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The Functional Heterogeneity of Type 1 Effector T Cells in Response to Infection Is Related to the Potential for IFN- Production¹

Katrin D. Mayer^*, Katja Mohrs^*, Sherry R. Crowe^*, Lawrence L. Johnson^*, Paul Rhyne, David L. Woodland^* and Markus Mohrs^2,*

^* Trudeau Institute, Saranac Lake, NY 12983; and Upstate Biotechnology, Lake Placid, NY 12946

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The expression of IFN- is a hallmark of Th1 cells and CD8⁺ effector T cells and is the signature cytokine of type 1 responses. However, it is not known whether T cells are homogeneous in their capacity to produce IFN-, whether this potential varies between tissues, and how it relates to the production of other effector molecules. In the present study we used bicistronic IFN--enhanced yellow fluorescent protein (IFN--eYFP) reporter mice (Yeti) and MHC class I tetramers to directly quantify IFN- expression at the single cell level. The eYFP fluorescence of Th1 cells and CD8⁺ effector T cells was broadly heterogeneous even before cell division and correlated with both the abundance of IFN- transcripts and the secretion of IFN- upon stimulation. CD4⁺ and CD8⁺ T cells of influenza-infected mice revealed a similarly heterogeneous IFN- expression, and eYFP^high cells were only found in the infected lung. Ag-specific T cells were in all examined tissues eYFP⁺, but also heterogeneous in their reporter fluorescence, and eYFP^high cells were also restricted to the infected lung. A similar heterogeneity was observed in Toxoplasma gondii-infected animals, but eYFP^high cells were restricted to different tissues. Highly eYFP fluorescent cells produced elevated levels of proinflammatory cytokines and chemokines in addition to IFN-, suggesting their coregulated expression as a functional unit in highly differentiated effector T cells.

	Introduction

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The expression of IFN- is a hallmark of Th1-polarized CD4⁺ T cells and effector CD8⁺ T cells and is the signature cytokine of type 1 responses induced by infection with intracellular bacteria, protozoa, and viruses (1, 2). In fact, IFN- was originally described as a soluble factor protecting cells from viral infection (2). Mice deficient for IFN- (3), its receptor (4), or the downstream Stat-1 (5, 6) have severely impaired type 1 immunity. CD4⁺ and CD8⁺ T cells are major sources of IFN- during adaptive immune responses (2), and recent data suggest that Th1 cells express IFN- monoallelically (7). The diverse biological functions of IFN- include the up-regulation of Ag processing and presentation by MHC classes I and II, the induction of class switch recombination in B cells, the priming of macrophages for NO production, the induction of immunomodulatory and proinflammatory cytokines, as well as the recruitment and death of activated T cells (2, 8).

Infection with the respiratory influenza virus or the protozoan parasite Toxoplasma gondii elicits a canonical type 1 response, with the production of IFN- as a signature cytokine (9, 10, 11, 12, 13). Numerous studies have analyzed the cellular response to infection, and the production of IFN- in response to in vitro peptide stimulation has been used to indirectly identify Ag-specific cells (9, 10, 14). The development of Ag-specific MHC class I tetramer reagents has contributed substantially to our understanding of the cellular response (15, 16). Despite the clear association of IFN- with these infectious disease models, none of the studies was tailored to specifically analyze the cells that elaborate the cytokine response. Thus, we know little about the phenotype of IFN--expressing cells in vivo; whether these cells have a homogeneous potential to produce IFN-, other cytokines, or chemokines; and whether these parameters vary with their anatomical locations. Commonly used methods to identify IFN- expression at the single cell level require in vitro restimulation, thereby altering the in situ phenotype (17, 18, 19). Recently generated bicistronic IFN- reporter mice (IFN--enhanced yellow fluorescent (IFN--eYFP)³ protein reporter mice (Yeti)) were specifically designed to overcome these limitations and allow the noninvasive detection and isolation of live, cytokine-expressing cells (20, 21). Yeti mice were generated by the targeted integration of an internal ribosomal entry site linked to an eYFP-encoding sequence into the 3'-untranslated region of the Ifn gene, thereby linking IFN- expression to eYFP fluorescence in any cell (20, 21). Using Yeti mice, we previously observed a substantial heterogeneity in the level of eYFP fluorescence in CD4⁺ T cells (20, 21).

