The Multifaceted Role of Tissue-Resident Memory T Cells.

Regionalized immune surveillance relies on the concerted efforts of diverse memory T cell populations. Of these, tissue-resident memory T (TRM) cells are strategically positioned in barrier tissues, where they enable efficient frontline defense against infections and cancer. However, the long-term persistence of these cells has been implicated in a variety of immune-mediated pathologies. Consequently, modulating TRM cell populations represents an attractive strategy for novel vaccination and therapeutic interventions against tissue-based diseases. Here, we provide an updated overview of TRM cell heterogeneity and function across tissues and disease states. We discuss mechanisms of TRM cell-mediated immune protection and their potential contributions to autoimmune disorders. Finally, we examine how TRM cell responses might be durably boosted or dampened for therapeutic gain.

Memory T cell subsets, migration patterns, and tissue residence.

Tissues such as the skin and mucosae are frequently exposed to microbial pathogens. Infectious agents must be quickly and efficiently controlled by our immune system, but the low frequency of naive T cells specific for any one pathogen means dependence on primary responses initiated in draining lymph nodes, often allowing time for serious infection to develop. These responses imprint effectors with the capacity to home to infected tissues; this process, combined with inflammatory signals, ensures the effective targeting of primary immunity. Upon vaccination or previous pathogen exposure, increased pathogen-specific T cell numbers together with altered migratory patterns of memory T cells can greatly improve immune efficacy, ensuring infections are prevented or at least remain subclinical. Until recently, memory T cell populations were considered to comprise central memory T cells (TCM), which are restricted to the secondary lymphoid tissues and blood, and effector memory T cells (TEM), which broadly migrate between peripheral tissues, the blood, and the spleen. Here we review evidence for these two memory populations, highlight a relatively new player, the tissue-resident memory T cell (TRM), and emphasize the potential differences between the migratory patterns of CD4(+) and CD8(+) T cells. This new understanding raises important considerations for vaccine design and for the measurement of immune parameters critical to the control of infectious disease, autoimmunity, and cancer.

Runx3 drives a CD8+ T cell tissue residency program that is absent in CD4+ T cells.

Tissue-resident memory T cells (TRM cells) provide rapid and superior control of localized infections. While the transcription factor Runx3 is a critical regulator of CD8+ T cell tissue residency, its expression is repressed in CD4+ T cells. Here, we show that, as a direct consequence of this Runx3-deficiency, CD4+ TRM cells lacked the transforming growth factor (TGF)-ß-responsive transcriptional network that underpins the tissue residency of epithelial CD8+ TRM cells. While CD4+ TRM cell formation required Runx1, this, along with the modest expression of Runx3 in CD4+ TRM cells, was insufficient to engage the TGF-ß-driven residency program. Ectopic expression of Runx3 in CD4+ T cells incited this TGF-ß-transcriptional network to promote prolonged survival, decreased tissue egress, a microanatomical redistribution towards epithelial layers and enhanced effector functionality. Thus, our results reveal distinct programming of tissue residency in CD8+ and CD4+ TRM cell subsets that is attributable to divergent Runx3 activity.

Effector and stem-like memory cell fates are imprinted in distinct lymph node niches directed by CXCR3 ligands.

T cells dynamically interact with multiple, distinct cellular subsets to determine effector and memory differentiation. Here, we developed a platform to quantify cell location in three dimensions to determine the spatial requirements that direct T cell fate. After viral infection, we demonstrated that CD8+ effector T cell differentiation is associated with positioning at the lymph node periphery. This was instructed by CXCR3 signaling since, in its absence, T cells are confined to the lymph node center and alternatively differentiate into stem-like memory cell precursors. By mapping the cellular sources of CXCR3 ligands, we demonstrated that CXCL9 and CXCL10 are expressed by spatially distinct dendritic and stromal cell subsets. Unlike effector cells, retention of stem-like memory precursors in the paracortex is associated with CCR7 expression. Finally, we demonstrated that T cell location can be tuned, through deficiency in CXCL10 or type I interferon signaling, to promote effector or stem-like memory fates.

