isolation-mouse-kidney-resident-cd8-t-cells-for-flow-cytometry

Isolation of Mouse Kidney-Resident CD8<sup>+</sup> T cells for Flow Cytometry Analysis

Tissue-resident memory T cell (TRM) is a rapidly expanding field of immunology research. Isolating T cells from various non-lymphoid tissues is one of the ...

University of Texas Health Science Center at San Antonio

The inhibitory receptor killer cell lectin-like receptor G1 (KLRG1) and the integrin αE (CD103) are expressed by CD8(+) T cells and both are specific for E-cadherin. However, KLRG1 ligation by E-cadherin inhibits effector T-cell function, whereas binding of CD103 to E-cadherin enhances cell-cell interaction and promotes target cell lysis. Here, we demonstrate that KLRG1 and CD103 expression in CD8(+) T cells from untreated and virus-infected mice are mutually exclusive. Inverse correlation of KLRG1 and CD103 expression was also found in human CD8(+) T cells-infiltrating hepatocellular carcinomas. As TGF-β is known to induce CD103 expression in CD8(+) T cells, we examined whether this cytokine also regulates KLRG1 expression. Indeed, our data further reveal that TGF-β signaling in mouse as well as in human CD8(+) T cells downregulates KLRG1 expression. This finding provides a rationale for the reciprocal expression of KLRG1 and CD103 in different CD8(+) T-cell subsets. In addition, it points to the limitation of KLRG1 as a marker for terminally differentiated CD8(+) T cells if lymphocytes from tissues expressing high levels of TGF-β are analyzed. 

TGF-β downregulates KLRG1 expression in mouse and human CD8(+) T cells.

The long-term maintenance of memory T cells is essential for successful vaccines. Both the quantity and the quality of the memory T-cell population must be maintained. The signals that control the maintenance of memory T cells remain incompletely identified. Here we used two genetic models to show that continuous transforming growth factor-β signaling to antigen-specific T cells is required for the differentiation and maintenance of memory CD8(+) T cells. In addition, both infection-induced and microbiota-induced inflammation impact the phenotypic and functional identity of memory CD8(+) T cells. 

Transforming growth factor-β signaling is constantly shaping memory T-cell population.

Chlamydia muridarum is known to colonize in the gastrointestinal tract for long periods of time, which has been hypothesized to serve as a reservoir for spreading to the genital tract. To test this hypothesis, a luciferase-expressing C. muridarum was used to establish a long-lasting infection in the mouse gastrointestinal tract following either intragastric or intrarectal inoculations. In vivo imaging revealed significant bioluminescent signals mainly in the mouse abdominal area throughout the experiments. Ex vivo imaging localized the signals to the mouse gastrointestinal tract, which was confirmed by monitoring the C. muridarum organisms in the mouse organs/tissues. Despite the long-lasting colonization in the gastrointestinal tract and active shedding of infectious organisms in the rectal swabs, the organisms did not cause any significant infection or pathology in the genital tract throughout the experiments, which was reproduced in multiple strains of mice and with an increased inoculation dose to the gastrointestinal tract. The above observations have demonstrated that the long-lasting C. muridarum organisms from the gastrointestinal tract are inefficient in auto-inoculating the genital tract, suggesting that the gastrointestinal tract Chlamydia may utilize an indirect mechanism to affect its pathogenicity in the genital tract.

The Chlamydia muridarum Organisms Fail to Auto-Inoculate the Mouse Genital Tract after Colonization in the Gastrointestinal Tract for 70 days.

Chlamydia has been detected in the gastrointestinal tracts of both animals and humans. However, the mechanism by which Chlamydia colonizes the gut remains unclear. Chlamydia muridarum is known to spread from the genital to the gastrointestinal tracts hematogenously. The C. muridarum plasmid is a key pathogenic determinant in the mouse upper genital tract although plasmid-deficient C. muridarum is still able to colonize the upper genital tract. We now report that plasmid-deficient C. muridarum exhibits significantly delayed/reduced spreading from the mouse genital to the gastrointestinal tracts. C. muridarum with or without plasmid maintained similar levels in the mouse circulatory system following intravenous inoculation but the hematogenous plasmid-deficient C. muridarum was significantly less efficient in colonizing the gastrointestinal tract. Consistently, plasmid-deficient C. muridarum failed to restore normal colonization in the gastrointestinal tract even after intragastric inoculation at a high dose. Thus, we have demonstrated a plasmid-dependent colonization of C. muridarum in the gastrointestinal tract, supporting the concept that C. muridarum may have acquired the plasmid for adaptation to the mouse gastrointestinal tract during oral-fecal transmission. Since the plasmid is more important for C. muridarum to colonize the gastrointestinal tract than to infect the genital tract, the current study has laid a foundation for further defining the host pathways targeted by the plasmid-encoded or -regulated chlamydial effectors.

