Effector differentiation downstream of lineage commitment in ILC1s is driven by Hobit across tissues.

Innate lymphoid cells (ILCs) participate in tissue homeostasis, inflammation, and early immunity against infection. It is unclear how ILCs acquire effector function and whether these mechanisms differ between organs. Through multiplexed single-cell mRNA sequencing, we identified cKit+CD127hiTCF-1hi early differentiation stages of T-bet+ ILC1s. These cells were present across different organs and had the potential to mature toward CD127intTCF-1int and CD127-TCF-1- ILC1s. Paralleling a gradual loss of TCF-1, differentiating ILC1s forfeited their expansion potential while increasing expression of effector molecules, reminiscent of T cell differentiation in secondary lymphoid organs. The transcription factor Hobit was induced in TCF-1hi ILC1s and was required for their effector differentiation. These findings reveal sequential mechanisms of ILC1 lineage commitment and effector differentiation that are conserved across tissues. Our analyses suggest that ILC1s emerge as TCF-1hi cells in the periphery and acquire a spectrum of organ-specific effector phenotypes through a uniform Hobit-dependent differentiation pathway driven by local cues.

Tissue-resident memory CD8+ T cells shape local and systemic secondary T cell responses.

Tissue-resident memory CD8+ T cells (TRM cells) are crucial in protecting against reinvading pathogens, but the impact of reinfection on their tissue confinement and contribution to recall responses is unclear. We developed a unique lineage tracer mouse model exploiting the TRM-defining transcription factor homolog of Blimp-1 in T cells (Hobit) to fate map the TRM progeny in secondary responses. After reinfection, a sizeable fraction of secondary memory T cells in the circulation developed downstream of TRM cells. These tissue-experienced ex-TRM cells shared phenotypic properties with the effector memory T cell population but were transcriptionally and functionally distinct from other secondary effector memory T cell cells. Adoptive transfer experiments of TRM cells corroborated their potential to form circulating effector and memory cells during recall responses. Moreover, specific ablation of primary TRM cell populations substantially impaired the secondary T cell response, both locally and systemically. Thus, TRM cells retain developmental plasticity and shape both local and systemic T cell responses on reinfection.

Author Correction: Hobit- and Blimp-1-driven CD4+ tissue-resident memory T cells control chronic intestinal inflammation.

In the version of this article initially published, a portion of the Acknowledgements section ("the Clinical Research Group CEDER of the German Research Council (DFG)") was incorrect. The correct statement is as follows: "...the Collaborative Research Center TRR241 of the German Research Council (DFG)...". The error has been corrected in the HTML and PDF version of the article.

Hobit- and Blimp-1-driven CD4+ tissue-resident memory T cells control chronic intestinal inflammation.

Although tissue-resident memory T cells (TRM cells) have been shown to regulate host protection in infectious disorders, their function in inflammatory bowel disease (IBD) remains to be investigated. Here we characterized TRM cells in human IBD and in experimental models of intestinal inflammation. Pro-inflammatory TRM cells accumulated in the mucosa of patients with IBD, and the presence of CD4+CD69+CD103+ TRM cells was predictive of the development of flares. In vivo, functional impairment of TRM cells in mice with double knockout of the TRM-cell-associated transcription factors Hobit and Blimp-1 attenuated disease in several models of colitis, due to impaired cross-talk between the adaptive and innate immune system. Finally, depletion of TRM cells led to a suppression of colitis activity. Together, our data demonstrate a central role for TRM cells in the pathogenesis of chronic intestinal inflammation and suggest that these cells could be targets for future therapeutic approaches in IBD.

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.

Protective function and differentiation cues of brain-resident CD8+ T cells during surveillance of latent Toxoplasma gondii infection.

Chronic Toxoplasma gondii infection induces brain-resident CD8+ T cells (bTr), but the protective functions and differentiation cues of these cells remain undefined. Here, we used a mouse model of latent infection by T. gondii leading to effective CD8+ T cell-mediated parasite control. Thanks to antibody depletion approaches, we found that peripheral circulating CD8+ T cells are dispensable for brain parasite control during chronic stage, indicating that CD8+ bTr are able to prevent brain parasite reactivation. We observed that the retention markers CD69, CD49a, and CD103 are sequentially acquired by brain parasite-specific CD8+ T cells throughout infection and that a majority of CD69/CD49a/CD103 triple-positive (TP) CD8+ T cells also express Hobit, a transcription factor associated with tissue residency. This TP subset develops in a CD4+ T cell-dependent manner and is associated with effective parasite control during chronic stage. Conditional invalidation of Transporter associated with Antigen Processing (TAP)-mediated major histocompatibility complex (MHC) class I presentation showed that presentation of parasite antigens by glutamatergic neurons and microglia regulates the differentiation of CD8+ bTr into TP cells. Single-cell transcriptomic analyses revealed that resistance to encephalitis is associated with the expansion of stem-like subsets of CD8+ bTr. In summary, parasite-specific brain-resident CD8+ T cells are a functionally heterogeneous compartment which autonomously ensure parasite control during T. gondii latent infection and which differentiation is shaped by neuronal and microglial MHC I presentation. A more detailed understanding of local T cell-mediated immune surveillance of this common parasite is needed for harnessing brain-resident CD8+ T cells in order to enhance control of chronic brain infections.