Discrete tissue microenvironments instruct diversity in resident memory T cell function and plasticity.

Tissue-resident memory T (TRM) cells are non-recirculating cells that exist throughout the body. Although TRM cells in various organs rely on common transcriptional networks to establish tissue residency, location-specific factors adapt these cells to their tissue of lodgment. Here we analyze TRM cell heterogeneity between organs and find that the different environments in which these cells differentiate dictate TRM cell function, durability and malleability. We find that unequal responsiveness to TGFß is a major driver of this diversity. Notably, dampened TGFß signaling results in CD103- TRM cells with increased proliferative potential, enhanced function and reduced longevity compared with their TGFß-responsive CD103+ TRM counterparts. Furthermore, whereas CD103- TRM cells readily modified their phenotype upon relocation, CD103+ TRM cells were comparatively resistant to transdifferentiation. Thus, despite common requirements for TRM cell development, tissue adaptation of these cells confers discrete functional properties such that TRM cells exist along a spectrum of differentiation potential that is governed by their local tissue microenvironment.

Single-cell protein expression profiling resolves circulating and resident memory T cell diversity across tissues and infection contexts.

Memory CD8+ T cells can be broadly divided into circulating (TCIRCM) and tissue-resident memory T (TRM) populations. Despite well-defined migratory and transcriptional differences, the phenotypic and functional delineation of TCIRCM and TRM cells, particularly across tissues, remains elusive. Here, we utilized an antibody screening platform and machine learning prediction pipeline (InfinityFlow) to profile >200 proteins in TCIRCM and TRM cells in solid organs and barrier locations. High-dimensional analyses revealed unappreciated heterogeneity within TCIRCM and TRM cell lineages across nine different organs after either local or systemic murine infection models. Additionally, we demonstrated the relative effectiveness of strategies allowing for the selective ablation of TCIRCM or TRM populations across organs and identified CD55, KLRG1, CXCR6, and CD38 as stable markers for characterizing memory T cell function during inflammation. Together, these data and analytical framework provide an in-depth resource for memory T cell classification in both steady-state and inflammatory conditions.

Local proliferation maintains a stable pool of tissue-resident memory T cells after antiviral recall responses.

Although tissue-resident memory T cells (TRM cells) are critical in fighting infection, their fate after local pathogen re-encounter is unknown. Here we found that skin TRM cells engaged virus-infected cells, proliferated in situ in response to local antigen encounter and did not migrate out of the epidermis, where they exclusively reside. As a consequence, secondary TRM cells formed from pre-existing TRM cells, as well as from precursors recruited from the circulation. Newly recruited antigen-specific or bystander TRM cells were generated in the skin without displacement of the pre-existing TRM cell pool. Thus, pre-existing skin TRM cell populations are not displaced after subsequent infections, which enables multiple TRM cell specificities to be stably maintained within the tissue.

Adrenergic regulation of the vasculature impairs leukocyte interstitial migration and suppresses immune responses.

The sympathetic nervous system (SNS) controls various physiological functions via the neurotransmitter noradrenaline. Activation of the SNS in response to psychological or physical stress is frequently associated with weakened immunity. Here, we investigated how adrenoceptor signaling influences leukocyte behavior. Intravital two-photon imaging after injection of noradrenaline revealed transient inhibition of CD8+ and CD4+ T cell locomotion in tissues. Expression of ß-adrenergic receptor in hematopoietic cells was not required for NA-mediated inhibition of motility. Rather, chemogenetic activation of the SNS or treatment with adrenergic receptor agonists induced vasoconstriction and decreased local blood flow, resulting in abrupt hypoxia that triggered rapid calcium signaling in leukocytes and halted cell motility. Oxygen supplementation reversed these effects. Treatment with adrenergic receptor agonists impaired T cell responses induced in response to viral and parasitic infections, as well as anti-tumor responses. Thus, stimulation of the SNS impairs leukocyte mobility, providing a mechanistic understanding of the link between adrenergic receptors and compromised immunity.