In the present study we used Yeti mice (20, 21) in combination with MHC class I tetramers (15, 16) to visualize, phenotype, and functionally characterize IFN--expressing cells in various tissues in vitro and in response to infection with the respiratory influenza virus and the protozoan parasite T. gondii.

	Materials and Methods

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Mice and viral infections

Bicistronic IFN- reporter (Yeti) mice (20) were backcrossed to C57BL/6 for a minimum of five generations. All Yeti mice were heterozygous for the bicistronic reporter knockin, and wild-type (wt) littermates were used as controls. Experimental animals were kept under specific pathogen-free conditions in filter-top cages at the animal facility of Trudeau Institute. Mice, 8–12 wk of age, were anesthetized with 2,2,2-tribromoethanol i.p. and infected intranasally (i.n.) with 300 50% egg infectious doses of the A/HK-x31 (x31, H3N2) (22) influenza A virus in 30 µl of PBS. The T. gondii strain ME49 was obtained from brains of chronically infected mice, and infections were initiated by oral gavage of 10 cysts in 0.1 ml of diluted brain suspension (23). All experimental procedures involving mice were approved by the institutional animal care and use committee at Trudeau Institute.

Tissue sampling and preparation

Peripheral blood was collected into heparin before pleural exudates cells were isolated by lavage through the diaphragm. Next, bronchoalveolar lavage (BAL) cells were collected by five consecutive washes with 1 ml each. Mice were perfused through the heart after the portal vein was cut for drainage. Perfused lung tissue was dispersed by passage through a 70-µm pore size cell strainer and subsequently digested with collagenase IV (100 U/ml; Sigma-Aldrich) and DNase I (10 U/ml; Sigma-Aldrich). Lymphocytes were enriched in the interphase of a discontinuous 60%/40% Percoll (Amersham Biosciences) gradient spun at 1200 x g for 20 min at room temperature. Single cell suspensions were prepared from spleen and mediastinal lymph nodes (medLN) by mechanical disruption through a 70-µm pore size cell strainer. Erythrocytes were removed from blood and spleen by ammonium chloride lysis. Adherent cells were depleted from BAL, pleural exudates cells, and spleen by incubation in complete RPMI 1640 (cRPMI; 10% heat-inactivated FCS, 50 µM 2-ME, 2 mM L-glutamine, and 100 U/ml penicillin/streptomycin) for 2 h in tissue culture dishes at 37°C in 5% CO₂.

Flow cytometry

mAbs were purchased from Caltag Laboratories, eBioscience, or BD Biosciences, if not stated otherwise, and clones are noted in parentheses. Single cell suspensions were kept on ice at all times and were stained in 1% BSA/0.1% azide in PBS (FACS buffer). All samples were first incubated with anti-CD16/32 (2.4G2) to block nonspecific binding of Abs to FcRIII/II. mAbs were directly conjugated to PE, PerCP, allophycocyanin, or tandem conjugates of PE-Texas Red, PE-Cy7, and allophycocyanin-Cy7. The following mAbs were used: CD3 (145-2C11), CD4 (RM4-5), CD8(CT-CD8), CD11a (M17/4), CD25 (PC61 5.3), CD44 (IM7), CD62L (MEL-14), CD69 (H1.2F3), CD122 (TM-1), and NK1.1 (PK136). MHC class I peptide tetramers specific for nucleoprotein NP_366–374/D^b and the acid polymerase protein PA_224–233/D^b were generated by the Molecular Biology Core facility at Trudeau Institute according to previously described methods (22, 24). Tetramer staining was performed for 1 h at room temperature before staining of surface Ags. Dead cells were identified by addition of 4',6-diamidino-2-phenylindole (0.1 µg/ml; Sigma-Aldrich) or propidium iodide (3 µg/ml; Sigma-Aldrich).

Four-color samples were acquired on a FACSCalibur (BD Biosciences) cytometer with CellQuest software. Samples stained with tandem-conjugated fluorochromes were acquired on a CyAn (DakoCytomation) flow cytometer equipped with Summit 3.3 software (DakoCytomation).

