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. Author manuscript; available in PMC: 2018 Jan 15.
Published in final edited form as: J Immunol. 2016 Nov 30;198(2):749–756. doi: 10.4049/jimmunol.1601500

TGF-β controls the formation of kidney-resident T cells via promoting effector T cell extravasation

Chaoyu Ma 1, Shruti Mishra 1,#, Erika L Demel 1,#, Yong Liu 2,1, Nu Zhang 1,*
PMCID: PMC5225110  NIHMSID: NIHMS829699  PMID: 27903738

Abstract

Tissue-resident memory T (TRM) cells, a population of non-circulating memory T cells, are one of the essential components of immunological memory in both mouse and human. While CD69+CD103+ TRM cells represent a major TRM cell population in barrier tissues including the mucosal surface and the skin, CD69+CD103− TRM cells dominate most non-barrier tissues, such as the kidney. Transforming grow factor-β (TGF-β) is required for the differentiation of CD69+CD103+ TRM cells in barrier tissues. However, the developmental control of CD69+CD103− TRM cells in non-barrier tissues remains largely unknown and the involvement of TGF-β signaling is less clear. Here, we demonstrated that TGF-β promoted the formation of kidney-resident T cells via enhancing the tissue entry of effector T cells. Mechanistically, TGF-β enhanced E/P-selectin and inflammatory chemokine-mediated extravasation of effector T cells. Thus, TGF-β controls the first developmental checkpoint of TRM cell differentiation in non-barrier tissues.

Introduction

TRM cells, a recently identified non-circulating memory T cell population, are one of the major components of adaptive immune surveillance(1-6). It has been estimated that the number of TRM cells exceeds the number of T cells in all lymphoid organs and entire blood volume combined in both immunized mouse and human(2, 7, 8). TRM cells are required for optimal protection against subsequent local reinfections(9-14). Absent from most circulating effector and memory T cells, CD69 and CD103 are commonly used surface markers for TRM cells. At least two populations of TRM cells have been identified. CD69+CD103+ TRM cells mainly reside in barrier tissues including the gastrointestinal tract, skin, lung and reproductive tract. CD69+CD103− TRM cells are found in both barrier tissues and non-barrier tissues. TGF-β signaling is required for CD103 induction and essential for the differentiation of CD69+CD103+ TRM cells in various tissues(15-21). However, TGF-β is not required for CD69 up-regulation and the differentiation of CD69+CD103− TRM cells in the gut and salivary gland(22, 23). Thus, the signals that control the development of CD69+CD103− TRM cells in non-barrier tissues remain to be determined.

During an immune response, circulating effector T cells migrate from the blood into peripheral tissues to fight local infections. The same population of effector T cells may further differentiate into TRM cells(3). Thus, the signals that regulate the extravasation of effector T cells control the first step of TRM cell differentiation. However, these signals are not entirely clear. The molecules that mediate the interaction between leukocytes and blood wall endothelia have been documented(24). CD44, integrins, selectin ligands and inflammatory chemokine receptors on activated T cells cooperate to mediate the engagement with endothelia. However, the involvement of these molecules in TRM cell development has not been well characterized.

In addition to its function as a local signal that induces CD103+ TRM cell differentiation, we have previously shown that TGF-β signaling inhibits the expression of integrin α4β7 and dampens the migration of effector CD8+ T cells to the gut(19). Integrin α4β7 is a gut-specific homing molecule due to the restricted expression pattern of its ligand MAdCAM-1 (Mucosal Vascular Addressin Cell Adhesion Molecule 1). Thus, the roles of TGF-β signaling in the migration of effector T cells into non-barrier tissues remain unexplored.

Here, using the kidney as an example of non-barrier and non-mucosal tissue, we examined the molecular mechanisms that control the formation of kidney-resident T cells during viral infection and the involvement of TGF-β signaling. Although TGF-β plays diverse functions during the differentiation of CD4+ T cells, it is generally considered as an anti-inflammatory and inhibitory cytokine for effector CD8+ T cells(25-27). Unexpectedly, we found that TGF-β was required for efficient trans-endothelial migration of effector CD8+ T cells into the kidney. Mechanistically, TGF-β induced E/P-selectin ligands via promoting the expression of O-glycan synthesis enzymes in effector CD8+ T cells. In addition, TGF-β enhanced the expression of inflammatory chemokine receptor CXCR3. TGF-β-dependent expression of selectin ligands and CXCR3 cooperated to facilitate the trans-endothelial migration of effector CD8+ T cells into the kidney. Therefore, TGF-β controls the first developmental step of kidney-resident T cells.