Redefining the human corneal immune compartment using dynamic intravital imaging.

The healthy human cornea is a uniquely transparent sensory tissue where immune responses are tightly controlled to preserve vision. The cornea contains immune cells that are widely presumed to be intraepithelial dendritic cells (DCs). Corneal immune cells have diverse cellular morphologies and morphological alterations are used as a marker of inflammation and injury. Based on our imaging of corneal T cells in mice, we hypothesized that many human corneal immune cells commonly defined as DCs are intraepithelial lymphocytes (IELs). To investigate this, we developed functional in vivo confocal microscopy (Fun-IVCM) to investigate cell dynamics in the human corneal epithelium and stroma. We show that many immune cells resident in the healthy human cornea are T cells. These corneal IELs are characterized by rapid, persistent motility and interact with corneal DCs and sensory nerves. Imaging deeper into the corneal stroma, we show that crawling macrophages and rare motile T cells patrol the tissue. Furthermore, we identify altered immune cell behaviors in response to short-term contact lens wear (acute inflammatory stimulus), as well as in individuals with allergy (chronic inflammatory stimulus) that was modulated by therapeutic intervention. These findings redefine current understanding of immune cell subsets in the human cornea and reveal how resident corneal immune cells respond and adapt to chronic and acute stimuli.

Moving beyond velocity: Opportunities and challenges to quantify immune cell behavior.

The analysis of cellular behavior using intravital multi-photon microscopy has contributed substantially to our understanding of the priming and effector phases of immune responses. Yet, many questions remain unanswered and unexplored. Though advancements in intravital imaging techniques and animal models continue to drive new discoveries, continued improvements in analysis methods are needed to extract detailed information about cellular behavior. Focusing on dendritic cell (DC) and T cell interactions as an exemplar, here we discuss key limitations for intravital imaging studies and review and explore alternative approaches to quantify immune cell behavior. We touch upon current developments in deep learning models, as well as established methods from unrelated fields such as ecology to detect and track objects over time. As developments in open-source software make it possible to process and interactively view larger datasets, the challenge for the field will be to determine how best to combine intravital imaging with multi-parameter imaging of larger tissue regions to discover new facets of leukocyte dynamics and how these contribute to immune responses.

The developmental pathway for CD103(+)CD8+ tissue-resident memory T cells of skin.

Tissue-resident memory T cells (T(RM) cells) provide superior protection against infection in extralymphoid tissues. Here we found that CD103(+)CD8(+) T(RM) cells developed in the skin from epithelium-infiltrating precursor cells that lacked expression of the effector-cell marker KLRG1. A combination of entry into the epithelium plus local signaling by interleukin 15 (IL-15) and transforming growth factor-ß (TGF-ß) was required for the formation of these long-lived memory cells. Notably, differentiation into T(RM) cells resulted in the progressive acquisition of a unique transcriptional profile that differed from that of circulating memory cells and other types of T cells that permanently reside in skin epithelium. We provide a comprehensive molecular framework for the local differentiation of a distinct peripheral population of memory cells that forms a first-line immunological defense system in barrier tissues.

Liver-Resident Memory CD8+ T Cells Form a Front-Line Defense against Malaria Liver-Stage Infection.

In recent years, various intervention strategies have reduced malaria morbidity and mortality, but further improvements probably depend upon development of a broadly protective vaccine. To better understand immune requirement for protection, we examined liver-stage immunity after vaccination with irradiated sporozoites, an effective though logistically difficult vaccine. We identified a population of memory CD8+ T cells that expressed the gene signature of tissue-resident memory T (Trm) cells and remained permanently within the liver, where they patrolled the sinusoids. Exploring the requirements for liver Trm cell induction, we showed that by combining dendritic cell-targeted priming with liver inflammation and antigen recognition on hepatocytes, high frequencies of Trm cells could be induced and these cells were essential for protection against malaria sporozoite challenge. Our study highlights the immune potential of liver Trm cells and provides approaches for their selective transfer, expansion, or depletion, which may be harnessed to control liver infections or autoimmunity.

