Generation of effector cells that localize to mucosal tissues and form resident memory CD8 T cells is controlled by mTOR

Abstract

Mucosal tissues are subject to frequent pathogen exposure and major sites for transmission of infectious disease. CD8 T cells play a critical role in controlling mucosa-acquired infections though their migration into mucosal tissues is tightly regulated. The mechanisms and signals that control the formation of tissue-resident memory CD8 T cells are poorly understood however, one key regulator of memory CD8 T cell differentiation, mTOR kinase, can be inhibited by rapamycin. We report that despite enhancing the formation of memory CD8 T cells in secondary lymphoid tissues, rapamycin inhibits the formation of resident memory CD8 T cells in the intestinal and vaginal mucosa. The ability of rapamycin to block formation of functional resident CD8 T cells in mucosal tissues protected mice from a model of CD8 T cell mediated lethal intestinal autoimmunity. These findings demonstrate an opposing role for mTOR in the formation of resident versus non-resident CD8 T cell immunity.

Introduction

Protective CD8 T cell immunity in part requires positioning memory T cells in locations of recurrent pathogen exposure. The mucosal tissues are key transmission sites for many microbes. Vaccines that favor the generation of effector memory CD8 T cells, which reside primarily in non-lymphoid compartments, have been shown to protect macaques against SIV (1), presumably by maintaining a supply of resident memory CD8 T cells at the mucosal sites. Resident mucosal memory CD8 T cells are phenotypically and functionally distinct from their blood and secondary lymphoid counterparts (2, 3) in which a large population of these cells are CD103⁺ and constitutively express CD69. CD8 T cell migration and retention in the intestinal mucosa is critically dependent on the expression of gut-specific homing molecules (i.e. CCR9(4), α4β7(5), CD103(6)). Furthermore, migration to the intestine is limited to early effectors, and resident memory CD8 T cells in the small intestine (SI) do not recirculate back into secondary lymphoid tissues (7, 8). Thus the localization of highly responsive CD8 T cells within close proximity to barriers of initial pathogen exposure could be poised to control local infections prior to dissemination through secondary lymphoid tissues. Despite this, the imprinting events necessary for CD8 T cells to localize to mucosal tissues such as the intestine are poorly understood.

The mammalian target of rapamycin (mTOR) is a regulator of cell proliferation, differentiation, survival and its activity is selectively inhibited by the drug rapamycin (9). While originally used as an immunosuppressant in prevention of allograft rejection, it is now understood that rapamycin also influences many facets of immune function through modulation of mTOR activity (10). In particular, mTOR has been shown to regulate memory CD8 T cell differentiation (11, 12). Inhibition of mTOR by rapamycin during the priming and expansion of virus-specific CD8 T cells increases the number of memory precursors and subsequent memory CD8 T cells in secondary lymphoid tissues. However, some vaccines despite generating large numbers of memory CD8 T cells in secondary lymphoid tissues, fail to provide protective immunity (13). Given that mTOR controls the generation memory CD8 T cells (11), and may influence T cell trafficking (10), we wanted to determine if mTOR has a role in the generation and maintenance of tissue resident mucosal CD8 T cells.

Material and Methods

Mice and infection

6–8 week old female C57Bl/6J mice were purchased from NCI. iFABP-ova mice were previously described (14). Mice were maintained SPF at Rush University or the University of Minnesota in compliance with IACUC. Rapamycin was administered at a dosage of 75ug kg⁻¹ i.p. days −1 to 7 post-immunization (unless otherwise noted) or vehicle alone (PBS +5% DMSO). Mice were immunized with 10⁶ pfu VSV-Indiana i.v, VSV-Indiana-ova i.v., or 10⁹ cfu Listeria monocytogenes-ova (LM-ova) orally. In adoptive transfer experiments, 5×10³ naïve OT-I cells were injected intravenously and mice were immunized 24 hours later.

Isolation and analysis of antigen-specific CD8 T cells

Spleens, peripheral lymph nodes (axillary, brachial, and inguinal were pooled), mesenteric lymph nodes, lung and small intestine lamina propria were harvested and lymphocytes were isolated as previously described (2, 15). Vaginal mucosa lymphocytes were isolated from cervical-vaginal tissue that was cut into small pieces and digested for 1hour 300U/mL type IV collagenase followed by percoll gradient centrifugation. In some cases lymphocytes were stained with VSV-N (RGYVYQGL) tetramer (provided by NIH Tetramer Facility). Cells were analyzed using a flow cytometer.