The cryptic plasmid is more important for Chlamydia muridarum to colonize the mouse gastrointestinal tract than to infect the genital tract.

Systemic lupus erythematosus (SLE) is an autoimmune disease that is characterized by lymphocytic infiltration and overproduction of autoantibodies, leading to significant morbidity and mortality. However, the pathogenesis of this disorder has not yet been completely elucidated. It has been reported that CD70, a B cell costimulatory molecule encoded by the gene  (tumor necrosis factor ligand superfamily member 7), is overexpressed in CD4 T cells from patients with SLE due to the demethylation of its promoter. We aimed to investigate the expression patterns of MBD4 (methyl-CpG binding domain protein 4) in CD4 T cells and its contribution to the pathogenesis of SLE by increasing CD70 expression through epigenetic regulation.

Down-regulation of MBD4 contributes to hypomethylation and overexpression of CD70 in CD4 T cells in systemic lupus erythematosus.

 has been detected in the gastrointestinal tracts of humans and animals. We now report that gastrointestinal  is able to induce robust transmucosal protection in mice.  colonization in the gastrointestinal tract correlated with both a shortened course of  genital tract infection and stronger protection against subsequent genital tract challenge infection. Mice preinoculated intragastrically with  became highly resistant to subsequent  infection in the genital tract, resulting in prevention of pathology in the upper genital tract. The transmucosal protection in the genital tract was rapidly induced, durable, and dependent on major histocompatibility complex (MHC) class II antigen presentation but not MHC class I antigen presentation. Although a deficiency in CD4 T cells only partially reduced the transmucosal protection, depletion of CD4 T cells from B cell-deficient mice completely abolished the protection, suggesting a synergistic role of both CD4 T and B cells in the gastrointestinal -induced transmucosal immunity. However, the same protective immunity did not significantly affect  colonization in the gastrointestinal tract. The long-lasting colonization with  was restricted to the gastrointestinal tract and was nonpathogenic to either gastrointestinal or extragastrointestinal tissues. Furthermore, gastrointestinal  did not alter the gut microbiota or the development of gut mucosal resident memory T cell responses to a nonchlamydial infection. Thus,  may be developed into a safe and orally deliverable replicating vaccine for inducing transmucosal protection.

Nonpathogenic Colonization with Chlamydia in the Gastrointestinal Tract as Oral Vaccination for Inducing Transmucosal Protection.

Tissue-resident memory T (TRM) cells have emerged as a major component of T cell biology. Recent investigations have greatly advanced our understanding of TRMs. Common features have been discovered to distinguish memory T cells residing in various mucosal and non-mucosal tissues from their circulating counterparts. Given that most organs and tissues contain a unique microenvironment, local signal-induced tissue-specific features are tightly associated with the differentiation, homeostasis, and protective functions of TRMs. Here, we discuss recent advances in the TRM field with a special emphasis on the interaction between local signals and TRMs in the context of individual tissue environment.

Tissue-Specific Control of Tissue-Resident Memory T Cells.