Materials and Methods

Mice and Viruses

Tgfbr2f/f dLck-cre mice were as described before(30). Cxcr3−/− (stock no. 005796) mice were purchased from The Jackson Laboratory. C57BL/6 (stock no. 000664) mice were obtained from The Jackson Laboratory and a colony of Db-GP33-41 TCR transgenic (P14) mice was maintained at our specific pathogen-free animal facilities at the University of Texas Health Science Center at San Antonio (San Antonio, Texas). All recipient mice were used at 6 to 12 wk of age. All experiments were done in accordance with the University of Texas Health Science Center at San Antonio Institutional Animal Care and Use Committee guidelines. Mice were infected intraperitoneally by 2×105 pfu LCMV Armstrong or intravenously by 2×106 pfu LCMV Clone 13. Viruses were grown and quantified as described (51).

Naïve T Cell Isolation and Adoptive Transfer

Naïve CD8+ T cells were isolated from pooled spleen and lymph nodes using MojoSort™ mouse CD8 T cell isolation kit (Biolegend) following manufacturer’s instruction. During the first step of biotin antibody cocktail incubation, biotin-αCD44 (IM7, Biolegend) was added to label and deplete effector and memory T cells. Isolated naïve CD8+ T cells were numerated, 1:1 mixed when indicated, 104 cells adoptively transferred into each sex-matched un-manipulated B6 recipient via an intravenous route.

Intra-vascular Labeling of CD8+ T Cells

3μg biotin-αCD8α (53-6.7, Tonbo Biosciences) was injected intravenously into each mouse 3-5 minutes before euthanasia. After lymphocyte isolation, fluorescence labeled streptavidin (Thermo Fisher) was used during surface FACS staining to identify blood-borne CD8+ T cells.

Lymphocyte Isolation from the Kidney

Kidney was minced and digested with 1mg/ml collagenase B (Roche) in RPMI 1640/3% FBS at 37C for 30 minutes with gentle shaking. Digested kidney was further mashed and washed with RPMI 1640/10% FBS. Lymphocytes were enriched by density gradient centrifugation (67% Percoll overlaid with 44% Percoll, Percoll medium was from GE Healthcare). Enriched lymphocytes were washed with PBS/5% FCS and subjected to FACS staining.

Retrovirus Transduction

Murine Gcnt1 cDNA was cloned into MSCV-IRES-Thy1.1 (pMit) vector. pMit was a gift from Dr. Anjana Rao (Addgene plasmid#17442). Helper plasmid pCL-Eco was a gift from Dr. Inder Verma (Addgene plasmid#12371). pMit and pCL-Eco were co-transfected into 293T cells by FuGENE 6 (Promega). Retrovirus was harvested 48 hours after transfection and used freshly. Similar to a published protocol(52), naïve P14 T cells were isolated and stimulated with 10nM GP33-41 peptide plus soluble 1μg/ml αCD28 (E18, Biolegend) in the presence of 5ng/ml IL-2 (eBioscience) overnight. Activated P14 T cells were spin infected with retrovirus at 3,000rpm 30C for 1.5 hours in the presence of 8μg/ml polybrene (Sigma) and 5ng/ml IL-2. After spin infection, P14 T cells were incubated with retrovirus for another hour at 37C. After extensive wash, P14 T cells were counted and 105 cells adoptively transferred into each B6 recipient followed by LCMV Arm infection. Leftover P14 T cells were cultured in the presence of 5ng/ml IL-2 and 2.5ng/ml hTGF-β1 (R&D system) for another 3-4 days before in vitro analysis.

