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. Author manuscript; available in PMC: 2014 Mar 1.
Published in final edited form as: J Immunol. 2013 Jan 21;190(5):2178–2187. doi: 10.4049/jimmunol.1201945

Critical role for all-trans retinoic acid for optimal effector and effector memory CD8 T cell differentiation1

S Rameeza Allie *, Weijun Zhang *, Ching-Yi Tsai *, Randolph J Noelle *,†, Edward J Usherwood *,‡
PMCID: PMC3578118  NIHMSID: NIHMS432532  PMID: 23338237

Abstract

A plethora of work implicates important effects of the Vitamin A derivative, retinoic acid (RA), in myeloid differentiation, while fewer studies explore the role of RA on lymphoid cells. Most work on lymphoid cells has focused on the influence of RA on CD4 T cells. There is little information about the role of RA in CD8 T cell differentiation, and even less on cell-intrinsic effects in the CD8 T cell. This study explores the role of RA on effector and memory differentiation in a cell intrinsic manner in the context of vaccinia virus infection. We observed the loss of the short-lived effector cell phenotype (reduced KLRG1+, T-bethi, granzyme Bhi), accompanied by an enhanced memory precursor phenotype at the effector (increased CD127hi, IL-2+) and contraction phase (increased CD127hi, IL-2+, eomesoderminhi) of the CD8 response in the absence of RA signaling. The lack of RA also increased the proportion of central memory CD8s. Collectively; these results introduce RA in a new role in CD8 T cell activation and differentiation. This may have significant implication in optimal vaccine design where Vitamin A supplementation is used to augment effector responses, but this may be to the detriment of the long-term central memory response.

Introduction

The morphogenic role of all trans retinoic acid (RA), a vitamin A derivative, in development and differentiation was confirmed by White et al. in 2007, using a zebra fish model to confirm RA patterning the hindbrain (1). However, immunologists have studied it in various contexts of immune cell differentiation as early as the 1980s. Amongst myeloid cells RA has been shown to allow for differentiation into mature macrophage or antigen presenting cells (2). This RA mediated differentiation of dendritic cells (DCs) has been shown to skew them towards IL-12 producing DCs (3). RA also regulates isotype switching and plasma cell formation by B cells (4–6).

In the adaptive immune compartment RA has been shown to promote regulatory CD4 T and CD8 T cell differentiation and stabilization (7–9). Further, RA has been shown to enhance inflammatory effector responses by CD4 helper T cells (10, 11). In CD8 T cells, an early study showed that increased expression of RA receptor gamma increased the number of CD8 T cells (12). To our knowledge, no previous studies have looked at the cell intrinsic role of RA signaling in CD8 T cell effector and memory formation in the context of virus infection.

Paramount to eliciting optimal protective immunity to infections is the generation of high quality memory cells. Superior memory generation is a key component of vaccine design, as these cells can elicit optimal protection. In response to an acute viral insult, CD8 T cell responses go through three phases: the primary acute expansion phase to resolve the infection; the contraction phase to eliminate potentially harmful cytotoxic effectors and a memory phase, where self-renewing antigen (Ag) specific cells are maintained at low frequencies for extended periods of time (13). Upon activation in mice and humans after infection, CD8 T cells form highly differentiated short-lived effector cell (SLEC) and a memory-precursor effector cell (MPEC) populations (14–16). The SLEC population is driven by inflammatory cytokines like IL-12 or type-I interferons and characterized by high T-bet expression, compared to MPEC, which have high eomesodermin expression, recently shown to be driven by Forkhead Box Protein 01 (FOX01) expression (17–23). SLECs are identified by surface expression of high killer cell lectin-like receptor subfamily G member 1 (KLRG1) and low IL-7Rα (CD127) while MPECs are identified by the expression of low KLRG1 and high IL-7Rα (17). The terminally differentiated SLEC population is the desired population to resolve a viral infection with its high cytotoxic potential, while the MPEC population is thought to differentiate into the long-lived memory population (24). Amongst the memory population, central memory cells (Tcm) are the most long-lived, and are characterized by robust recall potential, capacity for homeostatic proliferation and homing to lymphoid organs. Effector-memory cells (Tem) are characterized by homing to peripheral sites and lower homeostatic turnover, while being the first to respond after re-exposure to infection (16, 25–27).

Acknowledging the role of RA in differentiation as seen by studies in development and in other immune cell types, we hypothesized that RA would promote the differentiation of CD8 T cells to their terminally differentiated phenotype, SLECs. To test this hypothesis, we used a mouse model expressing a dominant negative RA receptor alpha (RARαDN) in the T cell compartment, and mixed bone marrow (BM) chimeric mice to measure CD8 T cell intrinsic effects. To determine the effect of the absence of RA signaling in CD8 T cell differentiation, mice were infected with vaccinia virus, which induces a strong memory CD8 T cell response (28).