Revealing the mechanisms by which bacteria establish long-lasting colonization in the gastrointestinal tract is an area of intensive investigation. The obligate intracellular bacterium  is known to colonize mouse colon for long periods. A colonization-deficient mutant strain of this intracellular bacterium is able to regain long-lasting colonization in gamma interferon (IFN-γ) knockout mice following intracolon inoculation. We now report that mice deficient in conventional T lymphocytes or recombination-activating gene (Rag) failed to show rescue of mutant colonization. Nevertheless, antibody depletion of IFN-γ or genetic deletion of interleukin 2 (IL-2) receptor common gamma chain in Rag-deficient mice did rescue mutant colonization. These observations suggest that colonic IFN-γ, responsible for inhibiting the intracellular bacterial mutant, is produced by innate lymphoid cells (ILCs). Consistently, depletion of NK1.1 cells in Rag-deficient mice both prevented IFN-γ production and rescued mutant colonization. Furthermore, mice deficient in transcriptional factor RORγt, but not chemokine receptor CCR6, showed full rescue of the long-lasting colonization of the mutant, indicating a role for group 3-like ILCs. However, the inhibitory function of the responsible group 3-like ILCs was not dependent on the natural killer cell receptor (NCR1), since NCR1-deficient mice still inhibited mutant colonization. Consistently, mice deficient in the transcriptional factor T-bet only delayed the clearance of the bacterial mutant without fully rescuing the long-lasting colonization of the mutant. Thus, we have demonstrated that the obligate intracellular bacterium  maintains its long-lasting colonization in the colon by evading IFN-γ from group 3-like ILCs.

Evasion of Innate Lymphoid Cell-Regulated Gamma Interferon Responses by Chlamydia muridarum To Achieve Long-Lasting Colonization in Mouse Colon.

Sexually transmitted , which can cause fibrotic pathology in women's genital tracts, is also frequently detected in the gastrointestinal tract. However, the medical significance of the gastrointestinal  remains unclear. A murine  readily spreads from the mouse genital tract to the gastrointestinal tract while inducing oviduct fibrotic blockage or hydrosalpinx. We previously proposed a two-hit model in which the mouse gastrointestinal  might induce the second hit to promote genital tract pathology, and we are now providing experimental evidence for testing the hypothesis. First, chlamydial mutants that are attenuated in inducing hydrosalpinx in the genital tract also reduce their colonization in the gastrointestinal tract, leading to a better correlation of chlamydial induction of hydrosalpinx with chlamydial colonization in the gastrointestinal tract than in the genital tract. Second, intragastric coinoculation with a wild-type  rescued an attenuated  mutant to induce hydrosalpinx, while the chlamydial mutant infection in the genital tract alone was unable to induce any significant hydrosalpinx. Finally, the coinoculated gastrointestinal  failed to directly spread to the genital tract lumen, suggesting that gastrointestinal  may promote genital pathology via an indirect mechanism. Thus, we have demonstrated a significant role of gastrointestinal  in promoting pathology in the genital tract possibly via an indirect mechanism. This study provides a novel direction/dimension for further investigating chlamydial pathogenic mechanisms.

Article	Year
TGF-β downregulates KLRG1 expression in mouse and human CD8(+) T cells. European journal of immunology\| PubMed ID: 26014037	2015
Transforming growth factor-β signaling is constantly shaping memory T-cell population. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 26283373	2015
The Chlamydia muridarum Organisms Fail to Auto-Inoculate the Mouse Genital Tract after Colonization in the Gastrointestinal Tract for 70 days. PloS one\| PubMed ID: 27192556	2016
The cryptic plasmid is more important for Chlamydia muridarum to colonize the mouse gastrointestinal tract than to infect the genital tract. PloS one\| PubMed ID: 28542376	2017
Down-regulation of MBD4 contributes to hypomethylation and overexpression of CD70 in CD4 T cells in systemic lupus erythematosus. Clinical epigenetics\| PubMed ID: 29018507	2017
Nonpathogenic Colonization with Chlamydia in the Gastrointestinal Tract as Oral Vaccination for Inducing Transmucosal Protection. Infection and immunity\| PubMed ID: 29133348	2018
Tissue-Specific Control of Tissue-Resident Memory T Cells. Critical reviews in immunology\| PubMed ID: 29953389	2018
Evasion of Innate Lymphoid Cell-Regulated Gamma Interferon Responses by Chlamydia muridarum To Achieve Long-Lasting Colonization in Mouse Colon. Infection and immunity\| PubMed ID: 31818961	2020
Gastrointestinal Coinfection Promotes Chlamydial Pathogenicity in the Genital Tract. Infection and immunity\| PubMed ID: 31988173	2020

Nu Zhang

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