Memory Subsets Research Articles

Towards a better preclinical cancer model – human immune aging in humanized mice

BackgroundPreclinical models are often used for cancer studies and evaluation of novel therapeutics. The relevance of these models has vastly improved with mice bearing a human immune system, especially in the context of immunotherapy. Nonetheless, cancer is an age-related disease, and studies often overlook the effects of aging. Here we have established a humanized mouse model of human immune aging to investigate the role of this phenomenon on liver tumor dynamics.MethodsMultiple organs and tissues (blood, thymus, lung, liver, spleen and bone marrow) were harvested from NOD-scid IL2rγ−/− (NIKO) mice reconstituted with human immune cells, over a period of 60 weeks post-birth, for immune profiling. Young and aging immune cells were compared for transcriptomic changes and functional differences. Effect of immune aging was investigated in a liver cancer humanized mouse model.ResultsFocusing on the T cell population, which is central to cancer immunosurveillance and immunotherapy, we showed that the proportion of naïve T cells declined while memory subsets and senescent-like cells increased with age. RNA-sequencing revealed that downregulated genes were related to immune responses and processes, and this was corroborated by reduced cytokine production in aging T cells. Finally, we showed faster liver tumor growth in aging than younger humanized mice, which could be attributed to specific pathways of aging T cell exhaustion.ConclusionOur work improves on existing humanized (immune) mouse model and highlights the importance of considering immune aging in liver cancer modeling.

T cell memory revisited using single telomere length analysis.

The fundamental basis of T cell memory remains elusive. It is established that antigen stimulation drives clonal proliferation and differentiation, but the relationship between cellular phenotype, replicative history, and longevity, which is likely essential for durable memory, has proven difficult to elucidate. To address these issues, we used conventional markers of differentiation to identify and isolate various subsets of CD8+ memory T cells and measured telomere lengths in these phenotypically defined populations using the most sensitive technique developed to date, namely single telomere length analysis (STELA). Naive cells were excluded on the basis of dual expression of CCR7 and CD45RA. Memory subsets were sorted as CD27+CD45RA+, CD27intCD45RA+, CD27-CD45RA+, CD27+CD45RAint, CD27-CD45RAint, CD27+CD45RA-, and CD27-CD45RA- at >98% purity. The shortest median telomere lengths were detected among subsets that lacked expression of CD45RA, and the longest median telomere lengths were detected among subsets that expressed CD45RA. Longer median telomere lengths were also a feature of subsets that expressed CD27 in compartments defined by the absence or presence of CD45RA. Collectively, these data suggested a disconnect between replicative history and CD8+ memory T cell differentiation, which is classically thought to be a linear process that culminates with revertant expression of CD45RA.

Hub Genes and Immune Cell Infiltration in Hypoxia-Induced Pulmonary Hypertension: Bioinformatics Analysis and In Vivo Validation.

Hypoxia-induced pulmonary hypertension (HPH) represents a severe pulmonary disorder with high morbidity and mortality, which necessitates identifying the critical molecular mechanisms underlying HPH pathogenesis. The mRNA expression microarray GSE15197 (containing 8 pulmonary tissues from HPH and 13 normal controls) was downloaded from Gene Expression Omnibus (GEO). Gene ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) were executed by RStudio software. The Protein-Protein Interaction (PPI) network was visualized and established using Cytoscape, and the cytoHubba app from Cytoscape was used to pick out the hub modules. The infiltration of immune cells in HPH was analyzed using the CIBERSORTx. To confirm the potential hub genes, real-time quantitative reverse transcription PCR (qRT-PCR) was conducted using lung tissues of rat HPH models and controls. A total of 852 upregulated and 547 downregulated genes were identified. The top terms in biological processes were apoptosis, proliferation, and regulation of the MAPK cascade, including ERK1/2. Cytoplasm, cytosol, and membrane were enriched in cellular component groups. Molecular functions mainly focus on protein binding, protein serine/threonine kinase activity and identical protein binding. KEGG analysis identified pathways in cancer, regulation of actin cytoskeleton and rap1 signaling pathway. There was significantly different immune cell infiltration between HPH and normal control samples. High proportions of the memory subsets of B cells and CD4 cells, Macrophages M2 subtype, and resting Dendritic cells were found in HPH samples, while high proportions of naive CD4 cells and resting mast cells were found in normal control samples. The qRT-PCR results showed that among the ten identified hub modules, FBXL3, FBXL13 and XCL1 mRNA levels were upregulated, while NEDD4L, NPFFR2 and EDN3 were downregulated in HPH rats compared with control rats. Our study revealed the key genes and the involvement of immune cell infiltration in HPH, thus providing new insight into the pathogenesis of HPH and potential treatment targets for patients with HPH.