Vitamin A is used in conjunction with various vaccines (29, 30), so it is imperative to understand how its biologically active metabolite, retinoic acid, stimulates the T cell response. These studies will elucidate the effect of RA in shaping the effector and memory response helping to tailor the outcome of vaccination, in addition to providing essential information regarding optimal CD8 T cell activation and differentiation.

Materials & Methods

Mice

C57BL/6 and congenic B6-Ly5.2-Cr mice (Ly5.1/CD45.1+) were purchased from The National Cancer Institute (Bethesda, MD). C57BL/6 R26RAR403 (from this point referred to as dominant negative RARα mice (RARαDN) were used by permission from Dr. Shanthini Sockanathan (Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD) (28). WT and RARαDN mice used in all experiments are C57BL/6 strain Mouse experiments were performed under the guidelines and approval of Dartmouth College institutional animal care and use committee.

Aldefluor Assay to measure RA expression

We measured the levels of an enzyme necessary for ATRA production - Aldehyde dehydrogenase (ALDH). ALDH is a cytosolic isoenzyme that contributes to the oxidation of retinol to retinoic acid. The Aldefluor assay measures the activity of ALDH, which is a surrogate measure for RA in the cytosol (STEMCELL Technologies Inc. – Vancouver, BC, Canada). Living cells through passive diffusion take up uncharged ALDH-substrate bound to a green fluorophore. The substrate is converted by intracellular ALDH into a negatively charged reaction product, which is retained inside cells, causing the cells expressing ALDH to become brightly fluorescent. The brightly fluorescent ALDH-expressing cells are detected by flow cytometer in the green channel (520–540 nm). As a control half of each sample has an inhibitor of ALDH added to determine the background.

Chemical inhibition of RA signaling

A pan retinoic acid antagonist (NRX194310) was obtained from Randolph J. Noelle (Department of Immunology, Dartmouth Medical School, Hanover, NH. USA) (11). 30 μg per mouse was injected intra peretonealy (i.p.) every other day, starting at day-1, until the mice were euthanized at day 10 post infection.

Vaccinia and Listeria infection

Vaccinia virus, Western Reserve strain (VV-WR) was obtained from Dr. William Green (Dartmouth Medical School, Lebanon, NH). Primary infection (1000 PFU) was administered via the intranasal route (i.n.). Listeria Monocytogenes (LM) expressing the VV-WR CD8 dominant epitope, B8R, was kindly provided by Dr. Ross Kedl (University of Colorado), and administered (2×106 PFU) via the intravenous (i.v.) route.

Generation of mixed bone marrow chimeric mice

Bone marrow (BM) from donor B6-Ly5.2-Cr mice (1×106 cells) and C57BL/6 DN RARα mice (1×106 cells) were mixed 1:1 and transferred intravenous into recipient B6-Ly5.2-Cr mice. The recipient mice had been lethally irradiated (1050 Rads) the day before BM transfer with a split dose (525 Rads twice) given 24 hours apart. 50 days post BM transfer the reconstitution of the BM was checked by staining blood cells for the congenic marker CD45.2 (BioLegend – San Diego, CA). The mice that had an approximate 1:1 reconstitution of CD45.2+: CD45.2− were used in the study.

Tissue preparation

Blood was processed and the red blood cells lysed. The spleen and lymph nodes were mashed and the red blood cells were lysed. The lungs were digested with Collagenase (2.33 mg/ml Sigma-Aldrich – St. Louis, MO) and DNAse (0.2 mg/ml Roche Diagnostics – Indianapolis, IN) for 30min, then they were mashed and the red blood cells were lysed.

Flow cytometry

An MHC/peptide tetramer for the VV-WR epitope B8R20–27 (TSYKFESV)/Kb was obtained from the NIH Tetramer Core Facility (Emory University, Atlanta, GA). Cells were stained for 1 hour at room temperature with tetramer then washed and stained for surface markers for 30min at 4°C. The surface markers used were PerCP CD8 (BioLegend), FITC CD27 (BioLegend), PE CD62L (Invitrogen - Carlsbad, CA), FITC CD127 (BioLegend), PE KLRG1 (Abcam - Cambridge, MA), PE CD25 (BioLegend), PE IL-21R (BioLegend) and FITC CD44 (BioLegend). For T-bet and eomesodermin staining, the cells were fixed and permeabilized after tetramer staining and stained with FITC T-bet (Santa Cruz Biotechnology - Santa Cruz, CA), FITC mIgG1 isotype (BD Biosciences – San Jose, CA), PE eomesodermin (eBiosciences - San Diego, CA) and PE Rat IgG2a isotype (BioLegend). The fixation and permeabilization was done using the FoxP3/Transcription Factor Staining Buffer Set from eBiosciences (San Diego, CA).