A central role for Notch in effector CD8(+) T cell differentiation.

Activated CD8(+) T cells choose between terminal effector cell (TEC) or memory precursor cell (MPC) fates. We found that the signaling receptor Notch controls this 'choice'. Notch promoted the differentiation of immediately protective TECs and was correspondingly required for the clearance of acute infection with influenza virus. Notch activated a major portion of the TEC-specific gene-expression program and suppressed the MPC-specific program. Expression of Notch was induced on naive CD8(+) T cells by inflammatory mediators and interleukin 2 (IL-2) via pathways dependent on the metabolic checkpoint kinase mTOR and the transcription factor T-bet. These pathways were subsequently amplified downstream of Notch, creating a positive feedback loop. Notch thus functions as a central hub where information from different sources converges to match effector T cell differentiation to the demands of an infection.

Intestinal tissue-resident memory T cells maintain distinct identity from circulating memory T cells after in vitro restimulation.

Resident memory T (TRM) cells have been recently established as an important subset of memory T cells that provide early and essential protection against reinfection in the absence of circulating memory T cells. Recent findings showing that TRM expand in vivo after repeated antigenic stimulation indicate that these memory T cells are not terminally differentiated. This suggests an opportunity for in vitro TRM expansion to apply in an immunotherapy setting. However, it has also been shown that TRM may not maintain their identity and form circulating memory T cells after in vivo restimulation. Therefore, we set out to determine how TRM respond to antigenic activation in culture. Using Listeria monocytogenes and LCMV infection models, we found that TRM from the intraepithelial compartment of the small intestine expand in vitro after antigenic stimulation and subsequent resting in homeostatic cytokines. A large fraction of the expanded TRM retained their phenotype, including the expression of key TRM markers CD69 and CD103 (ITGAE). The optimal culture of TRM required low O2 pressure to maintain the expression of these and other TRM-associated molecules. Expanded TRM retained their effector capacity to produce cytokines after restimulation, but did not acquire a highly glycolytic profile indicative of effector T cells. The proteomic analysis confirmed TRM profile retention, including expression of TRM-related transcription factors, tissue retention factors, adhesion molecules, and enzymes involved in fatty acid metabolism. Collectively, our data indicate that limiting oxygen conditions supports in vitro expansion of TRM cells that maintain their TRM phenotype, at least in part, suggesting an opportunity for therapeutic strategies that require in vitro expansion of TRM.

Resident Memory T Cells in the Atherosclerotic Lesion Associate With Reduced Macrophage Content and Increased Lesion Stability.

BACKGROUND: Tissue resident memory T (TRM) cells are a T-cell subset that resides at the site of prior antigen recognition to protect the body against reoccurring encounters. Besides their protective function, TRM cells have also been implicated in inflammatory disorders. TRM cells are characterized by the expression of CD69 and transcription factors Hobit (homolog of Blimp-1 [B lymphocyte-induced maturation protein 1] in T cells) and Blimp-1. As the majority of T cells in the arterial intima expresses CD69, TRM cells may contribute to the pathogenesis of atherosclerosis as well. Here, we aimed to assess the presence and potential role of TRM cells in atherosclerosis. METHODS: To identify TRM cells in human atherosclerotic lesions, a single-cell RNA-sequencing data set was interrogated, and T-cell phenotypes were compared with that of integrated predefined TRM cells. The presence and phenotype of TRM in atherosclerotic lesions was corroborated using a mouse model that enabled tracking of Hobit-expressing TRM cells. To explore the function of TRM cells during atherogenesis, RAG1-/- (recombination activating gene 1 deficient) LDLr-/- (low-density lipoprotein receptor knockout) mice received a bone marrow transplant from HobitKO/CREBlimp-1flox/flox mice, which exhibit abrogated TRM cell formation, whereafter the mice were fed a Western-type diet for 10 weeks. RESULTS: Human atherosclerotic lesions contained T cells that exhibited a TRM cell-associated gene signature. Moreover, a fraction of these T cells clustered together with predefined TRM cells upon integration. The presence of Hobit-expressing TRM cells in the atherosclerotic lesion was confirmed in mice. These lesion-derived TRM cells were characterized by the expression of CD69 and CD49α. Moreover, we demonstrated that this small T-cell subset significantly affects lesion composition, by reducing the amount of intralesional macrophages and increasing collagen content. CONCLUSIONS: TRM cells, characterized by the expression of CD69 and CD49α, constitute a minor population in atherosclerotic lesions and are associated with increased lesion stability in a Hobit and Blimp-1 knockout mouse model.