Data were analyzed using FlowJo (TreeStar) software. Electronic compensation matrices for data acquired on the CyAn cytometer were calculated and verified using the FlowJo compensation platform based on proper single-stain controls. All analyses were gated on lymphocytes within a live (propidium iodide^– or 4',6-diamidino-2-phenylindole^–) gate.

Cell sorting

For in vitro priming, CD8⁺ or CD4⁺ T cells were purified from the lymph nodes of naive mice using negative selection with magnetic microbeads according to the manufacturer’s instructions (Miltenyi Biotec). The cultured CD8⁺ and CD4⁺ cells were sorted into eYFP^neg, eYFP^int, and eYFP^high populations on days 1 and 5, respectively.

On day 9 after influenza infection, T cells from BAL, lung, and medLN from 12–15 mice were prepared as described using buffers without azide. Samples were stained with CD4-PE, CD8-PE-Cy5, and CD62L-allophycocyanin and sorted on a FACSVantage flow cytometer equipped with DiVa electronics. MedLN were sorted into eYFP^neg or eYFP^int populations within a CD62L^low, CD4⁺, or CD8⁺ lymphocyte gate. Pooled BAL/lung samples were first sorted into total CD8⁺ cells and CD62L^lowCD4⁺ cells with eYFP^neg, eYFP^int, and eYFP^high phenotypes. CD8⁺ cells were subsequently resorted and separated according to eYFP fluorescence within a CD62L^low gate.

Cell culture

Purified T cells (1 x 10⁶/ml) were primed as previously described (25) in cRPMI with anti-CD3 (145-2C11; 2 µg/ml) and anti-CD28 (37.51; 5 µg/ml) in the presence of IL-2 (5 ng/ml) and irradiated (3000 rad) APC (5 x 10⁶/ml). Cultures were supplemented with IL-12 (5 ng/ml) and anti-IL-4 (11B11; 20 µg/ml) or IL-4 (50 ng/ml) and anti-IFN- (XMG1.2; 20 µg/ml) where indicated. Some cells were labeled with the vital red fluorescent dye SNARF-1 (Molecular Probes).

T cells sorted from influenza-infected animals were cultured at 5 x 10⁵/ml in flat-bottom, 96-well plates for 24 h in cRPMI at 37°C in 5% CO₂. Cells were activated by plate-bound anti-CD3 (10 µg/ml) where indicated.

IFN- secretion assay

Splenocytes from day 9 influenza-infected mice were cultured at 5 x 10⁶/ml in the presence or the absence of PMA (50 ng/ml; Sigma-Aldrich) and ionomycin (500 ng/ml; Sigma-Aldrich) in cRPMI for 4 h. The IFN- secretion assay was performed according to the manufacturer’s instructions (Miltenyi Biotec). A high control (26) was included by the addition of mouse rIFN- (100 ng/ml; eBioscience), and the staining specificity was confirmed using a PE-labeled rat IgG1 isotype control (R3-34).

Cytokine and chemokine quantification

For RT-PCR, RNA was extracted using the RNAqueous-4PCR kit (Ambion) and reverse transcribed with the SuperScript II RNase H^– kit (Invitrogen Life Technologies) using oligo(dT)₁₈ priming. Quantitative real-time RT-PCR was performed using gene-specific primers and probes (23) and the ABI PRISM 7700 Sequence BioDetector (Applied Biosystems) according to the manufacturer’s instructions (TaqMan; PerkinElmer). Threshold cycle values for GAPDH were routinely between 15 and 18 cycles, and normalization to ₂-microglobulin gave similar results.

Cytokines and chemokines in culture supernatants were quantified using a multiplex flow cytometric suspension microbead array (27) according to the manufacturer’s instructions (Beadlyte MultiCytokine/Chemokine Flex-Kit; Upstate Biotechnology). The following mouse Beadmates were combined in a multiplex analysis: IL-2, IFN-, GM-CSF, TNF-, RANTES (CCL5), stromal cell-derived factor (SDF) (CXCL12), and MIP-1 (CCL4). The median fluorescence intensity (MFI) was measured from at least 50 beads/set using the Luminex xMAP system. The concentrations of cytokines and chemokines were determined using a five-parameter curve-fit generated from the MFIs of the respective mouse cytokine and chemokine standards.