Intracellular cytokine staining

Splenocytes were re-stimulated with 1 μg/ml of B8R peptide, 10 U/ml IL-2, and 10μg/ml Brefeldin A for 5 hours at 37°C. Unstimulated splenocytes were used as a negative control, and the background subtracted. In the presence of 10 μg/ml of Brefeldin A, the cells were stained for surface expression of PerCP CD8 (BioLegend) for 20min on ice. Following the surface staining the cells were fixed with 2% formaldehyde (Ted Pella, Inc. – Redding, CA) for 20min at room temperature and permeabilized with 1X permeabilization buffer (eBiosciences) for 10min. Washed and permeabilized cells were stained with APC-labeled anti-IFNγ (BioLegend), PE-labeled anti-IL-2 (BioLegend), PE-labeled anti-TNFα (Invitrogen), PE-labeled anti-granzyme B (Invitrogen) and isotype for granzyme B - PE-labeled mouse anti-IgG1 (Invitrogen).

BrdU incorporation and staining

Mice were provided with 0.8 mg/ml of 5-Bromo-2′-deoxyuridine (BrdU) (Sigma-Aldrich) in the drinking water 10 days prior to staining for BrdU incorporation. Splenocytes were stained with MHC/peptide tetramer for the VV-WR epitope B8R20–27 (TSYKFESV)/Kb and surface stained for CD8 and CD44 as described above, followed by fixation and permeabilization for BrdU staining. The BrdU staining kit from BD Biosciences (San Jose, CA) was used to stain with anti-BrdU antibody, and measured by flow cytometry.

Secondary responses

At greater than 60 days post primary infection the chimeric mice were challenged with 1×106 CFU of replication defective Listeria Monocytogenes (LM) expressing the VV-WR CD8 dominant epitope - B8R- via the intravenous (i.v.) route. At 6 days post challenge the splenocytes were analyzed for to measure cell expansion. Ag specific CD8 T cell proportions and numbers were determined along with their phenotype using the surface and intracellular markers described previously.

Statistical analysis

The distribution was analyzed for normality and then p values were determined using a Student’s t-test or a Mann-Whitney U test. Paired t-tests were used in the BMC model to account for the same internal environment of the WT and RARαDN CD8 T cell populations. A one-way ANOVA followed by Dunn’s post-test was carried out when greater than two variables were compared. p<0.05 was considered significant.

Results

Measuring all-trans retinoic acid production following infection

We infected wild type C57BL/6 (WT B6) mice intranasally (i.n.) with 1000 PFU of vaccinia virus Western Reserve (VV-WR) strain and measured the levels of an enzyme necessary for RA production. Aldehyde dehydrogenase (ALDH) is a cytosolic isoenzyme that contributes to the oxidation of retinol to RA. The Aldefluor assay measures the activity of ALDH, which is a surrogate measure for RA in the cytosol. We used this assay to measure the level of RA in the spleen, lung and mediastinal (lung draining) lymph node (mLN), and in T cells, B cells, DCs and macrophages. We detected increased RA expression upon infection with vaccinia virus at day 4 post-infection in the lung and mLN (Supplementary Figure 1A). Increased expression of RA was also observed in B cells (CD19+) after infection in the mLN and lungs (Supplementary Figure 1B). CD11b+ cells increased RA production after infection in the spleen and lungs (Supplementary Figure 1C). CD11c+ cells showed expression of RA but only marginally increased production after infection (Supplementary Figure 1D).

Reduced Ag specific response in the absence of RA signaling in dominant negative RARα transgenic mice

To determine the effect of RA signaling on the Ag specific CD8 response, we infected WT B6 and dominant negative RARα expressing mice crossed with CD4cre mice, DNRARα*CD4cre (hereafter referred to as RARαDN), i.n. with 1000 PFU VV-WR and measured the Ag specific response at day 9 post infection, by staining with MHC/peptide tetramer folded with the VV-WR epitope B8R20–27 (TSYKFESV)/Kb (hereafter referred to as B8R). We saw a decrease in the B8R specific CD8 populations both proportionally and by total cell number per spleen (Figure 1A).

Figure 1. Significantly reduced Ag specific CD8 response, which is skewed towards an MPEC phenotype, in the absence of RA signaling.

Figure 1

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9 days post i.n. VV-WR (1000 PFU) infection, splenocytes from WT and RARαDN mice were stained with MHC/peptide tetramer for the VV-WR B8R epitope and analyzed by flow cytometry. (A) Top – representative plot showing B8R gate in the CD8 T cell population, bottom – percentages and cell count per spleen.