Negative Influence of Aging on Differentiation and Proliferation of CD8+ T-Cells in Dogs.

Immunosenescence is an age-related change in the immune system characterized by a reduction in naïve T-cells and an impaired proliferative capacity of CD8+ T-cells in older individuals. Recent research revealed the crucial impact of immunosenescence on the development and control of cancer, and aging is one of the causes that diminish the therapeutic efficacy of cancer immunotherapies targeting CD8+ T-cell activation. Despite dog cancer being defined as an age-related disease, there are few fundamental understandings regarding the relationship between aging and the canine immune system. Therefore, we aimed to elucidate the characteristics of immunosenescence in dogs and analyzed the effects of aging on the differentiation status and proliferation of canine CD8+ T cells using T-cell specific stimulation with anti-canine CD3/CD28 antibody-coated beads and interleukin-2. As a result, we found that older dogs have a lower proliferative capacity of CD8+ T-cells and a reduction in the naïve subset in their peripheral blood. Further analysis showed that older dogs had attenuated proliferation of the effector and central memory subsets. These results indicate the importance of maintaining less differentiated subsets to expand CD8+ T-cells in dogs and provide helpful insight into the development of dog immune therapies that require T-cell expansion ex vivo.

Insulin-like Growth Factor-1 Synergizes with IL-2 to Induce Homeostatic Proliferation of Regulatory T Cells.

IL-2 has been proposed to restore tolerance via regulatory T cell (Treg) expansion in autoimmunity, yet off-target effects necessitate identification of a combinatorial approach allowing for lower IL-2 dosing. We recently reported reduced levels of immunoregulatory insulin-like growth factor-1 (IGF1) during type 1 diabetes progression. Thus, we hypothesized that IGF1 would synergize with IL-2 to expand Tregs. We observed IGF1 receptor was elevated on murine memory and human naive Treg subsets. IL-2 and IGF1 promoted PI3K/Akt signaling in Tregs, inducing thymically-derived Treg expansion beyond either agent alone in NOD mice. Increased populations of murine Tregs of naive or memory, as well as CD5lo polyclonal or CD5hi likely self-reactive, status were also observed. Expansion was attributed to increased IL-2Rγ subunit expression on murine Tregs exposed to IL-2 and IGF1 as compared with IL-2 or IGF1 alone. Assessing translational capacity, incubation of naive human CD4+ T cells with IL-2 and IGF1 enhanced thymically-derived Treg proliferation invitro, without the need for TCR ligation. We then demonstrated that IGF1 and IL-2 or IL-7, which is also IL-2Rγ-chain dependent, can be used to induce proliferation of genetically engineered naive human Tregs or T conventional cells, respectively. These data support the potential use of IGF1 in combination with common γ-chain cytokines to drive homeostatic T cell expansion, both invitro and invivo, for cellular therapeutics and ex vivo gene editing.

A novel prosthesis presentation test to screen for cognitive and functional decline.

To validate a novel screening test for cognitive and functional decline in older patients rehabilitated with complete removable dental prostheses (CRDPs). Edentate old in-patients rehabilitated with CRDPs were included in this study. Participants were requested to remove their prostheses before their intraoral examinations. The prostheses were then presented in an inverted orientation. Participants had to correct the orientation of the prostheses and insert them in the appropriate jaws. The test was repeated after the intraoral exam. Appropriate statistical models were used (⍺ = .05) to associate the test results with the participants' mini-mental state examination (MMSE) score, functional independence measure (FIM), age and sex. Among the 86 participants (mean-age: 85.4 ± 6.4 years; mean MMSE: 19.8 ± 5.5; mean FIM: 77.9 ± 20.8), 21 (24.4%) failed to correctly insert the prosthesis. The prosthesis presentation test (PPT) was associated with the FIM but not the MMSE. Regression models further confirmed an association with age (P = .043), but not sex. Additional analyses revealed the PPT test is associated with the FIM's cognitive sub-sets of memory, problem solving and social interaction. The PPT is a novel, simple and quick screening tool that can help detect functional difficulties in older people. It can easily be performed during an oral examination. Future studies are needed to determine whether the PPT can be used to detect deficits in executive function, as a complement to the MMSE and also as a first assessment of a patient's ability to manage dentures independently.