No-shows in T-cell responses are frequent for clones of low T-cell receptor affinity.

The magnitude of CD8 T-cell responses against intracellular pathogens is thought to primarily depend on the expansion capacity of naïve T cells, given that their recruitment is considered optimal. In the current issue of the European Journal of Immunology [Eur. J. Immunol. 2023. 53: 000-000], Leube et al. challenge these concepts and show that the recruitment of naïve T-cell clones into primary responses can be far from complete. The failure to efficiently recruit T-cell clones occurs more frequently in case of low-affinity interactions of the T-cell receptor with cognate antigen of the pathogen. Using single-cell fate-mapping in the Lm-OVA model, the authors demonstrate that naïve T-cell clones of low affinity in contrast to those of high affinity often do not expand after pathogen encounter. These low-affinity clones are maintained as naïve CD8 T cells that can robustly respond upon secondary encounter with the same pathogen, in particular when the reencountered pathogen contains modifications resulting in improved recognition. Thus, this study indicates that the regulation of the response size of CD8 T cells is yet more elaborate than anticipated and involves control at the level of recruitment and expansion of naïve CD8 T cells.

ILC1: Development, maturation, and transcriptional regulation.

Type 1 Innate Lymphoid cells (ILC1s) are tissue-resident cells that partake in the regulation of inflammation and homeostasis. A major feature of ILC1s is their ability to rapidly respond after infections. The effector repertoire of ILC1s includes the pro-inflammatory cytokines IFN-γ and TNF-α and cytotoxic mediators such as granzymes, which enable ILC1s to establish immune responses and to directly kill target cells. Recent advances in the characterization of ILC1s have considerably furthered our understanding of ILC1 development and maintenance in tissues. In particular, it has become clear how ILC1s operate independently from conventional natural killer cells, with which they share many characteristics. In this review, we discuss recent developments with regards to the differentiation, polarization, and effector maturation of ILC1s. These processes may underlie the observed heterogeneity in ILC1 populations within and between different tissues. Next, we highlight transcriptional programs that control each of the separate steps in the differentiation of ILC1s. These transcriptional programs are shared with other tissue-resident type-1 lymphocytes, such as tissue-resident memory T cells (TRM ) and invariant natural killer T cells (iNKT), highlighting that ILC1s utilize networks of transcriptional regulation that are conserved between lymphocyte lineages to respond effectively to tissue-invading pathogens.

Memory CD8+ T cells upregulate glycolysis and effector functions under limiting oxygen conditions.

Memory CD8+ T cells are indispensable for maintaining long-term immunity against intracellular pathogens and tumors. Despite their presence at oxygen-deprived infected tissue sites or in tumors, the impact of local oxygen pressure on memory CD8+ T cells remains largely unclear. We sought to elucidate how oxygen pressure impacts memory CD8+ T cells arising after infection with Listeria monocytogenes-OVA. Our data revealed that reduced oxygen pressure during in vitro culture switched CD8+ T cell metabolism from oxidative phosphorylation to a glycolytic phenotype. Quantitative proteomic analysis showed that limiting oxygen conditions increased the expression of glucose transporters and components of the glycolytic pathway, while decreasing TCA cycle and mitochondrial respiratory chain proteins. The altered CD8+ T cell metabolism did not affect the expansion potential, but enhanced the granzyme B and IFN-γ production capacity. In vivo, memory CD8+ T cells cultured under low oxygen pressure provided protection against bacterial rechallenge. Taken together, our study indicates that strategies of cellular immune therapy may benefit from reducing oxygen during culture to develop memory CD8+ T cells with superior effector functions.

Mouse Hobit is a homolog of the transcriptional repressor Blimp-1 that regulates NKT cell effector differentiation.

