Abstract
Recall responses by memory CD8 T cells are impaired in the absence of CD4 T cells. Although several mechanisms have been proposed, the molecular basis is still largely unknown. Using a local influenza virus infection in the respiratory tract and the lung of CD4−/− mice, we show that memory CD8 T cell impairment is limited to the lungs and the lung-draining lymph nodes, where viral Ags are unusually persistent and abundant in these mice. Persistent Ag exposure results in prolonged activation of the AKT–mTORC1 pathway in Ag-specific CD8 T cells, favoring their development into effector memory T cells at the expense of central memory T cells, and inhibition of mTORC1 by rapamycin largely corrects the impairment by promoting central memory T cell development. The findings suggest that the prolonged AKT–mTORC1 activation driven by persistent Ag is a critical mechanism underlying the impaired memory CD8 T cell development and responses in the absence of CD4 T cells.
Introduction
Long-lived memory CD8 T cells provide immune defenses against pathogens such as viruses and intracellular bacteria, and likely some cancers. The mechanisms underlying the development and function of these T cells, following infection or vaccination, has thus been extensively studied. Among the factors shown to have a significant impact on memory CD8 T development are: 1) CD4 T cell help (1–4); 2) duration of stimulation by Ag (peptide–MHC class I complexes) (5, 6); 3) mutation in the TCR β-chain transmembrane domain (7, 8); 4) activation of AKT, a serine/threonine protein kinase (9); and 5) the mechanistic target of rapamycin complex 1 (mTORC1), as indicated by the effect of rapamycin on CD8 memory development (10–12). Despite such progress, an integrated molecular and cellular mechanism that can account for the diverse contributing factors to memory CD8 T cell development and recall response remains incomplete.
Based on cell surface marker expression, anatomic localization, persistence, and function, memory CD8 T cells are often classified into two major types, central (TCM) and effector (TEM) memory T cells (13). TCM exhibit a phenotype of CD62LhiCCR7hi and reside primarily in lymphoid tissues, whereas TEM are CD62LloCCR7lo and widely dispersed in nonlymphoid tissues. The numbers of TCM are stably maintained over long periods by slow homeostatic proliferation. TEM, however, are not maintained by homeostatic proliferation and gradually decline over time. Compared to TEM, which rapidly respond to Ag restimulation to express effector functions, but proliferate poorly, TCM respond to Ag restimulation by proliferating before exhibiting effector functions (13, 14). Experimentally, recall responses of memory CD8 T cells usually measure the proliferative capacity of TCM by quantifying the number of Ag-specific T cells (14).
CD4 T cells are thought to play a critical role in the recall responses of memory CD8 T cells. Compared to wild-type (WT) mice, CD4−/− mice produce relatively normal primary CD8 T cell responses and similar numbers of memory CD8 T cells following a viral or bacterial infection (1–3), but they are deficient in recall responses. Although several mechanisms have been proposed to explain the observed defect, including the contribution of CD4 Th cells to CD8 T cell activation, expansion, contraction, memory CD8 T cell maintenance, and their response to restimulation (15, 16), the question remains unresolved. The fact that CD4−/− mice generate normal numbers of memory CD8 T cells during a primary response but do not produce significant recall responses following restimulation (17) suggests that the memory CD8 T cells in CD4−/− mice are of TEM type. However, the molecular mechanism underlying the TCM versus TEM development in the absence of CD4 T cells has not been examined.
Ag has also been found to play a critical role in the outcome of TCM versus TEM development. Following influenza A virus infection in the respiratory tract and the lung in mice, viral Ag is abundant and persists in the lung and lung-draining lymph nodes (DLN) but is scant and transient in non–draining lymph nodes (NDLN). Correspondingly, most persisting CD8 T cells in the lung and DLN exhibit the TEM phenotype, whereas those persisting in NDLN exhibit the TCM phenotype. The importance of Ag is further demonstrated by manipulating Ag persistence: prolonged exposure to Ag leads to TEM development whereas shortened exposure enhances TCM development (5). However, how Ag regulates TCM versus TEM development at the molecular level has not been elucidated.
Furthermore, studies have shown that alterations in TCR and its signaling pathway also dramatically affect TCM versus TEM development. Replacement of the most C-terminal tyrosine with leucine in the TCR β transmembrane domain has only minimal effect on TCR–CD3 interaction but impairs TCR polarization and NF-κB signal in the immunological synapse. Transgenic mice expressing the mutant TCR β-chain produce normal primary CD8 T cell responses but have an impaired development and function of memory CD8 T cells (8). Following binding to cognate peptide–MHC complexes, TCR signals through many proteins (18), including AKT kinase (9) and mTORC1 (12). Persistent activation of AKT promotes development of short-lived effector cells, but inhibits development of memory precursor cells (9). Inhibition of mTORC1 by rapamycin greatly improves the quality of memory CD8 T cells and their recall responses by increasing the number of memory precursors during the expansion phase and accelerating memory T cell development during the contraction phase (10, 11). However, whether the effect of Ag and CD4 T cells on memory CD8 T cell development is through TCR, AKT, and mTOR at the molecular level has not been determined.