A data-driven Boolean model explains memory subsets and evolution in CD8+ T cell exhaustion

T cells play a key role in a variety of immune responses, including infection and cancer. Upon stimulation, naïve CD8+ T cells proliferate and differentiate into a variety of memory and effector cell types; however, failure to clear antigens causes prolonged stimulation of CD8+ T cells, ultimately leading to T cell exhaustion (TCE). The functional and phenotypic changes that occur during CD8+ T cell differentiation are well characterized, but the underlying gene expression state changes are not completely understood. Here, we utilize a previously published data-driven Boolean model of gene regulatory interactions shown to mediate TCE. Our network analysis and modeling reveal the final gene expression states that correspond to TCE, along with the sequence of gene expression patterns that give rise to those final states. With a model that predicts the changes in gene expression that lead to TCE, we could evaluate strategies to inhibit the exhausted state. Overall, we demonstrate that a common pathway model of CD8+ T cell gene regulatory interactions can provide insights into the transcriptional changes underlying the evolution of cell states in TCE.

Peripheral Blood CD8+ T-Lymphocyte Subsets Are Associated with Prognosis in Prostate Cancer Patients

Background: Various studies have reported associations between frequencies of total peripheral blood lymphocytes and prostate cancer prognosis, but none so far has addressed the prognostic role of CD8+ T-lymphocyte subsets. Methods: A total of 43 prostate cancer patients with metastatic disease and 81 patients with non-metastatic disease were included in this study. Flow cytometry analyses were employed for determining the frequencies of peripheral CD8+ T-lymphocyte subsets. Results: Statistically significant lower levels of terminally differentiated effector (TEMRA) cells in patients with non-metastatic disease vs. patients with metastatic disease were observed. Central memory (CM) and effector memory (EM) CD8+ subsets, were found to be significantly higher in patients with non-metastatic disease vs. patients with metastatic disease. A similar profile was revealed when these CD8+ subsets were analyzed based on the patients’ Gleason scores, as well as by combined disease stage (i.e., non-metastatic vs. metastatic disease) and Gleason score. Conclusions: Peripheral blood-derived CD8+ T-lymphocyte memory subsets could function as biomarkers for the prognosis of PCa.

Organizing memories for generalization in complementary learning systems

Memorization and generalization are complementary cognitive processes that jointly promote adaptive behavior. For example, animals should memorize safe routes to specific water sources and generalize from these memories to discover environmental features that predict new ones. These functions depend on systems consolidation mechanisms that construct neocortical memory traces from hippocampal precursors, but why systems consolidation only applies to a subset of hippocampal memories is unclear. Here we introduce a new neural network formalization of systems consolidation that reveals an overlooked tension—unregulated neocortical memory transfer can cause overfitting and harm generalization in an unpredictable world. We resolve this tension by postulating that memories only consolidate when it aids generalization. This framework accounts for partial hippocampal–cortical memory transfer and provides a normative principle for reconceptualizing numerous observations in the field. Generalization-optimized systems consolidation thus provides new insight into how adaptive behavior benefits from complementary learning systems specialized for memorization and generalization.

IKK2/NFkB signaling controls lung resident CD8+ T cell memory during influenza infection

CD8+ T cell tissue resident memory (TRM) cells are especially suited to control pathogen spread at mucosal sites. However, their maintenance in lung is short-lived. TCR-dependent NFkB signaling is crucial for T cell memory but how and when NFkB signaling modulates tissue resident and circulating T cell memory during the immune response is unknown. Here, we find that enhancing NFkB signaling in T cells once memory to influenza is established, increases pro-survival Bcl-2 and CD122 levels thus boosting lung CD8+ TRM maintenance. By contrast, enhancing NFkB signals during the contraction phase of the response leads to a defect in CD8+ TRM differentiation without impairing recirculating memory subsets. Specifically, inducible activation of NFkB via constitutive active IKK2 or TNF interferes with TGFβ signaling, resulting in defects of lung CD8+ TRM imprinting molecules CD69, CD103, Runx3 and Eomes. Conversely, inhibiting NFkB signals not only recovers but improves the transcriptional signature and generation of lung CD8+ TRM. Thus, NFkB signaling is a critical regulator of tissue resident memory, whose levels can be tuned at specific times during infection to boost lung CD8+ TRM.