Author Correction: Tissue-resident memory CD8+ T cells promote melanoma-immune equilibrium in skin.

Panel j was inadvertently labelled as panel k in the caption to Fig. 4. Similarly, 'Fig. 4k' should have been 'Fig. 4j' in the sentence beginning 'TNF-α-deficient gBT-I cells were…'. In addition, the surname of author Umaimainthan Palendira was misspelled 'Palendria'. These errors have been corrected online.

Tissue-resident memory CD8+ T cells promote melanoma-immune equilibrium in skin.

The immune system can suppress tumour development both by eliminating malignant cells and by preventing the outgrowth and spread of cancer cells that resist eradication1. Clinical and experimental data suggest that the latter mode of control-termed cancer-immune equilibrium1-can be maintained for prolonged periods of time, possibly up to several decades2-4. Although cancers most frequently originate in epithelial layers, the nature and spatiotemporal dynamics of immune responses that maintain cancer-immune equilibrium in these tissue compartments remain unclear. Here, using a mouse model of transplantable cutaneous melanoma5, we show that tissue-resident memory CD8+ T cells (TRM cells) promote a durable melanoma-immune equilibrium that is confined to the epidermal layer of the skin. A proportion of mice (~40%) transplanted with melanoma cells remained free of macroscopic skin lesions long after epicutaneous inoculation, and generation of tumour-specific epidermal CD69+ CD103+ TRM cells correlated with this spontaneous disease control. By contrast, mice deficient in TRM formation were more susceptible to tumour development. Despite being tumour-free at the macroscopic level, mice frequently harboured melanoma cells in the epidermal layer of the skin long after inoculation, and intravital imaging revealed that these cells were dynamically surveyed by TRM cells. Consistent with their role in melanoma surveillance, tumour-specific TRM cells that were generated before melanoma inoculation conferred profound protection from tumour development independently of recirculating T cells. Finally, depletion of TRM cells triggered tumour outgrowth in a proportion (~20%) of mice with occult melanomas, demonstrating that TRM cells can actively suppress cancer progression. Our results show that TRM cells have a fundamental role in the surveillance of subclinical melanomas in the skin by maintaining cancer-immune equilibrium. As such, they provide strong impetus for exploring these cells as targets of future anticancer immunotherapies.

CD169+ macrophages in lymph node and spleen critically depend on dual RANK and LTbetaR signaling.

CD169+ macrophages reside in lymph node (LN) and spleen and play an important role in the immune defense against pathogens. As resident macrophages, they are responsive to environmental cues to shape their tissue-specific identity. We have previously shown that LN CD169+ macrophages require RANKL for formation of their niche and their differentiation. Here, we demonstrate that they are also dependent on direct lymphotoxin beta (LTß) receptor (R) signaling. In the absence or the reduced expression of either RANK or LTßR, their differentiation is perturbed, generating myeloid cells expressing SIGN-R1 in LNs. Conditions of combined haploinsufficiencies of RANK and LTßR revealed that both receptors contribute equally to LN CD169+ macrophage differentiation. In the spleen, the Cd169-directed ablation of either receptor results in a selective loss of marginal metallophilic macrophages (MMMs). Using a RANKL reporter mouse, we identify splenic marginal zone stromal cells as a source of RANKL and demonstrate that it participates in MMM differentiation. The loss of MMMs had no effect on the splenic B cell compartments but compromised viral capture and the expansion of virus-specific CD8+ T cells. Taken together, the data provide evidence that CD169+ macrophage differentiation in LN and spleen requires dual signals from LTßR and RANK with implications for the immune response.

Fibroblastic reticular cells provide a supportive niche for lymph node-resident macrophages.