Retroviral transduction of shRNA in CD8 T cells

Activated OT-I cells were pooled from spleen and MLN, transduced with GFP-expressing retrovirus containing shRNA against mTOR, and adoptively transferred into congenic recipients, as previously described (11). OT-Is transduced with empty retrovirus was used as a control. shRNA sequences used against mTOR in CD8 T cells were previously described (11).

Statistical analysis

When means were compared between groups, error bars represent s.e.m. Differences (P values) between groups were calculated using unpaired, two-tailed t-test or as indicated analysis of variance (ANOVA). Survival curves were analyzed using a Log-Rank test. Statistical significance is represented as: *p<0.05, **p<0.005, ***p<0.0005. ns, not significant.

Results and Discussion

Rapamycin enhances memory CD8 T cell generation in lymphoid but not mucosal tissues

To establish whether intestinal resident memory CD8 T cell differentiation is modulated by mTOR, we utilized a model of acute virus infection with vesicular stomatitis virus (VSV) that leads to the generation of memory CD8 T cells that accumulate in both secondary lymphoid and non-lymphoid tissues such as the SI (16). We treated mice with a low dose of rapamycin during priming and expansion (days −1 – 7) and found no significant difference between the quantity of peripheral blood VSV-specific effector CD8 T cells generated in mice treated with rapamycin compared to untreated controls at day 7 (Fig. 1A). However, rapamycin increased the total number of virus-specific cells that persisted after contraction (Fig. 1A).

FIGURE 1.

Quantification of memory and effector VSV-specific CD8 T cells from tissues of mice treated with low-dose rapamycin (day −1 – 8) or vehicle alone. (A) Kinetics of a VSV-specific CD8 T cell response in peripheral blood. (B) Absolute number of N-Tet⁺ CD8 T cells in spleen, peripheral lymph nodes (PLN, pooled brachial, axillary, inguinal nodes), bone marrow (BM), lung, and small intestine lamina propria (SI LP) 64 days post-immunization. (C) Mean percent integrin β7⁺, CD103⁺, and CCR9⁺ of total CD44^hiN-tet⁺ CD8 T cells from spleen and SI LP. Results shown are from one experiment out of three, with three (A), four-five (B, C) or eight-nine (C) mice per experimental group.

We then determined the numbers of VSV-specific CD8 T cells from rapamycin and control mice 64 days post immunization. Consistent with our observations in the peripheral blood, rapamycin increased the quantity of memory CD8 T cells in the spleen (Fig. 1B). In contrast, rapamycin reduced the total number of virus-specific memory CD8 T cells in the small intestine lamina propria (SI LP). Rapamycin did not affect the formation of memory CD8 T cells in other secondary lymphoid tissues or in the vascular (“recirculating”) and tissue resident (”non-circulating”) memory populations within the lung (Fig. 1B, Supplemental Fig. 1A). These data show that rapamycin selectively attenuates intestinal CD8 T cell responses, indicating that mTOR plays different roles in the generation of intestinal and lymphoid memory CD8 T cells and/or their migration to the intestinal mucosa.

Rapamycin inhibits the generation of memory CD8 T cells with mucosal homing and retention markers

Expression of the integrin subunit β7, CD103 and the chemokine receptor CCR9, facilitate T cell trafficking and residence in the intestinal mucosa (4–6). To determine if rapamycin selectively inhibits the generation of memory CD8 T cells that express gut-specific homing markers we analyzed the expression of β7, CD103 and CCR9 on VSV-specific memory CD8 T cells. In the SI LP, rapamycin treatment decreased the frequencies of β7⁺, CD103⁺ and CCR9⁺ VSV-specific memory CD8 T cells, compared to vehicle (Fig. 1C), suggesting mTOR controls early signals that generate and/or maintain memory CD8 T cells able to traffic to and persist in the SI.

Rapamycin specifically inhibits the generation of intestinal and vaginal mucosal effector CD8 T cells

To determine if rapamycin treatment inhibited the generation of effector CD8 T cells in the SI and as a result decreased mucosal memory, we measured the total number of VSV-specific CD8 T cells in the SI LP and mesenteric lymph nodes (MLN) 7 days after immunization. We also established the effect of rapamycin on the vaginal mucosa (VM) of the female reproductive tract, another mucosal tissue where memory CD8 T cells do not recirculate. We found that the number of VSV N-tetramer⁺ CD8 T cells was decreased in the MLN, SI LP, and VM in rapamycin treated mice at the peak of the virus-specific CD8 T cell response, but not in the spleen or peripheral lymph nodes (PLN) (Supplemental Fig. 1B). Rapamycin is known to enhance the formation of memory precursor effector CD8 T cells, broadly defined as being CD127^hiKLRG1⁻, capable of developing into memory CD8 T cells through the modulation of the transcription factors T-bet and Eomes. Our data demonstrating reduced numbers of virus specific CD8 T cells in the SI LP could not be explained by differences in memory precursor formation, as rapamycin enhanced the frequency of CD127^hiKLRG1⁻ cells similarly in the spleen, PLN, and SI LP (Supplemental Fig. 1C–D).