We have studied memory CD8 T cell development in a localized influenza virus infection in the lung and respiratory tracts in mice and identified a critical role of Ag in regulating TEM versus TCM development (5). In this study, we show that following influenza virus infection of CD4−/− mice, the defective recall response of memory CD8 T cells is limited to the lung and DLN, where Ag is abundant and favors TEM development. In NDLN and spleen, where the Ag load is low and favors TCM development, the recall response is normal or even better. We further show that persistent Ag exposure leads to prolonged activation of the AKT pathway in CD8 T cells. Inhibition of mTORC1 by rapamycin restores TCM development and corrects the limited recall response of memory CD8 T cells in CD4−/− mice. These findings suggest that persistence of Ag in the lung and DLN of CD4−/− mice leads to a prolonged TCR engagement and persistent activation of the AKT–mTORC1 pathway, resulting in TEM development and therefore deficient recall responses of memory CD8 T cells in these tissues. Elucidation of Ag and CD4 T cells in regulating TEM versus TCM development through the AKT–mTORC1 pathway helps to develop an integrated mechanism of memory CD8 T cell development and identify possible approaches to improve memory CD8 T cell development in response to vaccination, especially in individuals with impaired or deficient CD4 T cells. Additionally, our findings suggest that CD4 T cells do not play a direct role, as several hypotheses postulated, in memory CD8 T cell development.
Materials and Methods
Mice and virus
2C TCR transgenic mice on the RAG1−/− and C57BL/6 background (2C+rag1−/−Thy1.2+) were used as donors and maintained in the animal facility at the Massachusetts Institute of Technology. These mice express the 2C TCR on CD8+ T cells specific for SIYRYYGL peptide in association with MHC class I Kb molecule (19). B6, B6 Thy1.1+, CD4−/−, and OTI+rag1−/−Thy1.1+ mice were from The Jackson Laboratory and bred in the animal facility at the Massachusetts Institute of Technology. Mice were used at 8–16 wk of age. All animal studies and procedures were approved by Massachusetts Institute of Technology’s Committee for Animal Care. Recombinant WSN-SIY virus encoding the SIY epitope in the neuraminidase stalk was constructed by plasmid-based reverse genetics and grown in Madin–Darby canine kidney cells (20).
Abs, staining, and flow cytometry
The Abs for CD62L (MEL-14), CD27 (LG. 3A10), KLRG1 (2F1/KLRG1), Thy1.1 (OX-7), Thy1.2 (53-2.1), and CD69 (H1.2F3) were from BioLegend. The Abs for pAKT308 (C31E5E) and pAKT473 (D9E) were from Cell Signaling Technology. The Abs for CD8 (53-6.7), IFN-γ (XMG1.2), and TNF-α (MP6-XT22) were from BD Biosciences. Single-cell suspensions were prepared from lung, mediastinal (draining) lymph nodes (DLN), spleens, and inguinal and mesenteric lymph nodes (NDLN). The procedures for preparing single-cell suspensions and for the cell surface staining were as described (21).
For SIY/Kb-specific CD8 T cell staining, we added SIY to DimerX-H-2Kb-PE per the manufacturer’s protocol (BD Biosciences). For the 2C TCR staining, clonotypic Ab 1B2 was used (5). For intracellular IFN-γ and TNF-α staining, cells were stimulated with SIY peptide for 4 h in the presence of GolgiPlug (BD Biosciences). The cells were then fixed and stained with labeled Abs using an intracellular staining kit (Cytofix/Cytoperm kit; BD Biosciences) according to the manufacturer’s instructions. For the intracellular pAKT308 and pAKT473 staining, the cells were fixed and stained following the description for FOXP3 Fix/Perm buffer set (BioLegend). Stained cells were analyzed on FACS LSR II HTS (BD Biosciences) flow cytometer. Data were analyzed with FlowJo software (Tree Star)
Infection, adoptive transfer, and preparation of peptide-loaded bone marrow–derived dendritic cells
For influenza virus infection, adoptive transfer, and peptide-loaded bone marrow–derived dendritic cell (BMDC) preparation, we followed the procedures described previously (5, 20). For infection, mice were anesthetized and intranasally infected with a sublethal dose (100 PFU) of WSN-SIY virus. For T cell transfer, naive 2C cells were isolated from 2C rag1−/− mice and adoptively transferred into CD4−/− and WT mice by i.v. injection (2 × 105 2C cells per recipient). For adoptive transfer of memory 2C cells or endogenous memory CD8 T cells, CD8 T cells were enriched following the protocol of an EasySep mouse CD8+ T cell isolation kit (StemCell Technologies). Then the enriched CD8 T cells containing 1 × 104 memory 2C cells or endogenous memory CD8 T cells were i.v. injected into B6 or B6 Thy1.1 mice, respectively. For preparation of peptide-loaded BMDCs, BMDCs were prepared and loaded with SIY peptide as earlier described (5) and i.v. injected into CD4−/− and WT mice (5 × 105 SIY peptide-loaded BMDCs per recipient).
Cells were isolated from lymph nodes of OTI rag1−/−Thy1.1+ mice and 2C rag1−/−Thy1.2+ mice. The lymphocytes were counted and mixed at a 1:1 ratio. The cells were incubated with peptides SIYRYYGL (SIY) or SIINEFKL (2 μg/ml) for 2 d and stained and analyzed as above by a flow cytometer.