Antibodies and Flow Cytometry

Single cell suspension from spleen and kidney was incubated with FcR blocker (clone 2.4G2, generated in the lab). Cells were typically stained with fluorescence labeled streptavidin (Thermo Fisher), CD8β (H35-17.2, eBioscience), CD162 (2PH1, BD), CD45.1 (A20, Tonbo), CD45.2 (104, Tonbo) and the following antibodies from BioLegend, CD127 (A7R34), KLRG1 (2F1/KLRG1), CD69 (H1.2F3), CD103 (2E7), CD90.1 (OX-7), CD43 (1B11), CXCR3 (CXCR3-173), mouse P-selectin/hIgG1 Fc chimera protein and mouse E-selectin/hIgG1 Fc chimera protein. For E/P-selectin binding experiments, fluorescence labeled anti-human IgG secondary antibody was purchased from Jackson ImmunoResearch. In some experiments, Fixable Viability Dye eFluor 506 (eBioscience) was used to identify live cells. Washed and fixed samples were analyzed by BD LSRII or BD FACSCelesta and analyzed by FlowJO (TreeStar) software.

Statistic Analysis

P value was calculated by two-tail paired or unpaired Student t-test using Prism 6 software.

Results

The formation of kidney-resident CD8+ T cells during viral infection

To examine the developmental control of kidney TRM cells, we employed a model system in which TRM cells, including kidney TRM cells, had been characterized extensively via parabiosis(7). Briefly, congenically marked P14 TCR transgenic CD8+ T cells, specific for the Db-GP33-41 LCMV (lymphocytic choriomeningitis virus) epitope, were adoptively transferred into un-manipulated C57BL/6 (B6) mice followed by acute LCMV Armstrong infection. To distinguish blood-borne versus kidney-resident CD8+ T cells, recipient mice were given 3μg fluorescence labeled anti-CD8α antibody via an intravenous route 3-5 minutes before euthanasia. Intact endothelia of blood vessels protected kidney-resident, but not vasculature-associated CD8+ T cells from intravenous antibody staining (28). As shown in Fig. 1A, in both CD8α staining positive (intravascular, i.v.) and CD8α staining negative (extravascular, e.v.) compartments, a donor derived P14 T cell population (distinguished by congenic marker CD45.1) was clearly identified after infection. At the early time points after infection, only extravascular P14 T cells received the signals to up-regulate the expression of TRM cell marker CD69. The restricted expression of CD69 further confirmed that intravascular labeling efficiently distinguished i.v versus e.v. CD8+ T cells in the kidney (Fig. 1A). Similar to the TRM cells in the skin, lung and small intestine(15-18), kidney-resident T cells were enriched for KLRG1− effector population (Fig. 1A). Interestingly, comparing with blood-borne cells (i.v.), kidney-resident T cells (e.v.) expressed substantially higher level of inflammatory chemokine receptor CXCR3 (Fig. 1A). Together, a phenotypically distinct population of virus-specific CD8+ T cells occupies the extravascular compartment of the kidney following viral infection.

Figure 1. The development of kidney-resident T cells.

Figure 1

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Naïve congenically marked P14 T cells were isolated from the spleen and lymph nodes. 104 P14 T cells were adoptively transferred into each B6 recipient followed by LCMV Arm infection. (A) D12 post infection, lymphocytes were isolated from the spleen and kidney. Representative FACS profiles are shown. (B) The ratios of extravascular (e.v.) over intravascular (i.v.) CD8+ T cells are shown. (C) The percentage of donor P14 T cells in the e.v. CD8+ T cell compartment is shown. (D) The percentage of CD69+ cells among e.v. P14 T cells is shown. Combined results from 4-5 independent experiments are shown for (B-D).

Further, in uninfected animals, the vast majority of CD8+ T cells resided in the vasculature of the kidney (Fig. 1B at day 0). Shortly after viral infection, e.v. compartment expanded (Fig. 1B), coinciding with the arrival of virus-specific P14 T cells (Fig. 1C). Comparing with the appearance of e.v. P14 T cells, the induction of CD69 on e.v. P14 T cells was slightly delayed (Comparing Fig. 1C and Fig. 1D). Interestingly, among e.v. P14 T cells, the percentage of CD69+ cells was stabilized shortly after infection and a population of CD69− cells persisted (Fig. 1A and 1D), consistent with a recent finding that CD69− CD8+ T cells could be tissue-resident(7). Together, during acute LCMV infection, the formation of kidney-resident CD8+ T cells is dynamically regulated.