To eliminate the possibility that these findings were unique to a systemic infection we orally infected rapamycin and vehicle treated mice with LM-ova. We found with rapamycin treatment decreased numbers of OVA-specific CD8 T cells in the MLN and SI LP 9 days post-immunization (Supplemental Fig. 1E) and delayed LM-ova clearance with higher bacteria burdens in the spleen and SI (Supplemental Fig. 1F).

To elucidate the temporal requirements of mTOR signaling in the generation of mucosal CD8 T cells, we treated mice with rapamycin before initial T cell priming (day −1–6) or after priming (day 2–6), but during clonal expansion, and migration to mucosal tissues. In these experiments we adoptively transferred a low number naïve CD45.1⁺ OT-I into congenic recipient mice, subsequently treated with rapamycin and immunized with VSV-ova. Using adoptive transfer of TCR transgenic CD8 T cells that recognize a high-affinity epitope of ovalbumin (OVA) eliminates the possibility that the effect of rapamycin on mucosal CD8 T cells is related to TCR affinity differences within polyclonal antigen-specific T cells and controls the naïve precursor frequency.

Rapamycin treatment from days −1 to 6 significantly decreased numbers of OT-I cells in the small intestine intraepithelial lymphocytes (SI IEL) and SI LP at 6 days post-immunization. The number of OT-I cells isolated from rapamycin and vehicle treated mice from days −1 to 6 were similar in the spleen, PLN, and slightly reduced in the VM and MLN, although not statistically significant (Fig. 2a). Rapamycin administered after priming, from days 2 to 6, inhibited the accumulation of OT-I cells in both the SI IEL and LP, similar to those treated from day –1. These data indicate that for the generation of mucosal CD8 T cells, mTOR signaling is important during differentiation and migration to mucosal sites and not during initial T cell priming. Consistent with endogenous virus specific CD8 T cells, we observed a down-regulation of β7 and CD103 on OT-Is isolated from the SI IEL and SI LP but few CD103⁺ or β7⁺ OT-I CD8 T cells from the VM of rapamycin treated mice 6 days post infection (Fig. 2b, Supplemental Fig. 1G). In the presence of rapamycin, fewer OT-Is isolated from the MLN and VM made IFN-γ (Fig. 2B). Expression of CD103, on resident mucosal memory CD8 T cells is essential for their long-term retention, however it is not a prerequisite for their migration into the SI. We detect decreased numbers of virus specific CD8 T cells in the mucosal tissues as early as 4.5 days post-infection (data not shown) which is prior to migrating CD8 T cells upregulation of CD103 in the SI. Together, these data point to rapamycin not directly inhibiting CD103 expression, but rather the decreased CD103 expression on CD8 T cells in the SI is a consequence of rapamycin inhibiting the formation of resident mucosal memory CD8 T cell precursors. In the SI CD103⁺ CD8 T cells concomitantly express CCR9 and upregulate α4β7 early after activation. However, not all α4β7⁺ CD8 T cells in the SI upregulate CD103 and persist as resident memory CD8 T cells. CD103⁺ CD8 T cells within the SI do not express KLRG1, although a significant population of CD103⁻α4β7⁺KLRG1⁺ CD8 T cells can be found within the SI and are likely lost during the contraction phase (data not shown).

FIGURE 2.

Rapamycin impairs the development of mucosal CD8 T cells. Adoptively transferred OT-I cells 6 days post-immunization with VSV-ova in mice treated with low-dose rapamycin or vehicle. 5×10³ naïve CD45.1⁺ OT-I CD8 T cells were transferred into CD45.2⁺ B6 mice, immunized with VSV-ova, and the mean absolute number of CD45.1⁺ OT-I in the spleen, PLN, MLN, SI IEL, SI LP, and VM of mice treated with either vehicle alone, low dose rapamycin from days −1 – 6, or low dose rapamycin from days 2 – 6 were determined. B) Percent β7⁺, CD103⁺, and IFN-γ⁺ of total OT-I cells 6 days post-immunization, treated with vehicle (white bar) or rapamycin days −1 – 6 (black bar). IFN-γ production measured by ex vivo re-stimulation with 1mg/ml SIINFEKL peptide for 5 hours in the presence of golgi transport inhibitor. Results are from one experiment out of three with three to five mice per experimental group, means were compared using one-way ANOVA.