The transcriptional repressor Blimp-1 mediates the terminal differentiation of many cell types, including T cells. Here we identified Hobit (Znf683) as a previously unrecognized homolog of Blimp-1 that was specifically expressed in mouse natural killer T cells (NKT cells). Through studies of Hobit-deficient mice, we found that Hobit was essential for the formation of mature thymic NKT cells. In the periphery, Hobit repressed the accumulation of interferon-γ (IFN-γ)-producing NK1.1(lo) NKT cells at steady state. After antigenic stimulation, Hobit repressed IFN-γ expression, whereas after innate stimulation, Hobit induced granzyme B expression. Thus, reminiscent of the function of Blimp-1 in other lymphocytes, Hobit controlled the maintenance of quiescent, fully differentiated NKT cells and regulated their immediate effector functions.

mRNA-1273 vaccinated inflammatory bowel disease patients receiving TNF inhibitors develop broad and robust SARS-CoV-2-specific CD8+ T cell responses.

SARS-CoV-2-specific CD8+ T cells recognize conserved viral peptides and in the absence of cross-reactive antibodies form an important line of protection against emerging viral variants as they ameliorate disease severity. SARS-CoV-2 mRNA vaccines induce robust spike-specific antibody and T cell responses in healthy individuals, but their effectiveness in patients with chronic immune-mediated inflammatory disorders (IMIDs) is less well defined. These patients are often treated with systemic immunosuppressants, which may negatively affect vaccine-induced immunity. Indeed, TNF inhibitor (TNFi)-treated inflammatory bowel disease (IBD) patients display reduced ability to maintain SARS-CoV-2 antibody responses post-vaccination, yet the effects on CD8+ T cells remain unclear. Here, we analyzed the impact of IBD and TNFi treatment on mRNA-1273 vaccine-induced CD8+ T cell responses compared to healthy controls in SARS-CoV-2 experienced and inexperienced patients. CD8+ T cells were analyzed for their ability to recognize 32 SARS-CoV-2-specific epitopes, restricted by 10 common HLA class I allotypes using heterotetramer combinatorial coding. This strategy allowed in-depth ex vivo profiling of the vaccine-induced CD8+ T cell responses using phenotypic and activation markers. mRNA vaccination of TNFi-treated and untreated IBD patients induced robust spike-specific CD8+ T cell responses with a predominant central memory and activated phenotype, comparable to those in healthy controls. Prominent non-spike-specific CD8+ T cell responses were observed in SARS-CoV-2 experienced donors prior to vaccination. Non-spike-specific CD8+ T cells persisted and spike-specific CD8+ T cells notably expanded after vaccination in these patient cohorts. Our data demonstrate that regardless of TNFi treatment or prior SARS-CoV-2 infection, IBD patients benefit from vaccination by inducing a robust spike-specific CD8+ T cell response.

Hobit and Blimp-1 instruct the differentiation of iNKT cells into resident-phenotype lymphocytes after lineage commitment.

iNKT cells are CD1d-restricted T cells that play a pro-inflammatory or regulatory role in infectious and autoimmune diseases. Thymic precursors of iNKT cells eventually develop into distinct iNKT1, iNKT2, and iNKT17 lineages in the periphery. It remains unclear whether iNKT cells retain developmental potential after lineage commitment. iNKT cells acquire a similar phenotype as tissue-resident memory T cells, suggesting that they also differentiate along a trajectory that enables them to persist in peripheral tissues. Here, we addressed whether lineage commitment and memory differentiation are parallel or sequential developmental programs of iNKT cells. We defined three subsets of peripheral iNKT cells using CD62L and CD69 expression that separate central, effector, and resident memory phenotype cells. The majority of iNKT1 cells displayed a resident phenotype in contrast to iNKT2 and iNKT17 cells. The transcription factor Hobit, which is upregulated in iNKT cells, plays an essential role in their development together with its homolog Blimp-1. Hobit and Blimp-1 instructed the differentiation of central memory iNKT cells into resident memory iNKT cells, but did not impact commitment into iNKT1, iNKT2, or iNKT17 lineages. Thus, we conclude that memory differentiation and the establishment of residency occur after lineage commitment through a Hobit and Blimp-1-driven transcriptional program.

Hobit and Blimp-1 regulate TRM abundance after LCMV infection by suppressing tissue exit pathways of TRM precursors.