TGF-β-dependent accumulation of CD8+ T cells in the extravascular compartment of the kidney

We and others have previously shown that TGF-β signaling to CD8+ T cells is essential for the differentiation and maintenance of CD103+ mucosal TRM cells (15-21, 29). The majority of non-mucosal TRM cells remain CD103− (Fig. 1A and (7)). Recent publications have revealed a TGF-β-independent developmental path for CD69+CD103− TRM cells in both the small intestine and salivary gland(22, 23). To further clarify the involvement of TGF-β in our system, we used previously described TGF-β unresponsive Tgfbr2−/− (Tgfbr2f/f dLck-cre) P14 TCR transgenic mice(30). Briefly, control and Tgfbr2−/− P14 T cells bearing different congenic markers were adoptively co-transferred into B6 recipients followed by LCMV Arm infection. Twelve days later (i.e. the peak of kidney-resident T cell response), compared with co-transferred control P14 T cells, we consistently observed that Tgfbr2−/− cells were significantly decreased in the e.v. compartment (i.e. kidney-resident), but not in the i.v. compartment (i.e. blood-borne) of the kidney (Fig. 2A). To quantitatively present this phenomenon, we defined Accumulation Index as the ratio of (Tgfbr2−/−/Control)e.v. over (Tgfbr2−/−/Control)i.v. (Fig. 2B). An Accumulation Index of 1 represents similar accumulation of Tgfbr2−/− cells as control cells in the e.v. compartment of the kidney. During the course of acute LCMV infection, the Accumulation Index was always significantly lower than one (Fig. 2B), suggesting that the formation of kidney-resident CD8+ T cells depended on TGF-β signaling. Moreover, this phenomenon was not kidney-specific because similar defects were observed in the brain in the absence of TGF-β signaling (Fig. S1). Thus, TGF-β promotes the formation of non-barrier tissue TRM cells in the extravascular compartment.

Figure 2. TGF-β-dependent accumulation of extravascular CD8+ T cells in the kidney.

Figure 2

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Naïve congenically marked P14 T cells were isolated from control and Tgfbr2−/− mice and 1:1 mixed. 104 mixed P14 T cells were adoptively co-transferred into each B6 recipient followed by LCMV Arm infection. (A) D12 post infection, representative FACS profiles are shown. (B) The A.I. (Accumulation Indexes) of control and Tgfbr2−/− P14 T cells are shown. To calculate A.I. of control P14 T cells, two populations of control P14 T cells bearing distinct congenic markers were 1:1 mixed and co-transferred into B6 recipients followed by LCMV infection. Each symbol in (B) represents the results from an individual recipient mouse. Combined results from 4-5 independent experiments are shown for (B). NS, not significant, **, p<0.01 Student t-test.

Defective trans-endothelial migration of CD8+ T cells in the absence of TGF-β signaling

Previously, we have demonstrated that TGF-β inhibits the migration of CD8+ T cells to the gut and promotes the retention of gut-resident T cell. Thus, in the absence of TGF-β signaling, the accumulation of gut-resident T cells is severely defective after acute infection, but not during chronic infection due to enhanced continuous recruitment of circulating effector T cells(19). In stark contrast to the situation at the intestinal mucosal surface, the accumulation of kidney-resident Tgfbr2−/− T cells was further decreased during chronic infection comparing with that after acute infection (Fig. 2B). Therefore, unique and previously unrecognized functions of TGF-β signaling regulate the accumulation of kidney-resident T cells.

Decreased accumulation of extravascular kidney T cells may be due to reduced trans-endothelial migration and/or defective homeostasis of kidney-resident T cells in the absence of TGF-β signaling. To directly test the involvement of TGF-β in the extravasation of effector T cells in the kidney, we performed a short-term migration assay. As illustrated in Fig. 3A, congenically marked control and Tgfbr2−/− P14 T cells were adoptively co-transferred into B6 recipients followed by LCMV Arm infection. Seven days later, splenic effector P14 T cells were isolated and transferred intravenously into infection matched B6 recipients. The distribution of donor P14 T cells was determined 16 hours later. Due to the nature of this short-term assay, the impacts of the homeostasis of kidney-resident T cells were minimal. Significantly defective accumulation of Tgfbr2−/− T cells in the e.v. compartment of the kidney still occurred (Fig. 3B and 3C). Together, these results demonstrate that TGF-β directly controls the trans-endothelial migration of effector T cells in the kidney.