B8R-tetramer+CD8+ splenocytes were surface stained for (B) CD127, (C) IL-21R. (D) Spleen cells were incubated for 5 hrs with or without B8R peptide in the presence of Brefeldin A. Intracellular staining was performed to determine IFNγ and IL-2 production by the CD8 T cells. The percent of IL-2+ reported is gated on the IFNγ+ and the MFI was calculated for IFNγ+IL-2+ cells. Filled circles are WT and open circles are RARαDN. T-tests were performed, * p <0.01 and *** p<0.0001. n of 6 per group tested, experiment repeated three times showing similar data.

To determine the effect of the reduced Ag specific CD8 response on the viral titer we measured the viral load in the lung (LG) 9 days post infection by plaque assay. There was no significant difference in viral load in the LGs of WT B6 or RARαDN mice (Supplementary Figure 2A). Since the dominant negative RARα was expressed in CD8 and CD4 T cells, we wanted to determine the effect of RARαDN expression on the CD4 response. Using an IFNγ ELISPOT to measure responses to two CD4 epitopes (I1L and L4R) of VV-WR we saw no significant difference in the CD4 response (Supplementary Figure 2B). We also analyzed the antibody response and saw similar levels of neutralizing antibody (data not shown).

Increased MPEC skewing with selective defects in effector phenotype in RARαDN mice during acute infection

At the acute stage of the VV-WR infection we determined the phenotype of the Ag specific CD8 response by staining for surface and intracellular markers in Ag specific CD8 T cells, identified by B8R tetramer or by IFNγ production following cognate peptide stimulation. We saw a statistically significant increases in surface expression of the IL-7Rα (CD127) and IL-21 receptor (IL-21R) in the B8R specific CD8 T cells in the RARαDN (Figure 1B & C). This phenotype is indicative of a memory precursor effector cell (MPEC) population that is high in CD127 and is responsive to IL-21. To further confirm the MPEC phenotype we measured IL-2 production in the IFNγ positive CD8 T cell population. We observed an increase in both the proportion of B8R-specific CD8 T cells producing IL-2 and the amount of IL-2 produced per cell in the RARαDN relative to the WT B6 group (Figure 1D). We observed a population of cells that were IFNγ-, which produced IL-2. We confirmed that this population was not an artifact but statistical analysis of the WT and RARαDN samples confirmed that this population was the same in both the tested groups. In addition, this population did not alter the observed higher proportion of IL-2+ cells in the B8R specific or total CD8 population. When we examined molecules associated with effector activity, we saw significant decreases in KLRG1 and granzyme B levels (Figure 2A & B). This was indicative of a reduced SLEC phenotype, but not all effector functions were impaired. IFNγ production (Figure 2C) was not impaired and TNFα production (Figure 2D) was significantly increased in the RARαDN compared to the WT group.

Figure 2. Ag specific CD8 T cells exhibited a reduced SLEC phenotype in the absence of RA signaling.

Figure 2

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VV-WR (i.n. 1000 PFU) infected WT and RARαDN mice were analyzed 9 days post infection. (A) B8R-tetramer+CD8+ splenocytes were surface stained for KLRG1. (B, C & D) Spleen cells were incubated for 5 hrs with or without B8R peptide in the presence of Brefeldin A. (B) Intracellular staining was performed to determine granzyme B (GrB) production in the IFNγ+ CD8+ cells. T-tests were performed on values subtracted for background using an isotype control for GrB (filled: WT, open bold: RARαDN, open thin: isotype). (C) Intracellular staining was performed to determine IFNγ production in the CD8+ cells. Statistics were performed on values subtracted for background using a no B8R peptide control IFNγ. (D) Intracellular staining was performed to determine TNFα production in the IFNγ+ CD8+ cells. The percent of TNFα+ reported is gated on the IFNγ+ and the MFI was calculated for IFNγ+TNFα+ cells. B8R-tetramer+CD8+ splenocytes were stained for expression of (E) T-bet, (F) eomesodermin (filled: WT, open bold: RARαDN, open thin: isotype).

Filled circles are WT and open circles are RARαDN. T-tests were performed, *p<0.01, *** p<0.0001. n of 6 per group tested and experiment repeated 3 times.

We wanted to determine the effect of the absence of RA signaling on transcription factors associated with effector and memory CD8 T cell differentiation, so we stained for T-bet and eomesodermin, respectively. We observed a reduction in both T-bet and eomesodermin in the RARαDN mice (Figure 2E & F).

Similar MPEC skewing observed after RA inhibitor treatment

The RARαDN mice expressed the dominant negative RARα in all CD4 and CD8 cells, as T cells will express the cre recombinase when they express CD4, at the double positive stage during thymic development. This raised the possibility that the MPEC phenotype was due to developmental defects during T cell development, rather than the absence of RA signaling specifically during the priming stage of the CD8 response. To rule out the effect of the absence of RA during development, we used an antagonist of RARα, β and γ (RA-I), which we administered before and during the priming stage (every other day from day -1 to day 9, post infection). We did not observe a significantly reduced B8R Ag specific response in the RA-I treated mice during the acute response (Figure 3A), unlike that observed in the RARαDN mice (Figure 1A).