Rapamycin treated CD8 T cells in mesenteric lymph nodes fail to migrate to the small intestine

To determine the mechanism by which rapamycin blocks the formation of CD103⁺ resident memory CD8 T cell precursors we examined the expression of α4β7 and CCR9, 5 days post-infection. We found that virus specific CD8 T cells in the MLN and SI tended to express lower levels of α4β7 and CCR9 in rapamycin treated mice, although not statistically significant (Supplemental Fig. 2B). While the expression of CCR9, and to a lesser degree α4β7, is induced by retinoic acid (RA), a vitamin A metabolite, produced by mucosal dendritic cells (17). RA is reported to activate components of the mTOR pathway (18) and thus rapamycin could block the formation of resident mucosal memory CD8 T cells through inhibiting responses to stimuli such as RA or TGF-β, which are critical for imprinting CD8 T cells with the capacity to migrate into mucosal tissues and be retained. Furthermore, CD103 expression on CD8 T cells in the small intestine is dependent on TGF-β (19). We next addressed whether CD8 T cells isolated from the MLN of rapamycin treated mice were capable of trafficking to the SI. 5 days post-infection effector OT-I cells were isolated from the MLNs of vehicle or rapamycin treated mice and transferred into congenic infection-matched hosts (Supplemental Fig. 2A). Two days after transfer (7 days post-infection) we determined the frequency of OT-Is in the spleen and SI. OT-Is from vehicle treated mice were detected in the spleen, SI IEL, and SI LP, however OT-I cells from rapamycin treated mice were only detectable in the spleen (Supplemental Fig. 2C). Thus, blocking mTOR during the differentiation of virus specific CD8 T cells results in effectors that are defective in their ability to traffic to the SI.

CD8 T cell intrinsic mTOR signals are required for the accumulation of CD8 T cells within the small intestine

mTOR regulates many vital cell processes and therefore rapamycin invariably has broad effects on many cell types. To determine if rapamycin inhibited the accumulation of CD8 T cells in the SI through either a direct effect on CD8 T cells or by indirect mechanisms we used shRNA to target mTOR in CD8 T cells. To knock down mTOR exclusively in CD8 T cells, we adoptively transferred, into congenic recipients, OT-Is transduced with GFP-expressing retrovirus containing shRNA against mTOR. OT-Is transduced with empty GFP-retrovirus were adoptively transferred into a control group. We then assessed the percentage of GFP⁺ OT-Is within the tissues 6 and 28 days post-immunization with VSV-ova. mTOR shRNA decreased the accumulation of effector CD8 T cells overall. 28 days post-immunization the percentage GFP⁺ of OT-Is was then similar to empty retrovirus controls. However the distribution of GFP⁺ OT-Is was significantly skewed towards the lymphoid tissues in both effector and memory CD8 T cells (Fig. 3A–B, Supplemental Fig. 2D–E). The lowest proportion of GFP⁺ OT-1 cells was found within the SI IEL and SI LP compared to other tissues, indicating that mTOR signals in CD8 T cells drive their accumulation in the SI.

FIGURE 3.

shRNA knockdown of mTOR in antigen-specific CD8 T cells. Retrovirally transduced OT-I with either empty GFP vector (RV-Empty) or GFP vector containing mTOR shRNA (RV-mTOR shRNA) were adoptively transferred into congenic VSV-ova immunized recipients. 6 and 28 days post-immunization, the percentage GFP⁺ of total OT-I cells were determined in the spleen, PLN, lung, MLN, SI IEL, and SI LP. Experiments were repeated twice with 3–5 mice per group. Results were compared using one-way ANOVA.

Rapamycin alters T cell responses to self-antigen expressed in the intestinal mucosa