Figure 3. Defective trans-endothelial migration of effector CD8+ T cells in the absence of TGF-β signaling.

Figure 3

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(A) Experimental design. Mixed P14 T cells from control and Tgfbr2−/− mice were adoptively co-transferred into B6 recipients followed by LCMV Arm infection. D7 post infection, splenic P14 T cells were isolated and transferred into infection-matched B6 mice. 16 hours later, the distribution of donor P14 T cells were determined. (B) Representative FACS profiles of the kidney are shown. (C) The ratios of control versus Tgfbr2−/− P14 T cells in the e.v. and i.v. compartments of the kidney are shown. Each pair of symbols represents results from an individual recipient. Representative data from two independent experiments are shown. **, p<0.01 by paired Student t-test.

TGF-β is required for the optimal expression of selectin ligands on effector T cells

Lymphocyte extravasation mainly depends on CD44, selectins, integrins and chemokine receptors(24). The expression of CD44 on T cells is not impaired in the absence TGF-β signaling(30). Further, the expression of the integrins that are commonly expressed on circulating effector T cells (including Itga1, Itga4, Itgb1, Itgb3 and Itgb7) is not decreased on Tgfbr2−/− T cells((19, 31) and data not shown). Thus, to elucidate the underlying mechanisms of TGF-β-controlled effector T cell extravasation in the kidney, we focused on selectins/selectin ligands and chemokine receptors.

Because the expression of L-selectin quickly declines upon naïve T cell activation, to examine effector T cell migration, we chose to examine E- and P-selectins. E- and P- selectins are expressed on endothelial cells under steady state conditions and further induced during inflammation(32-35). E/P-selectin ligands are highly O-glycosylated cell surface proteins expressed on activated T cells. Anti-CD43 antibody clone 1B11 has been demonstrated to recognize an O-glycosylated isoform of CD43 and used as a surrogate marker for E/P-selectin ligands(36-38). The staining of 1B11 was significantly decreased on splenic Tgfbr2−/− P14 T cells after viral infection (Fig. 4A and 4B). Further, defective P-selectin and E-selectin binding on Tgfbr2−/− T cells supported 1B11 as a valid marker for E/P-selectin ligands and directly approved that TGF-β was required for optimal expression of E/P-selectin ligands on effector T cells in vivo (Fig. 4A and 4B). Consistent with the enrichment of KLRG1− effector T cells in the extravascular compartment of the kidney (Fig. 1A), comparing with KLRG1+ counterparts, KLRG1− cells expressed higher levels of E/P-selectin ligands (Fig. 4A). Interestingly, 1B11 staining was elevated on the kidney-resident cells (identified as CD69+ cells) and Tgfbr2−/− cells that did migrate across blood vessel walls (identified as CD69+ cells) exhibited a similar level of 1B11 staining as control cells, suggesting a selection process that only 1B11hi cells efficiently crossed blood vessel walls (Fig. 4C and 4D). Together, TGF-β promotes the expression of E/P-selectin ligands on effector CD8+ T cells during viral infection in vivo.

Figure 4. Defective expression of E/P-selectin ligands on Tgfbr2−/− effector CD8+ T cells.

Figure 4

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Similar experimental setup as in Figure 2. D10 post infection, representative FACS profiles of splenic P14 T cells are shown in (A). (B) Mean Fluorescent Intensity (MFI) of 1B11 staining and P-selectin binding on KLRG1− P14 T cell is shown. (C) Representative FACS profiles of kidney P14 T cells are shown. (D) MFI of 1B11 staining on kidney P14 T cells is shown. Each pair of symbols in (B) and (D) represents data from an individual recipient mouse. Representative data from 2-4 independent experiments are shown. **, p<0.01 by paired Student t-test. NS, not significant.