Figure 3. Enhanced MPEC and reduced SLEC phenotype recapped in mice treated with a chemical RA antagonist.

Figure 3

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WT and RA antagonist (RA-I) treated mice were infected with 1000 pfu VV-WR i.n. At day 10-post infection splenocytes were stained with MHC/peptide tetramer for (A) the VV-WR epitope B8R and CD8 and analyzed by flow cytometry for the presence of B8R specific CD8 T cells. B8R-tetramer+CD8+ splenocytes were surface stained for (B) CD127 and (C), intracellular staining for B8R specific IFNγ and IL-2 production. The percent of IL-2+ reported is gated on the IFNγ+ and the MFI was calculated for IFNγ+IL-2+ cells. (D) B8R-tetramer+CD8+ splenocytes were surface stained for KLRG1. (E) Spleen cells were incubated for 5 hrs with or without B8R peptide in the presence of Brefeldin A. Intracellular staining was performed to determine GrB production in the IFNγ+ CD8+ cells (filled: WT, open bold: RA-I, open thin: isotype). T-tests were performed on values subtracted for background using an isotype control for GrB. Filled circles are WT and open circles are RA-I. T-tests were performed, **p<0.001, *** p <0.0001. (A–C) n of 5–6 per group tested, experiment repeated three times showing similar data. (D) Combined data from two experiments with n of 5–6, (E) n of 3–4 per group tested and experiment repeated 3 times showing similar data.

Ag specific cells had a statistically significant increase in CD127 expression (Figure 3B) and IL-2 production (Figure 3C) in the RA-I group. We also observed a statistically significant increase in IL-21R expression on a per cell basis (data not shown). Further examination of these cells showed a significant reduction in KLRG1 expression (Figure 3D) but an increase in granzyme B, IFNγ and TNFα production (Figure 3E, Supplementary Figure 2C & 2D) in the RA-I treated mice. These results indicate that the skewing toward an MPEC phenotype was not due to defects in CD8 T cell development caused by a lack of RA.

MPEC skewing and effector defects are CD8 T cell-intrinsic

In the systems analyzed above, we compared WT B6 to either RARαDN or RA-I treated mice. In neither case was the RA defect limited to the CD8 T cells. To determine the CD8 T cell-intrinsic effects of RA, we made mixed bone marrow chimeric mice. We reconstituted the hematopoietic compartments of lethally irradiated B6 Ly5.2 hosts with a 1:1 mix of WT B6 and RARαDN bone marrow. This experimental system resulted in the same mouse containing both WT and RARαDN CD8 T cells, identified by different congenic markers, so any differences observe could be considered cell-intrinsic. Following a 50-day reconstitution period we confirmed 1:1 reconstitution and these mice were used in the proceeding experiments, referred to from this point as RARαDN chimeric mice.

We infected the RARαDN chimeric mice with 1000 PFU of VV-WR i.n. and determined the Ag specific response at 10 days post infection. We did not see a significant difference in the total CD8 response (data not shown) or the B8R Ag specific response (Figure 4A). Analysis of surface markers by flow cytometry revealed an increase in CD127 and IL-21R (Figure 4B & C). Thus, the surface MPEC phenotype seen in Figure 1 and 3 in the previously used models was confirmed, showing this to be a cell intrinsic phenotype. This phenotype was further corroborated by the increase in IL-2 production by B8R specific IFNγ producing RARαDN CD8 T cells (Figure 4D). There was no significant difference in the expression of eomesodermin (Figure 4E).

Figure 4. Cell intrinsic skewing of CD8 T cells to MPEC in the absence of RA signaling.

Figure 4

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To test the cell intrinsic requirements of RA in CD8 function, mixed bone marrow chimeric mice (RARαDN) were infected with 1000 PFU VV-WR i.n. At day 10 post infection splenocytes were stained with (A) MHC/peptide tetramer for the VV-WR epitope B8Rand CD8 and analyzed by flow cytometry for B8R specific CD8 T cells. B8R-tetramer+CD8+ splenocytes were surface stained for (B) CD127, (C) IL-21R (filled: WT, open bold: RARαDN). (D) Spleen cells were incubated for 5 hrs with or without B8R peptide in the presence of Brefeldin A. Intracellular staining was performed for IFNγ and IL-2. The percent of IL-2+ was calculated for the IFNγ+ CD8 population. (E) B8R-tetramer+CD8+ splenocytes were stained for expression of eomesodermin (filled WT, open bold RARαDN, open thin isotype). Filled circles are WT and open circles are RARαDN. T-tests were performed, ***p <0.0001, ns – not significant. n of 4–5 per group tested experiment repeated two times showing similar data.