CLA+ memory T cells in atopic dermatitis

Circulating skin-homing cutaneous lymphocyte-associated antigen (CLA)+ T cells constitute a small subset of human memory T cells involved in several aspects of atopic dermatitis: Staphylococcus aureus related mechanisms, the abnormal Th2 immune response, biomarkers, clinical aspects of the patients, pruritus, and the mechanism of action of targeted therapies. Superantigens, IL-13, IL-31, pruritus, CCL17 and early effects on dupilumab-treated patients have in common that they are associated with the CLA+ T cell mechanisms in atopic dermatitis patients. The function of CLA+ T cells corresponds with the role of T cells belonging to the skin-associated lymphoid tissue and could be a reason why they reflect different mechanisms of atopic dermatitis and many other T cell mediated skin diseases. The goal of this review is to gather all this translational information of atopic dermatitis pathology.

T memory stem cell characteristics in autoimmune diseases and their promising therapeutic values.

Memory T cells are conventionally subdivided into T central memory (TCM) and T effector memory (TEM) cells. However, a new subset of memory T cells named T memory stem cell (TSCM) cells has been recognized that possesses capabilities of both TCM and TEM cells including lymphoid homing and performing effector roles through secretion of cytokines such as interleukin-2 (IL-2) and interferon-gamma (IFN-γ). The TSCM subset has some biological properties including stemness, antigen independency, high proliferative potential, signaling pathway and lipid metabolism. On the other hand, memory T cells are considered one of the principal culprits in the pathogenesis of autoimmune diseases. TSCM cells are responsible for developing long-term defensive immunity against different foreign antigens, alongside tumor-associated antigens, which mainly derive from self-antigens. Hence, antigen-specific TSCM cells can produce antitumor responses that are potentially able to trigger autoimmune activities. Therefore, we reviewed recent evidence on TSCM cell functions in autoimmune disorders including type 1 diabetes, systemic lupus erythematosus, rheumatoid arthritis, acquired aplastic anemia, immune thrombocytopenia, and autoimmune uveitis. We also introduced TSCM cell lineage as an innovative prognostic biomarker and a promising therapeutic target in autoimmune settings.

Dynamics of circulating lymphocytes responding to human experimental enterotoxigenic Escherichia coli infection.

Enterotoxigenic Escherichia coli (ETEC) is an important cause of children's and travelers' diarrhea, with no licensed vaccine. This study aimed to explore the role of cellular immunity in protection against human ETEC infection. Nine volunteers were experimentally infected with ETEC, of which six developed diarrhea. Lymphocytes were collected from peripheral blood buffy coats, before and 3, 5, 6, 7, 10, and 28 days after dose ingestion, and 34 phenotypic and functional markers were examined by mass cytometry. Thirty-three cell populations, derived by manually merging 139 cell clusters from the X-shift unsupervised clustering algorithm, were analyzed. Initially, the diarrhea group responded with increased CD56dim CD16+ natural killer cells, dendritic cells tended to rise, and mucosal-associated invariant T cells decreased. On day 5-7, an increase in plasmablasts was paralleled by a consistent rise in CD4+ Th17-like effector memory and regulatory cell subsets. CD4+ Th17-like central memory cells peaked on day 10. All Th17-like cell populations showed increased expression of activation, gut-homing, and proliferation markers. Interestingly, in the nondiarrhea group, these same CD4+ Th17-like cell populations expanded earlier, normalizing around day 7. Earlier development of these CD4+ Th17-like cell populations in the nondiarrhea group may suggest a recall response and a potential role in controlling ETEC infections.

Human peripheral blood age-associated (CD11c+Tbet+) B cells: No association with age.

Human peripheral blood age-associated (CD11c+Tbet+) B cells: No association with age.

Comprehensive immune landscape of lung-resident memory CD8+ T cells after influenza infection and reinfection in a mouse model.