TGF-β enhances O-glycosylation in activated CD8+ T cells

To exclude the possibility that TGF-β-dependent E/P-selectin ligand expression is LCMV-specific or P14 T cell-specific, polyclonal naïve CD8+ T cells from control and Tgfbr2−/− mice were activated in vitro. Consistent with our previous findings(30), the activation of CD8+ T cells was TGF-β-independent as demonstrated by increased cell size and up-regulation of CD69 to a comparable level in the presence and absence of TGF-β (Fig. 5A). Dramatically decreased 1B11 staining and E/P-selectin binding was observed on activated T cells in the absence of TGF-β (Fig. 5A). This defect was independent of growth cytokines added in the culture as similar trend was observed when IL-2, IL-7, IL-15 or IL-21 was used (Fig. S2A). Interesting, when added two days after T cell activation, TGF-β still promoted the expression of selectin ligands (Fig. S2B). Anti-CD162 recognizes the protein core of P-selectin glycoprotein ligand-1 (PSGL-1). Consistent with previous reports(39), minimal induction of PSGL-1 was observed on activated T cells and the expression of PSGL-1 was largely TGF-β-independent (Fig. 5A). Thus, TGF-β specifically promotes O-glycosylation in activated CD8+ T cells.

Figure 5. TGF-β signaling is required for the induction of O-glycosylation in activated CD8+ T cells.

Figure 5

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Naïve CD8+ T cells were isolated from control and Tgfbr2−/− mice and cultured in the presence of 1μg/ml coated αCD3, 1μg/ml soluble αCD28, 5ng/ml IL-2 and 2.5ng/ml hTGF-β1 for two to five days. (A) Representative FACS profiles of live CD8+ T cells (d5 for E-selectin binding and d2 for others) are shown. (B) Total RNA was isolated from live CD8+ T cells and subjected to qPCR analysis. Representative results from three independent experiments are shown. (C) Day 10 post LCMV infection, splenic KLRG1+ and KLRG1− P14 T cells (control and Tgfbr2−/−) were FACS sorted and subjected to qPCR analysis.

For in vitro activated CD4+ T cells, it has been demonstrated that TGF-β promotes the expression of O-glycan synthesis enzymes Fut7 (Fucosyltransferase 7) and Gcnt1 (Glucosaminyl (N-Acetyl) Transferase 1, core 2)(40-42). Gcnt1 is required for selectin ligand expression on various types of immune cells(43), including IL-15-dependent selectin ligand induction on memory CD8+ T cells(39). Indeed, the expression of both Fut7 and Gcnt1 was significantly reduced in activated CD8+ T cells in the absence of TGF-β signaling (Fig. 5B). Strikingly, the expression of Gcnt1 was almost completely abolished at later time point in the absence of TGF-β. Further, freshly isolated splenic effector CD8+ T cells exhibited decreased expression of Gcnt1 in the absence of TGF-β signaling (Fig. 5C). Together, TGF-β is required for optimal expression of O-glycan synthesis enzymes Fut7 and Gcnt1 in activated CD8+ T cells. Especially, TGF-β is essential to maintain the expression of Gcnt1 in effector CD8+ T cells.

Forced expression of Gcnt1 rescues the defective migration of Tgfbr2−/− CD8+ T cells

To further test the functional relevance of TGF-β-dependent O-glycosylation in effector CD8+ T cells, we generated a retrovirus carrying a murine cDNA of Gcnt1 using MSCV (Mouse Stem Cell Virus)-IRES-Thy1.1 vector. Briefly, activated P14 T cells from control and Tgfbr2−/− mice were spin infected with empty control or Gcnt1 carrying retrovirus. After retroviral transduction, P14 T cells were either left in vitro or adoptively transferred into B6 recipients followed by LCMV Arm infection. Indeed, overexpression of Gcnt1 in Tgfbr2−/− cells corrected the expression of CD43 and E/P-selectin ligands both in vitro and in vivo (Fig. 6A). Further, comparing with retrovirus-negative (Thy1.1−) and empty control virus transduced Tgfbr2−/− P14 T cells, overexpression of Gcnt1 significantly enhanced the Accumulation Index of Tgfbr2−/− P14 T cells in the kidney (Fig. 6B). Interestingly, for most mice bearing Gcnt1-correcting Tgfbr2−/− P14 T cells, the Accumulation Index was higher than one (Fig. 6B) probably because overexpression of Gcnt1 led to higher E/P-selectin ligand expression in Tgfbr2−/− cells than that of control cells (Fig. 6A). Thus, TGF-β-controlled Gcnt1 expression is one of the critical mechanisms underlying defective trans-endothelial migration of Tgfbr2−/− CD8+ effector T cells in the kidney.