As seen in the RARαDN mice, the chimeric mice showed a significant reduction in SLEC phenotype by KLRG1, granzyme B and T-bet expression in the RARαDN CD8 T cells (Figure 5A–C). Unlike in the RARαDN mice, the chimeric mice showed an increase in IFNγ (Figure 5D) along with the previously observed increase in TNFα in the RARαDN CD8 T cells (Figure 5E). We further examined these cells for direct ex vivo granzyme B production and CD107 expression as an indicator of degranulation. We saw significant impairments in both (data not shown).

Figure 5. Selective Reduction in SLEC phenotype in the absence of RA in chimeric mice.

Figure 5

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At day 10-post infection with VV-WR (i.n. 1000 PFU), splenocytes from RARαDN chimeric mice were analyzed. (A) B8R-tetramer+CD8+ splenocytes were surface stained for KLRG1. [B, D & E) Spleen cells were incubated for 5 hrs with or without B8R peptide in the presence of Brefeldin A. (B) Intracellular staining was performed to measure GrB production in the IFNγ+ CD8+ cells. T-tests were performed on values subtracted for background using an isotype control for GrB. (C) B8R-tetramer+CD8+ splenocytes were stained for expression of T-bet. (Filled WT, open bold RARαDN) (D) Intracellular staining was performed to determine IFNγ production in the CD8+ cells. Statistics were performed on values subtracted for background using a no B8R peptide control. (E) Intracellular staining was performed to determine TNFα production in the IFNγ+ CD8+ cells. **p<0.001, *** p<0.0001. (A) Combined data from two experiments with n of 5–6, others are n of 3–4 per group tested and experiment repeated 2 times showing similar data.

CD8 T cell intrinsic increase in MPEC skewing with selective defects in effector phenotype is maintained during the contraction phase

When the VV-WR RARαDN chimeric mice were examined at 21 days post-infection (contraction phase of the CD8 response) we saw a significant reduction in the B8R Ag specific CD8 T cell response among the RARαDN CD8 T cells (Supplementary Figure 3A). The B8R Ag specific RARαDN CD8 T cells maintained the increase in CD127and IL-21R (Supplementary Figure 3B & C) seen at 10 days (Figure 4) post infection. The increased production of IL-2 was also maintained during the contraction phase (Supplementary Figure 3D). At the contraction phase the expression of eomesodermin corroborated the MPEC phenotype seen by other markers by being significantly elevated in the absence of RA signaling (Supplementary Figure 3E).

The SLEC phenotype remained diminished at the contraction phase, as seen by the reduction in KLRG1, granzyme B and T-bet (Supplementary Figure 3F–H) but the increase in IFNγ (Supplementary Figure 3I) was not maintained at the contraction phase, unlike the increased TNFα expression (Supplementary Figure 3J). This may indicate a differential requirement for RA signaling in IFNγ production at the acute and contraction phases.

Enhanced central memory phenotype in the absence of RA signaling

We examined RARαDN chimeric mice at >60 days post infection (memory phase of the CD8 response) and saw a significant reduction in the proportion but not total number of the B8R-specific CD8 T cell response (Supplementary Figures 4A). These memory cells were further examined and revealed an increase in CD127, CD62L, CD27 and IL-2 production (Figure 6A–D), which are all indicative of a central memory T cell (Tcm) phenotype. Analysis of transcription factors revealed a significant decrease in T-bet (Figure 6E) along with an increase in eomesodermin expression (Figure 6F).

Figure 6. Enhanced Tcm phenotype in the absence of RA signaling in chimeric mice.

Figure 6

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B8R-tetramer+CD8+ splenocytes from RARαDN chimeric mice at >60 days post infection with VV-WR (i.n. 1000 PFU) were surface stained for (A) CD127, (B) CD62L and (C) CD27 (filled: WT, open bold: RARαDN). (D) Intracellular staining was performed to determine IL-2 production in the IFNγ+ CD8+ cells. The percent of IL-2+ reported is gated on the IFNγ+ and the MFI was calculated for IFNγ+IL-2+ cells. B8R-tetramer+CD8+ splenocytes were stained for expression of (E) T-bet and (F) eomesodermin (filled: isotype (arrow), open bold: RARαDN, open thin: WT). Filled circles are WT and open circles are RARαDN. T-tests were performed, **p <0.001 and ***p<0.0001. n of 4–5 per group tested, experiment repeated four times showing similar data.