To exclude the possibility that antigen specific CD8 T cells were being preferentially retained in secondary lymphoid tissues we used a system that enabled us to restrict antigen presentation to the intestinal mucosa. To do this we utilized a transgenic mouse model where OVA is expressed as a self-antigen produced in the gut epithelium (iFABP-ova mice) and antigen draining to gut-associated lymph nodes induces robust activation of naïve OT-Is (14). We adoptively transferred OT-Is into iFABP-ova mice treated with rapamycin (d-1–5) and 5 days post OT-I transfer, isolated lymphocytes from various tissues including the gut. Without the addition of inflammatory stimuli, we found increased numbers of OT-Is in the SI LP and SI IEL, compared to spleen, PLN, MLN, and Peyer's Patch (PP). Rapamycin treatment reduced the numbers of OT-I cells in the SI LP and SI IEL of iFABP-ova mice (Fig. 4A) and this corresponded with a decreased number of CCR9⁺CD103⁺ OT-Is within the SI IEL compared to control (Supplemental Fig. 2F). These data indicate that the effects of rapamycin are not dependent on pathogen exposure or inflammation per se. In this model, infection of iFABP-ova mice with VSV-ova and OT-I transfer results in an OVA-specific CD8 T cell mediated destruction of the intestinal epithelium that is lethal (14, 20). To determine if the decreased numbers of OT-I cells generated from rapamycin treatment were sufficient to prevent fatal intestinal disease, we challenged iFABP-ova mice, that were given OT-I cells and treated with rapamycin, with VSV-ova and monitored their survival. Rapamycin significantly increased the percent survival of iFABP-ova mice challenged with VSV-ova (Fig. 4B) and although epithelial damage was apparent in both groups, rapamycin treatment resulted in less blunting of the villi and shallower crypts, signifying less epithelial damage (Supplemental Fig. 2G). We propose the mechanism by which rapamycin enhanced survival is by inhibiting CD8 T cell migration and subsequent epithelial damage and thus could have clinical importance in the treatment of T cell mediated intestinal autoimmune disease in which CD8 T cells ultimately infiltrate the SI leading to destruction of the epithelium.

FIGURE 4.

CD8 T cell responses to self-antigen expressed exclusively in the intestinal mucosa are dampened by rapamycin. (A) Total numbers of CD45.1⁺ donor OT-I effector CD8 T cells in the spleen, PLN, MLN, PP, SI IEL, and SI LP. (B) Survival of IFABP-ova mice that were adoptively transferred with 5×10⁵ OT-Is, immunized with VSV-ova, and treated (days −1–7) with vehicle or rapamycin (vehicle 1/5 survivors at d22, rapamycin 4/4 survivors, P=0.0073 Log-Rank test). Experiments were repeated twice with three to eleven mice per group.

In summary, we show that the generation of effector and resident memory CD8 T cells in the intestinal and vaginal mucosa is abrogated by early low-dose rapamycin treatment, suggesting a requirement for mTOR in their development. Inhibition of mTOR activity by rapamycin during T cell proliferation and differentiation selectively disrupts the formation of effector CD8 T cell populations in the mucosal tissues, while enhancing the generation of memory CD8 T cells in secondary lymphoid tissues. We find that mTOR plays a central role in regulating the in situ induction of mucosal effector CD8 T cells, and disruption of this signal results in reduced responses to localized antigen and the ability to control a localized infection. Our results using iFABP-ova mice where rapamycin decreased the generation of antigen specific CD8 T cells in the small intestine even in absence of inflammation eliminates the possibility that rapamycin blocks the production of or response to inflammatory stimuli. RA, a critical inducer of α4β7 and CCR9 expression, can be produced by mucosal dendritic cells during inflammation and steady-state. Taken together with RA's ability to activate the mTOR pathway, our results showing mTOR-dependent induction of CCR9 and α4β7 provide a viable mechanism for rapamycin's effect on mucosal CD8 T cells. The suggested use of rapamycin to enhance CD8 T cell responses to vaccines (21) may not provide protection to localized infections and thus not prevent the establishment of mucosa-acquired infections such as HIV. Depending on location, antigen presentation to CD8 T cells is mediated by different DC populations, which provides a potential mechanism for tissue specific diversity and quality of effector/memory CD8 T cell generation. A major question that remains is whether resident memory CD8 T cells within mucosal tissues are sufficient for protection against mucosal pathogen exposure or if the recruitment of recirculating memory CD8 T cells into the site of exposure is necessary. Upon re-activation, circulating memory cells can upregulate mucosal homing markers and gain access to mucosal tissues. Thus, there is a great deal of interest in defining the contribution of resident versus recirculating memory CD8 T cells in protection from mucosal pathogens. Identifying upstream signals that augment mTOR activity may be an important therapeutic target for enhancing mucosal resident memory CD8 T cell formation and could provide insight for developing vaccine strategies that inhibit the establishment of mucosa-acquired infections.

Abbreviations

mTOR

mammalian target of rapamycin

VSV

vesicular stomatitis virus

SI LP

small intestine lamina propria

BM

bone marrow

MLN

mesenteric lymph nodes

PLN

peripheral lymph nodes

VM

vaginal mucosa

SI IEL

small intestine intraepithelial lymphocytes

RA

retinoic acid