Resident phenotypic memory CD8+ T cells are crucial for immune defense against pathogens. However, little is known about the potential transitions and regulation mechanisms of their function after influenza virus infection and reinfection. In this study, we utilized integrated transcriptome data and in vivo experiments to investigate the key characteristics behind it. Two single-cell RNA sequencing (scRNA-seq) datasets of lung CD8+ T cells and one RNA-seq dataset of lung tissue after infection or reinfection were included. After Seurat procedures classifying CD8+ T subsets, the scCODE algorithm was used to identify the differentially expressed genes for GSVA, GO, and KEGG pathway enrichment. Monocle 3 and CellChat were used to infer pseudotime cell trajectory and cell interactions. The ssGSEA method was used to estimate the relative proportions of immune cells. The findings were confirmed with a mouse model via flow cytometry and RT-PCR analysis. Our study refined the landscape of CD8+ T-cell subsets in the lung, showing that CD8+ Trm cells accumulated in the lung within 14 days after influenza infection. The classical CD8+ Trm cells co-expressed a high level of CD49a and even maintained 90 days after primary infection. The ratio of CD8+ Trm cells decreased 1 day after influenza reinfection, which may be parallel with their potential transition into effector types, as observed in trajectory inference analysis. KEGG analysis suggested that PD-L1 expression and PD-1 checkpoint pathway were upregulated in CD8+ Trm cells on day 14 after infection. GO and GSVA analyses revealed that PI3K-Akt-mTOR and type I interferon signaling pathways were enriched in CD8+ Tem and Trm cells after reinfection. Additionally, CCL signaling pathways were involved in cell interaction between CD8+ Trm cells and other cells, with Ccl4-Ccr5 and Ccl5-Ccr5 ligand/receptor pairs being important between CD8+ Trm and other memory subsets after infection and reinfection. Our data suggest that resident memory CD8+ T cells with CD49a co-expression account for a large proportion after influenza infection, and they can be rapidly reactivated against reinfection. Function differences exist in CD8+ Trm and Tem cells after influenza infection and reinfection. Ccl5-Ccr5 ligand/receptor pair is important in cell interactions between CD8+ Trm and other subsets.

The diverse landscape of memory T-cell heterogeneity.

The diverse landscape of memory T-cell heterogeneity.

Persistent Exhausted T-Cell Immunity after Severe COVID-19: 6-Month Evaluation in a Prospective Observational Study.

Severe COVID-19 can result in a significant and irreversible impact on long-term recovery and subsequent immune protection. Understanding the complex immune reactions may be useful for establishing clinically relevant monitoring. Hospitalized adults with SARS-CoV-2 between March/October 2020 (n = 64) were selected. Cryopreserved peripheral blood mononuclear cells (PBMCs) and plasma samples were obtained at hospitalization (baseline) and 6 months after recovery. Immunological components' phenotyping and SARS-CoV-2-specific T-cell response were studied in PBMCs by flow cytometry. Up to 25 plasma pro/anti-inflammatory cytokines/chemokines were assessed by LEGENDplex immunoassays. The SARS-CoV-2 group was compared to matched healthy donors. Biochemical altered parameters during infection were normalized at a follow-up time point in the SARS-CoV-2 group. Most of the cytokine/chemokine levels were increased at baseline in the SARS-CoV-2 group. This group showed increased Natural Killer cells (NK) activation and decreased CD16high NK subset, which normalized six months later. They also presented a higher intermediate and patrolling monocyte proportion at baseline. T cells showed an increased terminally differentiated (TemRA) and effector memory (EM) subsets distribution in the SARS-CoV-2 group at baseline and continued to increase six months later. Interestingly, T-cell activation (CD38) in this group decreased at the follow-up time point, contrary to exhaustion markers (TIM3/PD1). In addition, we observed the highest SARS-CoV-2-specific T-cell magnitude response in TemRA CD4 T-cell and EM CD8 T-cell subsets at the six-months time point. The immunological activation in the SARS-CoV-2 group during hospitalization is reversed at the follow-up time point. However, the marked exhaustion pattern remains over time. This dysregulation could constitute a risk factor for reinfection and the development of other pathologies. Additionally, high SARS-CoV-2-specific T-cells response levels appear to be associated with infection severity.

Distinct association between chronic Epstein-Barr virus infection and T cell compartments from pediatric heart, kidney, and liver transplant recipients