Tcm cells preferentially home to lymphoid organs so we determined the total B8R-specific CD8 T cell number in the spleen, lymph nodes and lungs and did not see preferential homing to lymphoid organs but impairment in homing to the lung (Figure 7A). Further, Tcm cells exhibit increased homeostatic proliferation, so we examined BrdU incorporation after 10 days of BrdU administration in the drinking water, at the memory stage. We saw a significant increase in the proportion of BrdU+ cells in the absence of RA signaling (Figure 7B).

Figure 7. Distribution, homeostatic turnover and recall responses in memory CD8 T cells.

Figure 7

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At day >60 post 1000 PFU VV-WR i.n infection RARαDN chimeric mice were analyzed. (A) Spleens, mediastinal lymph nodes and lungs were stained with MHC/peptide tetramer for the VV-WR epitope B8R and CD8 and analyzed by flow cytometry for B8R specific CD8 T cells. (B) B8R-tetramer+CD8+ splenocytes were stained for BrdU incorporation after 10 days of BrdU uptake from the drinking water (filled: WT, open bold: RARαDN). (C) To test the recall potential of the B8R-tetramer+CD8+ T cells, at day >60-post infection some mice remained unchallenged as the memory (MEM) mice and others were infected with 106 CFU of LM-B8R i.v. as the Recall mice. 6 days post challenge splenocytes were stained with B8R tetramer and anti-CD8 and analyzed by flow cytometry for B8R specific CD8 T cells. **p <0.001, ***p<0.0001 and ns - not significant. n of 4 per group tested, experiment (A) repeated four times, (B) two times (C) n of >5 per group tested, experiment repeated four times showing similar data.

A functional test of Tcm cells is their ability to recall in response to a secondary challenge. Thus we administered 2×106 CFU of LM expressing the B8R epitope, i.v. to a group of chimeric memory mice (labeled Recall) and left a group of mice unchallenged (labeled MEM). The Recall mice showed a robust expansion in the number of B8R Ag specific CD8 cells compared to the MEM mice, and both WT and RARαDN CD8 T cell compartments responded similarly (Figure 7C). Next we examined the phenotype of the secondary effector cells. The RARαDN CD8 T cells had an increase in CD127 and IL-2 expression (Supplementary Figure 4B & C) together with reduced KLRG1, granzyme B and T-bet expression (Supplementary Figure 4D–F), consistent with phenotypic differences observed prior to secondary challenge.

Discussion

We have shown that RA signaling is essential for optimal effector and effector memory differentiation by CD8 T cells. We see an increase in the MPEC phenotype at the expense of the SLEC phenotype in the primary effector and recall stages of the responses, as observed by increased CD127hi, eomesoderminhi, IL-2+ CD8 T cells and reduced KLRG1+, T-bethi and granzyme Bhi CD8 T cells. This suboptimal differentiation to effector T cells resulted in more MPECs and an enhanced central memory population. The central memory population was characterized by the surface expression of CD62L, CD27, and CD127, elevated IL-2 production and increased homeostatic proliferation, measured by BrdU incorporation.

Previous work using conditional (VavCre) knock out of RARγ showed that the absence of the RARγ signaling in the hematopoietic and endothelial compartments resulted in reduced Ag specific CD8 T cells at the effector and memory time points while the CD4 T cells were unaffected (31). A plethora of recent work allows for the appreciation of RA signaling in the functions of many other immune cells, which may influence the outcomes observed in CD8 T cells. Therefore some of the data from this previous study may be a result of extrinsic effects of the absence of RA signaling on other immune cell types. In addition, this study focused on a hematopoietic and endothelial knock out of the RARγ receptor mediated signaling which may enhance signaling through other RA receptors via the increase in the availability of retinoic X receptors (RXRs), that are necessary for all RA receptors to bind the response elements on DNA (32). In our model, the mice express a dominant negative RARα, thus a repressor is bound constitutively to the RARE, and inhibits signaling via all the RA receptors. Thus to our knowledge this is the first study to determine the cell intrinsic effect of RA signaling in effector and memory CD8 T cell differentiation.

Our hypothesis that RA is essential for optimal effector differentiation is supported by our observation that RA is expressed in the lung, lymph nodes and spleens and is increased after virus infection. This may indicate a requirement during the priming of the response. The expression was restricted to professional Ag presenting cells, DCs (CD11c+), Macrophages (CD11b+) and B cells (CD19+) indicating that it may augment the established three signals for T cell activation (TCR signal, costimulation and inflammatory signals) (33–36). Previous studies have shown that the nuclear factor of activated T cells (NFAT) family of transcription factors and calcium mobilization are enhanced by Vitamin A and RA signaling, respectively (10, 37, 38). NFAT and calcium mobilization are essential features of T cell activation, and their dependence on RA signaling supports the concept that RA is an important component of optimal T cell activation. Hall et al examined the role of RA in promoting the mTORC1 and AKT signaling pathways during T cell activation (10). They showed that the absence of RA diminished these pathways, cellular cytokine production and T cell proliferation. Their data is consistent with our findings of decreased SLECs, which is the expected result of weaker TCR signaling in the absence of RA responsiveness. Taken together, local upregulation of RA by APCs during the initiation of an immune response, and promotion of effector CD8 T cell generation act to enhance effector T cell generation, which is beneficial for the infected host.