Figure 6. Forced expression of Gcnt1 corrects the defects in E/P-selectin ligand expression and trans-endothelial migration for Tgfbr2−/− T cells.

Figure 6

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Activated P14 T cells from control and Tgfbr2−/− mice were spin infected by control retrovirus or Gcnt1 carrying retrovirus. Control P14s with control retrovirus were 1:1 mixed with either Tgfbr2−/− P14s with control retrovirus or Tgfbr2−/− P14s with Gcnt1 retrovirus. 105 mixed P14 T cells were adoptively co-transferred into each B6 recipient followed by LCMV Arm infection. Leftover P14 T cells were cultured in the presence of 5ng/ml IL-2 and 2.5ng/ml hTGF-β1 for another 3-4 days. (A) Representative FACS profiles of either live in vitro cultured P14 T cells (left) or d10 splenic P14 T cells post LCMV infection (right) are shown. (B) D10-14 post infection, kidney lymphocytes were analyzed by flow cytometry. Accumulation Indexes of Thy1.1− Tgfbr2−/− P14s (without retrovirus), control retrovirus infected Tgfbr2−/− P14s and Gcnt1 expressing Tgfbr2−/− P14s are shown. Each symbol represents data from an individual recipient. Combined results from three independent experiments are shown in (B). **, p<0.01 by unpaired Student t-test.

TGF-β-controlled CXCR3 expression promotes effector T cell trans-endothelial migration in the kidney

To fully elucidate the mechanisms underlying TGF-β-dependent effector T cell extravasation in the kidney, we also examined inflammatory chemokine receptors. We have previously shown that continuous TGF-β signaling is required to maintain the expression of chemokine receptor CXCR3 on circulating memory CD8+ T cells (44) and the expression of CXCR3 was differentially regulated in the intravascular and extravascular compartments of the kidney (Fig. 1A). Thus, we compared the expression of CXCR3 on effector CD8+ T cells in the presence and absence of TGF-β signaling. TGF-β promoted the expression of CXCR3 on effector CD8+ T cells isolated from both the spleen and kidney (Fig. 7A and 7B). To explore the functional relevance of CXCR3 expression in effector T cell extravasation, we generated congenically marked Cxcr3−/− P14 TCR transgenic mice. Similar to the experiments using Tgfbr2−/− P14 T cells, control and Cxcr3−/− P14 T cells were adoptively co-transferred into B6 recipients followed by LCMV Arm infection. Similar as Tgfbr2−/− cells, comparing with the intravascular compartment, the accumulation of Cxcr3−/− T cells was significantly reduced in the extravascular compartment of the kidney (Fig. 7D and 7E) while the splenic Cxcr3−/− T cells were not decreased in the same animals (Fig. 7C), consistent with previous findings that CXCR3 is required for effector CD8+ T cells to migrate to the lung, genital tract, epidermis, liver and brain(45-49). Further, in a short-term T cell migration assay, CXCR3 is required for the efficient extravasation of effector P14 T cells (Fig. S3). Thus, CXCR3 is required for the accumulation of kidney-resident T cells during viral infection. Taken together, TGF-β promotes the trans-endothelial migration of effector CD8+ T cells via enhancing the expression of both E/P-selectin ligands and chemokine receptor CXCR3.

Figure 7. TGF-β-dependent CXCR3 expression promotes trans-endothelial migration of effector CD8+ T cells.

Figure 7

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Similar experimental setup as Figure 2. D10 post infection, representative FACS profiles of splenic P14 T cells are shown in (A). (B) FACS plot (left) and MFI of CXCR3 (right) on P14 T cells isolated from the i.v. compartment of the kidney is shown. Each pair of symbols in (B) represents data from an individual recipient. (C), (D) and (E), congenically marked P14 T cells were isolated from control and Cxcr3−/− mice, 1:1 mixed and adoptively co-transferred into each B6 recipient followed by LCMV infection. The ratio of Cxcr3−/− over control P14 T cells in the spleen is shown in (C). D10 post infection, representative FACS profiles are shown in (D). Accumulation Indexes of Cxcr3−/− P14s are calculated as in Figure 2 and shown in (E). Each symbol in (E) represents an individual recipient mouse. **, p<0.01 by one sample Student t-test.