Rapamycin treatment
Rapamycin (Sirolimus, ChemieTek) was dissolved in DMSO to 4 mg/ml as stock and then diluted in PBS to 80 μg/ml for i.p. injection at 600 μg/kg/d (10). The control (without rapamycin) mice were injected with PBS with the same concentration of DMSO.
Statistical analysis
Unpaired two-tailed t tests were performed for statistical analyses of p values. Error bars represent the SEM.
Results
Recall responses of memory CD8 T cells in various tissues in CD4−/− mice
Naive CD8 T cells expressing the 2C TCR were adoptively transferred into WT and CD4−/− mice (2 × 105 2C cells per recipient) followed by intranasal infection with a sublethal dose (100 PFU) of influenza A virus, WSN-SIY, which expresses the SIYRYYGL (SIY) epitope recognized by the 2C TCR when presented by H-2Kb (20) (Fig. 1A). At the height of the primary response, 7 d postinfection (dpi), the number of 2C cells in the lung, DLN, spleen, and NDLN was quantified by flow cytometry staining for CD8 and the 2C TCR with a clonotypic Ab 1B2. The number of 2C cells and their frequency in the lung, DLN, spleen, and NDLN were similar in WT and CD4−/− mice (Fig. 1B, Supplemental Fig. 1A, 1B). Similarly, the proportion and the numbers of 2C cells that expressed IFN-γ and TNF-α in DLN and spleen were similar in WT and CD4−/− mice (Supplemental Fig. 1B, 1D). These results suggest that CD8 T cells do not require CD4 T cells to mount a normal primary response in a local respiratory tract infection, consistent with earlier findings with systemic infections (1–3).
CD8 T cell recall defects differ in various organs of CD4−/− mice. (A) Scheme of experimental procedures. (B and C) Numbers of 2C cells quantified with a clonotypic Ab (1B2) in the indicated sites in CD4−/− and WT mice at 7 and 30 dpi, respectively. (D and E) Number in BAL fluid in recall responses of adoptively transferred 2C cells (D) and endogenous Thy1.2+ SIY/Kb-specific CD8 T cells (SIY/Kb-dimer+ cells) (E). In (D) and (E), the donor tissues are labeled. Error bars indicate SEM from three to four mice per group from one of two independent experiments. *p < 0.05.
CD8 T cell recall defects differ in various organs of CD4−/− mice. (A) Scheme of experimental procedures. (B and C) Numbers of 2C cells quantified with a clonotypic Ab (1B2) in the indicated sites in CD4−/− and WT mice at 7 and 30 dpi, respectively. (D and E) Number in BAL fluid in recall responses of adoptively transferred 2C cells (D) and endogenous Thy1.2+ SIY/Kb-specific CD8 T cells (SIY/Kb-dimer+ cells) (E). In (D) and (E), the donor tissues are labeled. Error bars indicate SEM from three to four mice per group from one of two independent experiments. *p < 0.05.
At 30 dpi, the numbers of memory 2C cells persisting in the lung and DLN were similar in WT and CD4−/− mice, but the numbers of these cells were, respectively, ∼2.9- and 2.5-fold higher in the spleen and NDLN of WT mice (Fig. 1C, Supplemental Fig. 1E). To determine the recall potential of memory 2C cells, we isolated CD8 T cells at 30 dpi from the lung, DLN, spleen, and NDLN of WT and CD4−/− mice by negative depletion and adoptively transferred 1 × 104 memory 2C cells into naive WT mice. The recipient mice were then infected intranasally with 100 PFU WSN-SIY influenza virus and 7 d later the numbers of 2C cells in the bronchoalveolar lavage (BAL), the site of virus infection, were counted as a measure of recall potential of donor memory CD8 T cells. As shown in Fig. 1D, the recall response was similar when the transferred memory 2C cells were from spleen of WT and CD4−/− mice, but when they were from the lung and DLN, those from the WT mice gave a much stronger recall response as indicated by a 7- to 8-fold more 2C cells in the BAL. Unexpectedly, however, when transferred memory 2C cells were from NDLN, those from the CD4−/− mice made a 2-fold greater response. These data suggest that the recall response of memory CD8 T cells in different tissues of CD4−/− mice can differ in respiratory tract infection than in systemic infections.
To exclude any possible artifact due to the use of adoptively transferred transgenic T cells, we examined recall responses of the endogenous memory CD8 T cells that arise in the course of influenza virus infection. In this experiment, WT and CD4−/− mice on the Thy1.2 background were infected intranasally with 100 PFU WSN-SIY virus, and 30 dpi total CD8 T cells were isolated from the lung, DLN, spleen, and NDLN and transferred into C57BL/6 recipients on the Thy1.1 background (1 × 104 SIY/Kb-specific endogenous memory CD8 T cells per recipient). The recipient mice were then infected intranasally with 100 PFU WSN-SIY virus, and 7 d later the numbers of Thy1.2+SIY/Kb-dimer+ CD8 T cells were quantified in the BAL. Consistent with the transgenic 2C cell results, similar numbers of Thy1.2+SIY/Kb-dimer+ CD8 T cells were found in the BAL when the transferred endogenous memory CD8 T cells were from spleen of WT or CD4−/− mice (Fig. 1E). When, however, the memory CD8 T cells came from the lung and DLN those from the WT mice gave a 4- to 12-fold greater response, and when transferred memory CD8 T cells were from NDLN, those from the CD4−/− mice gave an ∼6-fold greater response. Taken together, these results suggest that the effect of CD4 T cells on recall responses of memory CD8 T cells following a localized respiratory tract infection differ in different tissues. The memory CD8 cell recall response is deficient in the lung and DLN, unaltered in spleen, but surprisingly better in NDLN of CD4−/− mice.