While we observed impairments in effector functions in the absence of RA signaling, they were limited to KLRG1 upregulation and granzyme B production. In contrast, TNFα production was significantly increased in the acute and memory phases. This outcome may not be surprising, as RA has been previously shown to negatively regulate TNFα mRNA stability (39). Thus the robust increase in TNFα production by the CD8 T cell, in our system, may be a result of the absence of post-transcriptional regulation of TNFα by RA. IFNγ was impaired in the absence of RA signaling during the acute phase of the response, which is supported by work in CD4 T cells showing impairment in IFNγ production in effector cells (10, 11). This impairment in IFNγ production was not carried forward into the contraction and memory phases showing that RA significantly influenced IFNγ production only in effector cells.

In the current study, data from the chimeric model shows that the influence of RA signaling on eomesodermin begins at the contraction phase and is carried into the memory phase. Work from the Reiner group has showed that CD8 T cells use T-bet and eomesodermin redundantly for effector functions but eomesodermin is essential to promote memory survival and the central memory phenotype (40). Therefore our observed increase in eomesodermin at the contraction phase and the memory phase may explain the enhanced MPEC and central memory phenotypes, respectively. As it is the absence of RA signaling that induces this increase in eomesodermin, there may be a direct or indirect role for RA in eomesodermin expression.

We observed an increase in IL-2 production by CD8 T cells from the acute to the memory phase in the absence of RA signaling. This is likely a direct effect of the absence of RA signaling as there is an RA response element (RARE) in the promoter region of IL-2, and studies have shown that RA suppresses expression of IL-2 expression (41, 42). This increase in IL-2 production combined with the increased eomesodermin further confirms the MPEC and central memory phenotype.

Although central memory cells are known to home preferentially to lymphoid organs, we do not see a significant accumulation of the CD8 T cells in the absence of RA signaling in the lymph nodes (25). CD62L expression by WT CD8 T cells may be sufficient for effective lymphoid homing, and further expression by the RARαDNs may not serve for better homing. In spite of similarities in lymphoid homing we see defective lung homing, as seen by the reduced Ag specific CD8 T cell numbers in the lung. Previous studies have shown that cells primed to a respiratory infection become imprinted to home to the lung (43). In addition, a recent study showed that CD8 T cells primed with RA supplementation resulted in increased memory cells in mucosal site, which supports our findings of defective homing to the lung in the absence of RA signaling (44).

Despite the significantly enhanced central memory phenotype of RARαDN CD8 T cells, wild type CD8 T cells expanded as robustly as the RARαDN during a recall response. During the primary response we observed a reduced number of effector cells, which implies that optimal RA signaling may be essential for robust effector differentiation, and this may also be true during secondary responses. Due to constitutive expression of the RARαDN construct in CD8 T cells, RA signaling was absent during the recall response, which may prevent the central memory skewed RARαDN memory cells from expanding to their full potential.

Collectively, our data shows that RA signaling is important during the priming of the response to promote effector differentiation. Deficiency in RA signaling may result in the effector population defaulting to the MPEC phenotype, leading to an increase in the central memory population, which also requires RA to differentiate to secondary effectors when re-challenged.

This is the first time that the effect of RA signaling has been isolated to the CD8 T cell compartment during a viral infection. These findings implicate RA in optimal effector function and these studies are consistent with findings from the Greenberg group showing the adjuvant effects of RA during vaccination against viral infections, which show enhanced effector responses (44). Therefore our study has important implications for vaccine trials involving supplementation of vitamin A or retinoic acid during priming, implying this may favor effector CD8 T cell differentiation but it may not result in an optimal central memory response.

Note added in proof. Another paper reporting similar findings was published while this paper was under review (45).

Supplementary Material

1
2

Acknowledgments

We thank Dr. Patricia Ernst (Department of Genetics at the Geisel School of Medicine at Dartmouth) who provided advice on improvements to the Bone Marrow Chimera protocol.

We are thankful to Jie Deng and Dr. James D. Gorham (Department of Microbiology & Immunology at the Geisel School of Medicine at Dartmouth) for the use of their Aldefluor Assay kit.

Footnotes

1

Funding was provided in part by National Institutes of Health grants AI069943 and CA103642.

The authors declare that they have no financial conflicts of interest.

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Supplementary Materials

1
NIHMS432532-supplement-1.doc (86KB, doc)
2
NIHMS432532-supplement-2.pdf (1.7MB, pdf)

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