TRM are not only important in combating secondary infections, but also for the elimination of tumor cells. In several solid cancers, intratumoral CD103+CD8+ tumor infiltrating lymphocytes (TILs), with TRM properties, are a positive prognostic marker. To better understand the role of TRM in tumors, we performed a detailed characterization of CD8+ and CD4+ TIL phenotype and functional properties in non-small cell lung cancer (NSCLC). Frequencies of CD8+ and CD4+ T cell infiltrates in tumors were comparable, but we observed a sharp contrast in TRM ratios compared to surrounding lung tissue. The majority of both CD4+ and CD8+ TILs expressed CD69 and a subset also expressed CD103, both hallmarks of TRM. While CD103+CD8+ T cells were enriched in tumors, CD103+CD4+ T cell frequencies were decreased compared to surrounding lung tissue. Furthermore, CD103+CD4+ and CD103+CD8+ TILs showed multiple characteristics of TRM, such as elevated expression of CXCR6 and CD49a, and decreased expression of T-bet and Eomes. In line with the immunomodulatory role of the tumor microenvironment, CD8+ and CD4+ TILs expressed high levels of inhibitory receptors 2B4, CTLA-4, and PD-1, with the highest levels found on CD103+ TILs. Strikingly, CD103+CD4+ TILs were the most potent producers of TNF-α and IFN-γ, while other TIL subsets lacked such cytokine production. Whereas, CD103+CD4+PD-1low TILs produced the most effector cytokines, CD103+CD4+PD-1++ and CD69+CD4+PD-1++ TILs produced CXCL13. Furthermore, a large proportion of TILs expressed co-stimulatory receptors CD27 and CD28, unlike lung TRM, suggesting a less differentiated phenotype. Agonistic triggering of these receptors improved cytokine production of CD103+CD4+ and CD69+CD8+ TILs. Our findings thus provide a rationale to target CD103+CD4+ TILs and add co-stimulation to current therapies to improve the efficacy of immunotherapies and cancer vaccines.

Tissue-resident memory T cells populate the human brain.

Most tissues are populated by tissue-resident memory T cells (TRM cells), which are adapted to their niche and appear to be indispensable for local protection against pathogens. Here we show that human white matter-derived brain CD8+ T cells can be subsetted into CD103-CD69+ and CD103+CD69+ T cells both with a phenotypic and transcription factor profile consistent with TRM cells. Specifically, CD103 expression in brain CD8+ T cells correlates with reduced expression of differentiation markers, increased expression of tissue-homing chemokine receptors, intermediate and low expression of the transcription factors T-bet and eomes, increased expression of PD-1 and CTLA-4, and low expression of cytolytic enzymes with preserved polyfunctionality upon activation. Brain CD4+ T cells also display TRM cell-associated markers but have low CD103 expression. We conclude that the human brain is surveilled by TRM cells, providing protection against neurotropic virus reactivation, whilst being under tight control of key immune checkpoint molecules.

Blimp-1 induces and Hobit maintains the cytotoxic mediator granzyme B in CD8 T cells.

CD8 T cells acquire cytotoxic molecules including granzyme B during effector differentiation. Both tissue-resident memory CD8 T cells (Trm) and circulating CD45RA+ effector-type T cells (Temra) cells have the ability to retain granzyme B protein expression into the memory phase, but it is unclear how this persistence of cytolytic activity is regulated during steady state. Previously, we have described that the transcriptional regulators Hobit and Blimp-1 have overlapping target genes that include granzyme B, but their impact on the regulation of cytotoxicity in Trm and Temra cells during homeostasis has remained unclear. We examined the expression regulation of Hobit and Blimp-1 in murine and human CD8 T-cells to determine their timeframe of activity. While Blimp-1 mRNA was expressed throughout effector and memory T cells, Blimp-1 protein, was only transiently expressed during the effector stage. In contrast, Hobit mRNA and protein expression was stably maintained during quiescence, but downregulated after activation. Notably, Blimp-1 was required for expression of granzyme B in murine effector T cells and Trm, while Hobit specifically regulated granzyme B in murine Trm during the memory phase. These findings suggest that Blimp-1 initiates cytotoxic effector function and that Hobit maintains cytotoxicity in a deployment-ready modus in Trm.

Tissue-resident memory T cells at the center of immunity to solid tumors.