Memory CD8 T cell development at various sites
To determine whether memory CD8 T cell development varies in different organs, we analyzed CD27hiCD62LloKLRG1lo effector memory precursors (TEMP) and CD27hiCD62LhiKLRG1lo central memory precursors (TCMP) (22–24) at 7 and 9 dpi. At 7 dpi, the percentages of 2C TEMP and TCMP cells in lung, DLN, spleen, and NDLN were similar in WT and CD4−/− mice (Fig. 2A, 2B). At 9 dpi, the percentages and absolute numbers of 2C TEMP cells in lung, DLN, spleen, and NDLN were not significantly different between WT and CD4−/− mice (Fig. 2C, 2E), probably because the high percentages of 2C TEMP masked the difference in WT and CD4−/− mice at this stage. However, the percentages and absolute numbers of 2C TCMP cells in the lung and DLN were significantly higher in WT mice than in CD4−/− mice (Fig. 2D, 2F). At 30 dpi, the percentages of 2C cells exhibiting the TCM phenotype (CD62Lhi) were significantly lower in DLN of CD4−/− than WT mice, but the percentages of 2C TCM were higher in NDLN of CD4−/− than WT mice (Fig. 2G, Supplemental Fig. 1F), consistent with greater recall responses in NDLN of CD4−/− than WT mice (Fig. 1D). The numbers of 2C TCM in DLN and spleen were significantly lower in CD4−/− mice than in B6 mice (Fig. 2H). These data are consistent with the observations that better development of TCMP and TCM is associated with better recall response (13, 14) and point to an effect of CD4 T cell deficiency on development of memory CD8 T cells.
Memory CD8 T cell development differs in WT and CD4−/− mice. The experimental procedures were the same as in Fig. 1A. (A and B) Percentage of TEMP (CD27hiCD62LloKLRG1lo) 2C cells (A) and TCMP (CD27hiCD62LhiKLRG1lo) 2C cells (B) in the indicated organs in CD4−/− and WT mice at 7 dpi. (C and D) Percentage of 2C TEMP (CD27hiCD62LloKLRG1lo) cells (C) and 2C TCMP (CD27hi CD62Lhi KLRG1lo) cells (D) in the indicated organs in CD4−/− and WT mice at 9 dpi. (E and F) Absolute numbers of 2C TEMP cells (E) and 2C TCMP cells (F) in the indicated organs in CD4−/− and WT mice at 9 dpi. (G and H) Percentage (G) and number (H) of 2C TCM (CD62Lhi) cells in the indicated organs in CD4−/− and WT mice at 30 dpi. Error bars indicate SEM from four mice per group from one of two experiments. *p < 0.05.
Memory CD8 T cell development differs in WT and CD4−/− mice. The experimental procedures were the same as in Fig. 1A. (A and B) Percentage of TEMP (CD27hiCD62LloKLRG1lo) 2C cells (A) and TCMP (CD27hiCD62LhiKLRG1lo) 2C cells (B) in the indicated organs in CD4−/− and WT mice at 7 dpi. (C and D) Percentage of 2C TEMP (CD27hiCD62LloKLRG1lo) cells (C) and 2C TCMP (CD27hi CD62Lhi KLRG1lo) cells (D) in the indicated organs in CD4−/− and WT mice at 9 dpi. (E and F) Absolute numbers of 2C TEMP cells (E) and 2C TCMP cells (F) in the indicated organs in CD4−/− and WT mice at 9 dpi. (G and H) Percentage (G) and number (H) of 2C TCM (CD62Lhi) cells in the indicated organs in CD4−/− and WT mice at 30 dpi. Error bars indicate SEM from four mice per group from one of two experiments. *p < 0.05.
Persistence of influenza virus and sustained AKT activation
As Ag abundance has been shown to affect TCM versus TEM development (5), it seemed possible that the different Ag levels in different tissues may be involved in the tissue-variable effect of CD4 T cell deficiency on memory CD8 T cell development and recall responses. To test this possibility, we quantified the duration of infectious influenza virus in BAL of WT and CD4−/− mice by plaque assay, as virus titer in BAL accurately reflects that in the lung parenchyma (25). As shown in Fig. 3A, at 5 dpi the virus level was similar in CD4−/− and WT mice, but in WT mice the virus level dropped precipitously by 7 dpi and was below detection by 8 dpi. In contrast, significant levels of virus persisted in CD4−/− mice and were detectable 10 dpi (Fig. 3A). These results show that infectious virus persists in the lungs of CD4−/− mice much longer than in WT mice, where viral Ag has been found to persist long after virus is cleared (25–27).