Statistical analysis

Statistical analysis was performed using PRISM 3.0c (GraphPad). Asterisks indicate statistical differences with p < 0.05, by Student’s t test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Heterogeneous IFN- expression of CD4⁺ and CD8⁺ T in vitro

To analyze the expression of IFN- during the activation of T cells, CD4⁺ and CD8⁺ T cells were purified from naive Yeti mice or wt littermates and stimulated with anti-CD3 and anti-CD28 in the presence of irradiated APC. The cultures were supplemented with IL-12 plus anti-IL-4 (Th1/CTL producing type-1 cytokines (Tc1) conditions) or IL-4 plus anti-IFN- (Th2/CTL producing type-2 cytokines (Tc2) conditions) as indicated. To assess cell division, some cells were labeled with the vital red-fluorescent dye SNARF-1 and cultured under the same conditions. As shown in Fig. 1A, CD8⁺ T cells expressed high levels of eYFP within 24 h of priming, although they had not yet divided. The eYFP fluorescence was broadly heterogeneous, and Tc1 conditions clearly increased IFN- expression, whereas Tc2 conditions reduced the frequency and brightness. Both effects were more apparent after 3 days of culture. In contrast to CD8⁺ T cells, CD4⁺ T cells required 2 days of priming to express IFN- and were only induced to do so under Th1 conditions (Fig. 2B). Although one cell division was apparent, even undivided cells were eYFP⁺, indicating that the time in culture, rather than cytokines, is the limiting factor for IFN- expression. Heterogeneous eYFP expression was maintained in Th1 cultures over the 5-day culture period. To confirm that eYFP fluorescence correlates with the expression of IFN-, we sorted CD8⁺ and CD4⁺ T cells into eYFP^neg, eYFP^int, and eYFP^high populations and determined the abundance of IFN- transcript by real-time RT-PCR. The eYFP brightness of both CD8⁺ (Fig. 1C) and CD4⁺ (Fig. 1D) T cells correlated directly with the abundance of IFN- transcripts of the respective population. Together, these data show that IFN- expression by CD4⁺ and CD8⁺ is broadly heterogeneous and can be directly quantified by the bicistronic eYFP reporter.

View larger version (34K): [in this window] [in a new window]   FIGURE 1. Heterogeneous fluorescence of the bicistronic IFN--eYFP reporter correlates with IFN- expression. CD8+ (A) or CD4+ (B) T cells were purified from the lymph nodes of naive Yeti mice (gray histograms) or wt littermate controls (bold line) and stimulated with anti-CD3 and anti-CD28 in the absence or the presence of polarizing cytokine conditions. Some cells were labeled before culture with the vital red-fluorescent dye SNARF-1. The cells were analyzed on the indicated days by FACS for eYFP expression and SNARF-1 fluorescence. Cultures of CD8+ (C) T cells cultured under neutral conditions and CD4+ (D) T cells cultured under Th1 conditions were sorted on days 1 and 5, respectively, into eYFPneg (open histogram and bar), eYFPint (gray histogram and bar), and eYFPhigh (black histogram and bar) populations (top panels). The abundance of IFN- transcripts in the sorted populations was determined by real-time RT-PCR and is depicted relative to the eYFPneg population. The frequency and MFI of the eYFP+ cells are noted in the histograms. All data are representative of two or more independent experiments.

View larger version (85K): [in this window] [in a new window]   FIGURE 2. Expression of the bicistronic IFN--eYFP reporter in naive and influenza-infected Yeti mice. A, Indicated organs from naive Yeti mice were analyzed by FACS for eYFP fluorescence of CD4+ and CD8+ T cells. B, Yeti mice were infected i.n. with influenza virus, and FACS was performed 9 days later together with the analysis of naive mice in A. C, Yeti mice and wt littermate controls were infected as described in B, and CD8+-gated T cells from the indicated organs were analyzed by flow cytometry for the expression of eYFP and NP366–374 tetramer staining. The vertical dashed line demarcates eYFPint and eYFPhigh cells. Data are representative of multiple independent experiments. In some of these experiments, three mice per cohort were analyzed with comparable results.