Immune responses in tissues are constrained by the physiological properties of the tissue involved. Tissue-resident memory T cells (TRM cells) are a recently discovered lineage of T cells specialized for life and function within tissues. Emerging evidence has shown that TRM cells have a special role in the control of solid tumors. A high frequency of TRM cells in tumors correlates with favorable disease progression in patients with cancer, and studies of mice have shown that TRM cells are necessary for optimal immunological control of solid tumors. Here we describe what defines TRM cells as a separate lineage and how these cells are generated. Furthermore, we discuss the properties that allow TRM cells to operate in normal and transformed tissues, as well as implications for the treatment of patients with cancer.

The Fate Choice Between Effector and Memory T Cell Lineages: Asymmetry, Signal Integration, and Feedback to Create Bistability.

CD8+ T cells clear primary infections with intracellular pathogens and provide long-term immunity against reinfection. Two different types of CD8+ T cells are responsible for these functions: short-lived effector T cells and memory T cells. The cellular relationship between these two types of CD8+ T cells has been subject to much investigation. Both cell types can derive from a single naïve CD8+ T cell precursor. Their generation requires a fate choice early during a T cell response. As a result, two populations of T cells emerge. One of these consists of terminally differentiated short-lived effector T cells. The other contains cells able to develop into long-lived memory T cells. A foundation for development of these two populations may be laid during the first division of an activated naïve T cell precursor, as a consequence of asymmetric segregation of fate-determining factors into the daughter cells. Nonetheless, the binary choice between the two lineages is strongly influenced by signals, which ensure that the differentiation process is matched with the needs posed by the infection. Here, we will discuss the genetic and metabolic programs governing differentiation of these two lineages as well as the processes leading to their induction and consolidation to create bistability. These processes involve extensive lateral inhibition between the programs as well as positive feedback between the genetic programs and the signaling pathways responsible for their induction. These features will be highlighted by discussing the role of the Notch signaling pathway in guiding the decision between the two lineages.

Specific T Cell Responses against Minor Histocompatibility Antigens Cannot Generally Be Explained by Absence of Their Allelic Counterparts on the Cell Surface.

Allogeneic stem cell transplantation has emerged as immunotherapy in the treatment of a variety of hematological malignancies. Its efficacy depends on induction of graft versus leukemia by donor lymphocytes. Both graft versus leukemia and graft versus host disease are induced by T cells reactive against polymorphic peptides, called minor histocompatibility antigens (MiHA), which differ between patient and donor and are presented in the context of self-HLA (where HLA is human leukocyte antigen). The allelic counterpart (AC) of the MiHA is generally considered to be absent at the cell surface, based on the absence of immune responses directed against the AC. To study this in detail, we evaluate the recognition, HLA-binding affinity, and cell surface expression of three selected MiHA. By quantitative MS, we demonstrate the similarly abundant expression of both MiHA and AC at the cell surface. We conclude that the absent recognition of the AC cannot generally be explained by insufficient processing and presentation at the cell surface of the AC.

The Transcription Factor Hobit Identifies Human Cytotoxic CD4+ T Cells.

The T cell lineage is commonly divided into CD4-expressing helper T cells that polarize immune responses through cytokine secretion and CD8-expressing cytotoxic T cells that eliminate infected target cells by virtue of the release of cytotoxic molecules. Recently, a population of CD4+ T cells that conforms to the phenotype of cytotoxic CD8+ T cells has received increased recognition. These cytotoxic CD4+ T cells display constitutive expression of granzyme B and perforin at the protein level and mediate HLA class II-dependent killing of target cells. In humans, this cytotoxic profile is found within the human cytomegalovirus (hCMV)-specific, but not within the influenza- or Epstein-Barr virus-specific CD4+ T cell populations, suggesting that, in particular, hCMV infection induces the formation of cytotoxic CD4+ T cells. We have previously described that the transcription factor Homolog of Blimp-1 in T cells (Hobit) is specifically upregulated in CD45RA+ effector CD8+ T cells that arise after hCMV infection. Here, we describe the expression pattern of Hobit in human CD4+ T cells. We found Hobit expression in cytotoxic CD4+ T cells and accumulation of Hobit+ CD4+ T cells after primary hCMV infection. The Hobit+ CD4+ T cells displayed highly overlapping characteristics with Hobit+ CD8+ T cells, including the expression of cytotoxic molecules, T-bet, and CX3CR1. Interestingly, γδ+ T cells that arise after hCMV infection also upregulate Hobit expression and display a similar effector phenotype as cytotoxic CD4+ and CD8+ T cells. These findings suggest a shared differentiation pathway in CD4+, CD8+, and γδ+ T cells that may involve Hobit-driven acquisition of long-lived cytotoxic effector function.