Comparison of virus persistence and AKT phosphorylation in WT and CD4−/− mice. (A) WT and CD4−/− mice were infected intranasally with 100 PFU WSN-SIY virus. BAL was harvested at the indicated dpi and virus titer was measured by plaque assays. (B–G) Naive 2C T cells were adoptively transferred into WT and CD4−/− mice followed by intranasal infection with WSN-SIY. At 7 and 9 dpi, cells from the lung and spleen were stained for CD8, 2C TCR, CD27, and pAKT308. Shown are CD27 versus pAKT308 staining profiles gating on CD8+ 2C TCR+ cells at 7 dpi (B) and 9 dpi (E), percentages of CD27hipAKT308+ 2C cells at 7 dpi (C) and 9 dpi (F), and MFI of pAKT308 of CD27hi 2C cells at 7 dpi (D) and 9 dpi (G). (H) Naive OTI and 2C cells were isolated from OTI rag1−/−Thy1.1+ and 2C rag1−/−Thy1.2+ mice, respectively. These cells were mixed at a 1:1 ratio and activated by either SIY or SIINFEKL peptides. Two days later, cells were stained for Thy1.1, Thy1.2, CD8 plus CD69 or pAKT308 or pAKT473. Shown are Thy1.1 versus CD69 or pAKT308 or pAKT473 staining profiles gating on CD8+ cells. Error bars indicate SEM from four mice per group in one of two experiments. *p < 0.05. ND, not detected.
Comparison of virus persistence and AKT phosphorylation in WT and CD4−/− mice. (A) WT and CD4−/− mice were infected intranasally with 100 PFU WSN-SIY virus. BAL was harvested at the indicated dpi and virus titer was measured by plaque assays. (B–G) Naive 2C T cells were adoptively transferred into WT and CD4−/− mice followed by intranasal infection with WSN-SIY. At 7 and 9 dpi, cells from the lung and spleen were stained for CD8, 2C TCR, CD27, and pAKT308. Shown are CD27 versus pAKT308 staining profiles gating on CD8+ 2C TCR+ cells at 7 dpi (B) and 9 dpi (E), percentages of CD27hipAKT308+ 2C cells at 7 dpi (C) and 9 dpi (F), and MFI of pAKT308 of CD27hi 2C cells at 7 dpi (D) and 9 dpi (G). (H) Naive OTI and 2C cells were isolated from OTI rag1−/−Thy1.1+ and 2C rag1−/−Thy1.2+ mice, respectively. These cells were mixed at a 1:1 ratio and activated by either SIY or SIINFEKL peptides. Two days later, cells were stained for Thy1.1, Thy1.2, CD8 plus CD69 or pAKT308 or pAKT473. Shown are Thy1.1 versus CD69 or pAKT308 or pAKT473 staining profiles gating on CD8+ cells. Error bars indicate SEM from four mice per group in one of two experiments. *p < 0.05. ND, not detected.
We hypothesized that persistent viral Ags may lead to persistent activation of the AKT–mTORC1 pathway and thereby affect development of memory precursors TCMP and TEMP. To determine AKT activation status we assayed phosphorylation of AKT at positions 308 (28) and 473 (29) in CD27+ 2C T memory precursors, because phosphorylation of Thr308 is required for activating AKT kinase activity to phosphorylate the downstream substrates (28, 30) and phosphorylation of Ser473 regulates AKT kinase activity (29, 30). At 7 dpi, the percentage of CD27+ 2C T cells positive for pAKT308 was higher in the lung than spleen of both CD4−/− and WT mice (Fig. 3B, 3C), although the mean fluorescence intensity (MFI) of pAKT308 was indistinguishable in the same tissues between 2C T cells from CD4−/− and WT mice (Fig. 3D). At 9 dpi, the percentage of CD27+ 2C T cells positive for pAKT308 increased significantly in the lung and spleen of both CD4−/− and WT mice (Fig. 3E, 3F). Furthermore, the MFI of pAKT308 was significantly higher in CD27+ 2C T memory precursors in the lung of CD4−/− mice than WT mice (Fig. 3G). Similarly, the percentage of CD27+ 2C T memory precursors positive for pAKT473 was higher in the lung than spleen of both CD4−/− and WT mice at both 7 and 9 dpi (Supplemental Fig. 2B–G). However, the MFI of pAKT473 did not change significantly at both 7 and 9 dpi (Supplemental Fig. 2D, 2G).
To determine whether Ag can directly activate AKT phosphorylation in the absence of CD4 T cells, naive 2C T cells (Thy1.2+) were mixed with naive OT1 T cells (Thy1.1+) at a 1:1 ratio and stimulated with either SIY peptide or SIINFEKL (recognized by the 2C or OT1 TCR, respectively). Two days later, the cells were analyzed for T cell activation marker CD69 and AKT phosphorylation. When the cell mixture was stimulated with SIY peptide, 2C T cells, but not OT1 T cells, were activated to express CD69 and phosphorylate AKT at the 308 and 473 positions (Fig. 3H, Supplemental Fig. 2H). When the cell mixture was stimulated with SIINFEKL peptide, OT1 T cells, but not 2C T cells, were activated to express CD69 and phosphorylate AKT at the 308 and 473 positions. Thus, Ag can specifically activate AKT phosphorylation in CD8 T cells in the absence of CD4 T cells.