Tissue-resident memory T cells (Trm) are retained in peripheral tissues after infection for enhanced protection against secondary encounter with the same pathogen. We have previously shown that the transcription factor Hobit and its homolog Blimp-1 drive Trm development after viral infection, but how and when these transcription factors mediate Trm formation remains poorly understood. In particular, the major impact of Blimp-1 in regulating several aspects of effector T-cell differentiation impairs study of its specific role in Trm development. Here, we used the restricted expression of Hobit in the Trm lineage to develop mice with a conditional deletion of Blimp-1 in Trm, allowing us to specifically investigate the role of both transcription factors in Trm differentiation. We found that Hobit and Blimp-1 were required for the upregulation of CD69 and suppression of CCR7 and S1PR1 on virus-specific Trm precursors after LCMV infection, underlining a role in their retention within tissues. The early impact of Hobit and Blimp-1 favored Trm formation and prevented the development of circulating memory T cells. Thus, our findings highlight a role of Hobit and Blimp-1 at the branching point of circulating and resident memory lineages by suppressing tissue egress of Trm precursors early during infection.

T-cell memory in tissues.

Immunological memory equips our immune system to respond faster and more effectively against reinfections. This acquired immunity was originally attributed to long-lived, memory T and B cells with body wide access to peripheral and secondary lymphoid tissues. In recent years, it has been realized that both innate and adaptive immunity to a large degree depends on resident immune cells that act locally in barrier tissues including tissue-resident memory T cells (Trm). Here, we will discuss the phenotype of these Trm in mice and humans, the tissues and niches that support them, and their function, plasticity, and transcriptional control. Their unique properties enable Trm to achieve long-lived immunological memory that can be deposited in nearly every organ in response to acute and persistent infection, and in response to cancer. However, Trm may also induce substantial immunopathology in allergic and autoimmune disease if their actions remain unchecked. Therefore, inhibitory and activating stimuli appear to balance the actions of Trm to ensure rapid proinflammatory responses upon infection and to prevent damage to host tissues under steady state conditions.

Circulating memory CD8+ T cells are limited in forming CD103+ tissue-resident memory T cells at mucosal sites after reinfection.

Tissue-resident memory CD8+ T cells (TRM ) localize to barrier tissues and mediate local protection against reinvading pathogens. Circulating central memory (TCM ) and effector memory CD8+ T cells (TEM ) also contribute to tissue recall responses, but their potential to form mucosal TRM remains unclear. Here, we employed adoptive transfer and lymphocytic choriomeningitis virus reinfection models to specifically assess secondary responses of TCM and TEM at mucosal sites. Donor TCM and TEM exhibited robust systemic recall responses, but only limited accumulation in the small intestine, consistent with reduced expression of tissue-homing and -retention molecules. Murine and human circulating memory T cells also exhibited limited CD103 upregulation following TGF-ß stimulation. Upon pathogen clearance, TCM and TEM readily gave rise to secondary TEM . TCM also formed secondary central memory in lymphoid tissues and TRM in internal tissues, for example, the liver. Both TCM and TEM failed to substantially contribute to resident mucosal memory in the small intestine, while activated intestinal TRM , but not liver TRM , efficiently reformed CD103+ TRM . Our findings demonstrate that circulating TCM and TEM are limited in generating mucosal TRM upon reinfection. This may pose important implications on cell therapy and vaccination strategies employing memory CD8+ T cells for protection at mucosal sites.

Murine iNKT cells are depleted by liver damage via activation of P2RX7.

Invariant natural killer T cells (iNKT) constitute up to 50% of liver lymphocytes and contribute to immunosurveillance as well as pathogenesis of the liver. Systemic activation of iNKT cells induces acute immune-mediated liver injury. However, how tissue damage events regulate iNKT cell function and homeostasis remains unclear. We found that specifically tissue-resident iNKT cells in liver and spleen express the tissue-damage receptor P2RX7 and the P2RX7-activating ectoenzyme ARTC2. P2RX7 expression restricted formation of iNKT cells in the liver suggesting that liver iNKT cells are actively restrained under homeostatic conditions. Deliberate activation of P2RX7 in vivo by exogenous NAD resulted in a nearly complete iNKT cell ablation in liver and spleen in a P2RX7-dependent manner. Tissue damage generated by acetaminophen-induced liver injury reduced the number of iNKT cells in the liver. The tissue-damage-induced iNKT cell depletion was driven by P2RX7 and localized to the site of injury, as iNKT cells in the spleen remained intact. The depleted liver iNKT cells reconstituted only slowly compared to other lymphocytes such as regulatory T cells. These findings suggest that tissue-damage-mediated depletion of iNKT cells acts as a feedback mechanism to limit iNKT cell-induced pathology resulting in the establishment of a tolerogenic environment.

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.