Discussion

During acute infections, effector T cells migrate across blood wall endothelia to enter peripheral tissues to fight against local infections. The same population of effector T cells will further differentiate into TRM cells after the clearance of the infection. Thus, the extravasation of effector T cells may be considered as the first step of TRM cell differentiation. Recent evidences have demonstrated that TGF-β is one of the essential local signals that induce the differentiation of CD69+CD103+ TRM cells(15-21), but not required for the formation of CD69+CD103− TRM cells(22, 23). Here, we show that TGF-β controls the first step of CD69+CD103− TRM cell differentiation by promoting the trans-endothelial migration of effector CD8+ T cells in the kidney. TGF-β promotes the expression of E/P-selectin ligands and chemokine receptor CXCR3, both of which are required for efficient extravasation of effector CD8+ T cells. Our results further suggest that in addition to the roles of local TGF-β in CD103+ TRM cell differentiation, systemic TGF-β signaling delivered in the secondary lymphoid organs during T cell priming controls the early differentiation steps of tissue-resident T cells.

TGF-β is a well-established anti-inflammatory cytokine that inhibits the effector function of CD8+ T cells(25-27). In contrast, our findings demonstrate that TGF-β promotes effector CD8+ T cell infiltration into peripheral non-lymphoid tissues via enhancing O-glycosylation and the expression of CXCR3. Glycosylation is a highly regulated process in effector/memory CD8+ T cells. IL-15 has been demonstrated to be essential for the induction of glycosylation in memory CD8+ T cells and trafficking of memory T cells into inflamed tissues(39), consistent with a recent proposed role for IL-15 as a “danger signal” to activate memory and tissue-resident CD8+ T cells(50). Interestingly, our results suggest that TGF-β plays a similar role in the similar pathway as IL-15 in effector CD8+ T cells. The comparison of TGF-β and IL-15 in this process warrants further investigation.

A recent elegant publication has documented that enzymatic digestion leads to significant cell loss during the preparation of kidney lymphocytes for flow cytometry analysis(7). In most of our experiments, we employed adoptive co-transfer system that control and genetic modified P14 T cells were compared side-by-side in the same tissue of the same animal. Whenever it was possible, the ratio of control versus genetic modified P14 T cells was used as the readout. Therefore, the impacts of tissue digestion-introduced cell loss are minimal in our system.

Even though a significant portion of blood and marginated pool associated T cells are tissue-resident(7), in most peripheral tissues, stromal and parenchyma associated T cells represent the major tissue-resident T cell population. Thus, the migration of T cells from blood vessels into the stromal and parenchyma tissues represents the initial developmental step for most TRM cells. Our results have emphasized the importance of tissue-entry as the first checkpoint during tissue-resident T cell differentiation.

Together, we have demonstrated that TGF-β promotes O-glycan synthesis and the expression of inflammatory chemokine receptor CXCR3. TGF-β enhances the migration of effector CD8+ T cells into non-barrier tissues. Considering our previous findings that TGF-β inhibits the migration of effector CD8+ T cells to the small intestine(19), TGF-β modifies the migration pattern of effector CD8+ T cells in a tissue-specific manner, which may provide an opportunity to manipulate tissue-specific immunity in the future.

Supplementary Material

1

Acknowledgments

We thank Dr. Benjamin Daniel and Karla Gorena for technical assistance in flow cytometry. We thank Jade Juarez, Richard Rodriguez and Jennifer Castro for general assistance. C.M. and N.Z. designed and performed the experiments, analyzed data and wrote the manuscript. S.M., E.D. and Y.L. performed the experiments and analyzed data.

N.Z. is supported by National Institute of Health Grant R01-AI125701, Young Investigator Award from Max and Minnie Tomerlin Voelcker Fund, University of Texas Rising STARS award and a pilot grant from School of Medicine at UT Health San Antonio.

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