To examine whether persistent Ag stimulation sustains AKT phosphorylation, naive OT1 T cells were stimulated with SIINFEKL (2 μg/ml) for 0, 15, 30, 60, or 120 min or 1 or 2 d. Upregulation of CD69 was rapidly induced between 30 and 120 min following stimulation (Supplemental Fig. 3A, 3B). The MFI of AKT phosphorylation at positions 308 and 473 on CD69+ OT1 T cells increased quickly within 30 min of stimulation, decreased by 90 and 120 min, and then increased by 1 and 2 d of stimulation (Supplemental Fig. 3A–D). To determine whether Ag dose affects AKT phosphorylation, naive OT1 T cells were stimulated for 1 d with SIINFEKL at concentrations from 0, 10−9, 10−8, 10−7, 10−6, 10−5, 10−4, 10−3, 10−2, 10−1, to 1 μg/ml. Upregulation of CD69 was induced in a dose-dependent manner from the concentration 10−8 to 10−3 μg/ml (Supplemental Fig. 3E, 3F). Similarly, higher Ag doses also induced higher MFI of AKT phosphorylation (Supplemental Fig. 3E, 3G, 3H). These data suggest that a persistent and higher dose of Ag stimulation induces sustained AKT phosphorylation.
Rescue of TCM development and recall responses by rapamycin
Phosphorylation of AKT at position 308 is known to lead to activation of mTORC1 (12). We reasoned that blocking the AKT–mTORC1 pathway with rapamycin should correct the impaired TCM development and CD8 T cell recall defects in CD4−/− mice. WT and CD4−/− mice were infected intranasally with WSN-SIY virus and given rapamycin (600 μg/kg) (10) daily between 8 and 37 dpi (Fig. 4A). SIY/Kb-specific endogenous CD8 T cells were quantified at 7 and 37 dpi. At 7 dpi just before starting rapamycin treatment, the numbers of SIY/Kb-specific CD8 T cells in the lung, DLN, spleen, and NDLN were similar in CD4−/− and WT mice (Supplemental Fig. 4A), as were the percentages of IFN-γ+ and TNF-α+ CD8 T cells (Supplemental Fig. 4B, 4C).
Rapamycin treatment enhances TCM development. (A) Scheme of experimental procedures. CD4−/− and WT mice were infected intranasally with 100 PFU WSN-SIY virus. The SIY/Kb-specific endogenous memory CD8 T cells in the indicated tissues were analyzed at 37 dpi by staining for CD8, CD62L, and SIY/Kb-dimer. (B–E) The percentages of CD8+CD62Lhi SIY/Kb-specific TCM among total CD8+ cells at 37 dpi in the lung (B), DLN (C), spleen (D), and NDLN (E) in CD4−/− and WT mice with and without rapamycin treatment are shown. Error bars indicate SEM from five to six mice per group from one of three representative experiments. *p < 0.05.
Rapamycin treatment enhances TCM development. (A) Scheme of experimental procedures. CD4−/− and WT mice were infected intranasally with 100 PFU WSN-SIY virus. The SIY/Kb-specific endogenous memory CD8 T cells in the indicated tissues were analyzed at 37 dpi by staining for CD8, CD62L, and SIY/Kb-dimer. (B–E) The percentages of CD8+CD62Lhi SIY/Kb-specific TCM among total CD8+ cells at 37 dpi in the lung (B), DLN (C), spleen (D), and NDLN (E) in CD4−/− and WT mice with and without rapamycin treatment are shown. Error bars indicate SEM from five to six mice per group from one of three representative experiments. *p < 0.05.
At 37 dpi, the numbers of SIY/Kb-specific CD8 T cells in various tissues were largely the same in CD4−/− and WT mice either with or without rapamycin treatment (Supplemental Fig. 4D, 4E). However, following rapamycin treatment the percentage of SIY/Kb-dimer+ TCM (CD62Lhi) in the lung and DLN of both CD4−/− and WT mice increased significantly (Fig. 4B, 4C, Supplemental Fig. 4D). However, there was no significant difference in the percentage of TCM in spleen and NDLN (Fig. 4D, 4E) of rapamycin-treated CD4−/− and WT mice.
To determine whether rapamycin inhibition of mTORC1 corrects the CD8 T cell recall defects in CD4−/− mice, we injected the rapamycin-treated mice i.v. with 5 × 105 BMDCs pulsed with SIY peptide (BMDC-SIY). The SIY/Kb-specific CD8 T cell recall responses were quantified 5 d later (Fig. 4A). Except in the lymph nodes (Fig. 5), significantly more SIY/Kb-specific CD8 T cells were recovered in the lung and spleen of rapamycin-treated CD4−/− and WT mice (Fig. 5A, 5B). More CD8 T cells were also induced to express IFN-γ in the spleen of both CD4−/− and WT mice following rapamycin treatment (Fig. 5E). Moreover, following rapamycin treatment the number of SIY/Kb-specific CD8 T cells in the lung of CD4−/− mice reached the same level as that in the lung of WT mice without rapamycin treatment (Fig. 5A). The numbers of SIY/Kb-specific CD8 T cells from the lung and spleen of rapamycin-treated WT mice were, however, still significantly higher than those of rapamycin-treated CD4−/− mice. Taken together, these results suggest that inhibition of the AKT–mTORC1 pathway by rapamycin restores TCM development and recall responses in CD4−/− mice to the levels found in WT mice not treated with rapamycin (Fig. 6).