The lymph node (LN) is home to resident macrophage populations that are essential for immune function and homeostasis, but key factors controlling this niche are undefined. Here, we show that fibroblastic reticular cells (FRCs) are an essential component of the LN macrophage niche. Genetic ablation of FRCs caused rapid loss of macrophages and monocytes from LNs across two in vivo models. Macrophages co-localized with FRCs in human LNs, and murine single-cell RNA-sequencing revealed that FRC subsets broadly expressed master macrophage regulator CSF1. Functional assays containing purified FRCs and monocytes showed that CSF1R signaling was sufficient to support macrophage development. These effects were conserved between mouse and human systems. These data indicate an important role for FRCs in maintaining the LN parenchymal macrophage niche.

The transcription factor SpiB regulates the fibroblastic reticular cell network and CD8+ T-cell responses in lymph nodes.

Fibroblastic reticular cells (FRCs) construct microanatomical niches that support lymph node (LN) homeostasis and coordination of immune responses. Transcription factors regulating the functionality of FRCs remain poorly understood. Here, we investigated the role of the transcription factor SpiB that is expressed in LN FRCs. Conditional ablation of SpiB in FRCs impaired the FRC network in the T-cell zone of LNs, leading to reduced numbers of FRCs and altered homeostatic functions including reduced CCL21 and interleukin-7 expression. The size and cellularity of LNs remained intact in the absence of SpiB but the space between the reticular network increased, indicating that although FRCs were reduced in number they stretched to maintain network integrity. Following virus infection, antiviral CD8+ T-cell responses were impaired, suggesting a role for SpiB expression in FRCs in orchestrating immune responses. Together, our findings reveal a new role for SpiB as an important regulator of FRC functions and immunity in LNs.

Hair follicles: gatekeepers to the epidermis.

Substantial depletion of Langerhans cells leads to their replenishment by bone marrow­derived precursors that access the epidermis through hair follicles, a site of crucial chemokine production.

Intravital Imaging of the Human Cornea Reveals the Differential Effects of Season on Innate and Adaptive Immune Cell Morphodynamics.

PURPOSE: Defining how the in vivo immune status of peripheral tissues is shaped by the external environment has remained a technical challenge. We recently developed Functional in vivo confocal microscopy (Fun-IVCM) for dynamic, longitudinal imaging of corneal immune cells in living humans. This study investigated the effect of seasonal-driven environmental factors on the morphodynamic features of human corneal immune cell subsets. DESIGN: Longitudinal, observational clinical study. PARTICIPANTS: Sixteen healthy participants (aged 18-40 years) attended 2 visits in distinct seasons in Melbourne, Australia (Visit 1, November-December 2021 [spring-summer]; Visit 2, April-June 2022 [autumn-winter]). METHODS: Environmental data were collected over each period. Participants underwent ocular surface examinations and corneal Fun-IVCM (Heidelberg Engineering). Corneal scans were acquired at 5.5 ± 1.5-minute intervals for up to 5 time points. Time-lapse Fun-IVCM videos were created to analyze corneal immune cells, comprising epithelial T cells and dendritic cells (DCs), and stromal macrophages. Tear cytokines were analyzed using a multiplex bead-based immunoassay. MAIN OUTCOME MEASURES: Difference in the density, morphology, and dynamic parameters of corneal immune cell subsets over the study periods. RESULTS: Visit 1 was characterized by higher temperature, lower humidity, and higher air particulate and pollen levels compared with Visit 2. Clinical ocular surface parameters and the density of immune cell subsets were similar across visits. At Visit 1 , corneal epithelial DCs were larger, with a lower dendrite probing speed (0.38 ± 0.21 vs. 0.68 ± 0.33 µm/min; P < 0.001) relative to Visit 2; stromal macrophages were more circular and had less dynamic activity (Visit 1, 7.2 ± 1.9 vs. Visit 2, 10.3 ± 3.7 dancing index; P < 0.001). Corneal T cell morphodynamics were unchanged across periods. Basal tear levels of interleukin 2 and CXCL10 were relatively lower during spring-summer. CONCLUSIONS: This study identifies that the in vivo morphodynamics of innate corneal immune cells (DCs, macrophages) are modified by environmental factors, but such effects are not evident for adaptive immune cells (T cells). The cornea is a potential in vivo window to investigate season-dependent environmental influences on the human immune system. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.