Chronic Epstein-Barr virus (EBV) infection after pediatric organ transplantation (Tx) accounts for significant morbidity and mortality. The risk of complications, such as posttransplant lymphoproliferative disorders, in high viral load (HVL) carriers is the highest in heart Tx recipients. However, the immunologic signatures of such a risk have been insufficiently defined. Here, we assessed the phenotypic, functional, and transcriptomic profiles of peripheral blood CD8+/CD4+ T cells, including EBV-specific T cells, in 77 pediatric heart, kidney, and liver Tx recipients and established the relationship between memory differentiation and progression toward exhaustion. Unlike kidney and liver HVL carriers, heart HVL carriers displayed distinct CD8+ T cells with (1) up-regulation of interleukin-21R, (2) decreased naive phenotype and altered memory differentiation, (3) accumulation of terminally exhausted (TEX PD-1+T-bet−Eomes+) and decrease of functional precursors of exhausted (TPEX PD-1intT-bet+) effector subsets, and (4) transcriptomic signatures supporting the phenotypic changes. In addition, CD4+ T cells from heart HVL carriers displayed similar changes in naive and memory subsets, elevated Th1 follicular helper cells, and plasma interleukin-21, suggesting an alternative inflammatory mechanism that governs T cell responses in heart Tx recipients. These results may explain the different incidences of EBV complications and may help improve the risk stratification and clinical management of different types of Tx recipients.

Therapeutic CAR T cells differentiate into diverse memory and exhausted T cell subsets in vivo to provide long-term protection from relapse and metastasis of solid tumors.

Abstract Canonical memory T cell differentiation is programmed during the early stages of priming by a multitude of variables such as TCR signal strength, duration of antigenic exposure, and the milieu of cytokines. A combination of such stochastic variables in vivo, drive a heterogeneity in T cell priming that leads to the generation of distinct subsets of T cell effector and memory lineages. Contrarily, CAR T cell priming is largely homogenous and the diversity of the ensuing memory CAR T cells following adoptive immunotherapy remains largely unknown. In this study, we show that following adoptive immunotherapy into solid tumor bearing mice, syngeneic CAR T cells differentiate into distinct memory T cell subsets. In the peripheral tissues and lymphoid organs, CAR T cells subsets of central memory (T CM), effector memory (T EM) and resident memory (T RM) were fully represented. Furthermore, within the tumor microenvironment, distinct lineages of exhausted T cells such as progenitor exhausted (T PEX) and terminally exhausted (T EX) CAR T cells were found. When benchmarked against gold-standard T cells programmed in vivo by viral infections, the subsets of CAR memory and exhausted T cells demonstrated the hallmark qualities of protection from metastasis, and responsiveness to checkpoint blockade, respectively. These findings are among the first to demonstrate that CAR T cells differentiate into a full-spectrum of memory and exhausted T cell subsets in therapeutic applications against solid tumors. Comprehensive understandings of CAR T cell memory and exhaustion programs would be critical in developing therapeutic CAR T cells that provide long-term protection from tumor relapse, tumor metastasis and responsiveness to checkpoint blockade. Research reported in this publication was supported by the National Cancer Institute of the National Institutes of Health under Award Number F32CA265056. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health

Small intestine and colon tissue-resident memory CD8 +T cells exhibit molecular heterogeneity and differential dependence on Eomesodermin

Abstract Tissue-resident memory CD8 +T (T RM) cells are a subset of memory T cells positioned at barrier sites, critical for host defense against infection. Recent studies have begun to reveal substantial heterogeneity in CD8 +T RMpopulations across different tissues. The gut consists of four anatomically distinct compartments, the epithelia and lamina propria in the small intestine (siIEL, siLPL) and colon (cIEL, cLPL). While CD8 +T RMin the small intestine epithelia are well-described, findings are often generalized to the whole gut, thus obscuring potential differences in CD8 +T RMin the much-less studied small intestine lamina propria and colon compartments. We thereby characterized CD8 +T RMin the four intestinal layers using single cell RNA-sequencing and flow cytometry. We show that CD8 +T RMacross the intestinal compartments exhibit differences in the expression of surface molecules, cytokines, granzymes, and transcription factors. For example, small intestine intraepithelial CD8 +T RMproduce the highest levels of granzymes, whereas colon intraepithelial CD8 +T RMhave the greatest capacity for cytokine production. We also uncover an unexpected, tissue-specific role for the T-box transcription factor Eomesodermin (Eomes), known to suppress T RMformation in the skin, liver, and kidney. Our data demonstrate that Eomes is dispensable for CD8 +T RMformation in both the small intestine and colon. By contrast, following CD8 +T RMformation, Eomes plays a critical role in promoting the maintenance of established CD8 +T RMin the small intestine, but not in the colon. Together, our study provides new insights into intestinal CD8 +T RMheterogeneity and differential transcriptional regulation of intestinal T RMformation vs. maintenance. Presenting author Y.H.L. is supported by the Canadian Institutes of Health Research (CIHR) Doctoral Foreign Study Award. This study is supported by grants from NIH (P01 AI132122).