Rapamycin treatment partially corrects CD8 T cell recall defects in CD4−/− mice. The mice were injected i.v. with BMDCs pulsed with SIY peptides (BMDC-SIY) at 37 dpi (Fig. 4A). The numbers of T cells specific for SIY/Kb and expressing IFN-γ were analyzed 5 d later. (A–E) Number of SIY/Kb-specific CD8 T cells in the lung (A), spleen (B), mediastinal lymph node (C), and inguinal lymph node (D) of CD4−/− and WT mice with or without rapamycin treatment. (E) Number of IFN-γ+ CD8 T cells in the spleen of CD4−/− and WT mice with or without rapamycin treatment. Error bars indicate SEM from five to six mice per group from one of three representative experiments. *p < 0.05.
Rapamycin treatment partially corrects CD8 T cell recall defects in CD4−/− mice. The mice were injected i.v. with BMDCs pulsed with SIY peptides (BMDC-SIY) at 37 dpi (Fig. 4A). The numbers of T cells specific for SIY/Kb and expressing IFN-γ were analyzed 5 d later. (A–E) Number of SIY/Kb-specific CD8 T cells in the lung (A), spleen (B), mediastinal lymph node (C), and inguinal lymph node (D) of CD4−/− and WT mice with or without rapamycin treatment. (E) Number of IFN-γ+ CD8 T cells in the spleen of CD4−/− and WT mice with or without rapamycin treatment. Error bars indicate SEM from five to six mice per group from one of three representative experiments. *p < 0.05.
Schematic comparison of memory CD8 T cell development and responses in WT and CD4−/− mice and how rapamycin restores memory CD8 T cell development and responses in CD4−/− mice.
Schematic comparison of memory CD8 T cell development and responses in WT and CD4−/− mice and how rapamycin restores memory CD8 T cell development and responses in CD4−/− mice.
Discussion
Various mechanisms have been proposed to explain CD4 T cell help in the development and recall response of memory CD8 T cells. Using influenza virus infection in the respiratory tract and examining memory CD8 T cells at various tissues, we show that the recall responses of memory CD8 T cells vary in different tissues. The recall responses of memory CD8 T cells from the lungs and DLN, but not spleen, are deficient in CD4−/− mice, as compared with those of WT mice. Surprisingly, memory CD8 T cells from NDLN of CD4−/− mice actually produce better recall responses than do those from WT mice. Thus, the impact of CD4 T cells on recall responses of memory CD8 T cells differs from that reported previously in CD4−/− mice following systemic infections (1–3), where recall responses of memory CD8 T cells were examined only in the spleen.
In our study, to compare the recall responses of memory CD8 T cells from different tissues, we isolated memory CD8 T cells from spleen, DLN, NDLN, and lung, adoptively transferred them into naive recipient mice, and then quantified the numbers of donor-derived influenza-specific CD8 T cells at the site of infection (BAL) at the peak of responses (7 dpi). One potential caveat is that the observed defect might be due to differential trafficking of the transferred memory CD8 T cells from the lung tissue into the airways. A recent study has shown that CXCR3 regulates memory CD8 T cell migration into the lung and BAL (31). Influenza-specific memory CD8 T cells in the lung uniformly express a high level of CXCL3, whereas those in the spleen exhibit a more heterogeneous CXCL3 expression. Transfer of memory CD8 T cells from the lung tissue would have favored their trafficking into the lung. Furthermore, following influenza virus challenge, Ag-specific CD8 T cells proliferate in the DLN, acquire effector function, and then migrate to the site of infection (lung). Any difference in trafficking of memory CD8 T cells from different tissues would be irrelevant following their reactivation, proliferation, and acquisition of effector function. Thus, trafficking is unlikely a significant factor contributing to the observed defect of memory CD8 T cells from the lung tissue.
The recall defects of memory CD8 T cells in CD4−/− mice have been attributed to various mechanisms, including 1) obligatory CD4 T cell involvement in memory CD8 T cell and APC interactions (32), 2) a critical role of IL-2, which is predominantly secreted by CD4 T cells (15), and 3) a requirement of CD4 T cells in the maintenance of memory CD8 T cells (17). Our findings suggest an alternative mechanism (Fig. 6). In the absence of CD4 T cells, clearance of influenza virus from the site of infection is delayed. The prolonged persistence of Ag in the respiratory tract and DLN (5) leads to persistent activation of the AKT–mTORC1 pathway, shifting local balance to favor TEM development at the expenses of TCM. Because TEM do not proliferate significantly upon restimulation (14), the recall responses of memory CD8 T cells, as measured by the numbers of Ag-specific T cells, are therefore limited.
Supporting this view, we show that influenza virus was detected for at least 3 more days in BAL of virus-infected CD4−/− mice than in similarly infected WT mice (Fig. 3A), consistent with previous observations showing a critical role of CD4 T cells in immune responses against various infections, including influenza viruses (16, 26). Note that CD4−/− mice have significant numbers of CD8 T cells that are restricted by MHC class II (33). However, these CD8 T cells do not substitute for CD4 T cell function in response to influenza virus infection. Consistently, MHC class II−/− mice also exhibit a defect in recall response of memory CD8 T cells as do the CD4−/− mice (2). We have shown previously that prolonged Ag exposure drives activated CD8 T cells to undergo further proliferation and differentiation into TEM (5). Consistently, in the lungs and DLN of CD4−/− mice, where viral Ag is abundant and persistent, the percentages of TCMP and TCM decreased significantly (Fig. 2), correlating with poor recall responses of memory CD8 T cells in these tissues (Fig. 1D, 1E). In contrast, in spleen and NDLN in CD4−/− mice, where viral Ag is scant, the percentages of TCMP and TCM are normal or elevated, correlating with normal or enhanced recall responses, respectively. These results suggest that the effect of CD4 T cells on memory CD8 T cell development and recall responses is likely indirect. It is the persistence of Ag, as a result of CD4 T cell deficiency, that alters memory CD8 T cell development and recall responses.
Our study has now directly addressed how Ag regulates TCM versus TEM development at the molecular level. We show that Ag stimulation of CD8 T cells in vitro leads to phosphorylation of AKT in the absence of CD4 T cells (Fig. 3H). More importantly, the persistence of viral Ag in CD4−/− mice also leads to a prolonged activation of AKT as indicated by the elevated levels of AKT phosphorylation in the lungs of CD4−/− mice (Fig. 3E–G). Prolonged activation of AKT is known to promote development of short-lived effector cells, but to inhibit development of memory precursors (9). Our results showing the impaired TCMP and TCM development and therefore the impaired memory responses in the lung and DLN of CD4−/− mice are consistent with this previous report. Furthermore, AKT exerts its effect partly through the mTORC1 pathway, the inhibition of which has been shown to improve the quality of memory CD8 T cells and their recall responses (10, 11). Similarly, inhibition of mTORC1 by rapamycin restores the CD8 T cell recall responses in the lung of CD4−/− mice to the same level as in the lung of the WT mice without rapamycin treatment (Fig. 5). Rapamycin has been shown to enhance TCM differentiation through several mechanisms independent of AKT. Although we could not exclude these mechanisms, our results are most consistent with the interpretation that Ag promotes TEM development at the expense of TCM through the prolonged activation of the AKT–mTORC1 pathway.
Modulation of TCM versus TEM development in different tissues by Ag may help to minimize the adverse effect associated with the great expansion of Ag-specific effector T cells during a recall response. For example, following respiratory influenza virus infection, TEM development is favored in the lung and DLN whereas TCM development predominates in the spleen and NDLN. With a subsequent influenza infection the expansion of memory CD8 T cells is limited in the lung and DLN and therefore avoids the potential massive destruction of the lung parenchyma. Alternatively, TCM in the spleen and NDLN can proliferate and help to eradicate the acute infection in the respiratory tract and the lung.
Our study also sheds light on the effect of rapamycin in reducing secondary cancers. Chronic immunosuppression by rapamycin in recipients of organ transplants, such as kidney allografts, is associated with a reduced frequency of secondary cancers, especially squamous cell and skin cancers (34). Chronic exposure to tumor Ag is known to favor effector CD8 T cell development and eventual exhaustion (35). The benefit of rapamycin treatment may be through promoting the development of tumor-specific TCM, which inhibit tumor development by mounting robust recall responses to newly emerged tumor cells. Similarly, rapamycin has been shown to restrict HIV and hepatitis C virus replication in AIDS patients (36). Again, rapamycin may promote TCM development following chronic exposure to HIV and hepatitis C virus Ags and therefore help to suppress viral replication.
Acknowledgements
We dedicate this paper to the memory of Herman N. Eisen, who died while working on the final draft of the manuscript. His passion for science, wisdom, and thoughtfulness in life will continue to inspire all of us. We thank Ling Wang, Glenn Paradis, and the Flow Cytometry Core facility of the Koch Institute Swanson Biotechnology Center at the Massacusetts Institute of Technology for technical support.
Footnotes
This work was supported in part by National Institutes of Health Grant AI69208, the National Research Foundation Singapore through the Singapore–MIT Alliance for Research and Technology’s Interdisciplinary Research Group in Infectious Disease research program, the Ivan R. Cottrell Professorship and Research Fund, and Koch Institute Support Grant P30-CA14051 from the National Cancer Institute.
The online version of this article contains supplemental material.
Abbreviations used in this article:
- BAL
bronchoalveolar lavage
- BMDC
bone marrow–derived dendritic cell
- DLN
lung-draining lymph node
- dpi
day postinfection
- MFI
mean fluorescence intensity
- mTORC1
mechanistic target of rapamycin complex 1
- NDLN
non–draining lymph node
- TCM
central memory T cell
- TCMP
central memory precursor
- TEM
effector memory T cell
- TEMP
effector memory precursor
- WT
wild-type.
References
Disclosures
The authors have no financial conflicts of interest.