Expression of IFN- in influenza virus-infected Yeti mice is broadly heterogeneous, and highly fluorescent cells are restricted to the infected lung

To investigate the expression of IFN- during infection, we analyzed the response to i.n. challenge with influenza virus. Yeti mice and wt littermates were infected i.n. with influenza virus and analyzed 9 days later at the peak of the cellular response by FACS for eYFP fluorescence. Consistent with published data, noninfected Yeti controls revealed only a background of eYFP fluorescence in all examined tissues (Fig. 2A) (20, 21). The background fluorescence intensity was low, and the number of eYFP⁺ cells in BAL and lung before infection was very small (Fig. 2A and data not shown). Upon infection, the number and frequency of eYFP⁺ cells increased substantially, mainly in BAL (>100-fold), lung (>6-fold), and pleural cavity (>7-fold; Fig. 2B and data not shown). Essentially all eYFP⁺ cells from all organs were contained within a forward/side scatter lymphocyte gate and were identified as CD4⁺ or CD8⁺ T cells (Fig. 2B and data not shown). T lymphocytes in the lung parenchyma and airways were almost exclusively eYFP⁺ (Fig. 2B), although only one Ifn allele was marked by the bicistronic reporter. In contrast, the frequency of eYFP⁺ T lymphocytes remained low in all secondary lymphoid organs, including the draining medLN, and in other peripheral tissues besides lung (Fig. 2B and data not shown).

The eYFP fluorescence intensity of eYFP⁺ CD4⁺ and CD8⁺ T cells was remarkably heterogeneous in all examined tissues, and highly fluorescent cells (eYFP^high) were present only in the lung airways and parenchyma of infected mice (Fig. 2B). Even the pleural cavity, which harbors the infected lung and had a similar increase and frequency of eYFP⁺ cell as the lung, did not contain cells of comparable brightness.

We next used NP_366–374 and PA_224–233 MHC class I tetramers to analyze IFN- expression in Ag-specific cells (15, 16). Both NP_366–374- and PA_224–233-specific CD8⁺ T cells disseminated into all examined tissues and were almost exclusively eYFP⁺, but, nonetheless, were highly heterogeneous in their fluorescence intensity (Fig. 2C and data not shown). Moreover, highly fluorescent Ag-specific cells were also only present in the lung airways and parenchyma. The fluorescence heterogeneity and anatomical restriction of eYFP^high cells were observed in both NP_366–374- and PA_224–233-specific cells and therefore are not due to differences in the Ag specificity of IFN--expressing cells (data not shown). The frequency of NP_366–374- or PA_224–233-specific cells was comparable between Yeti and wt littermate controls in all organs, demonstrating that insertion of the bicistronic reporter does not affect the cellular response to infection with the influenza virus (Fig. 2C).

eYFP fluorescence correlates directly with the expression of acute activation markers

Next, we investigated whether heterogeneous eYFP fluorescence intensity correlates with the expression of activation markers. Conventional methods to identify cytokine-producing cells at the single cell level require in vitro restimulation (17, 18), which alters the expression patterns of surface Ags and prevents their assessment ex vivo (28, 29). In contrast, bicistronic cytokine reporter mice allow direct phenotypic analysis of cytokine-expressing cells ex vivo (20, 25, 30, 31). Yeti mice were infected with influenza virus and analyzed 9 days later by FACS. T lymphocytes with a naive phenotype (CD62L^high, CD44^low, CD45RB^high, and CD11a^low) were eYFP-negative (Fig. 3A and data not shown). Conversely, eYFP⁺ CD4⁺ and CD8⁺ T cells were CD62L^low, CD44^high, CD45RB^low, and CD11a^high, consistent with an activated/memory phenotype. The activated/memory phenotype of eYFP⁺ cells was displayed on lymphocytes derived from either secondary lymphoid tissues or peripheral sites and was independent of eYFP brightness. In contrast, the surface expression of acute activation markers such as CD69, CD25, and CD122 on eYFP⁺ CD4⁺ and CD8⁺ T cells was clearly heterogeneous (Fig. 3B and data not shown). The expression of these markers on CD4⁺ and CD8⁺ T cells correlated positively with eYFP fluorescence. For example, eYFP^high cells in the lung expressed higher levels of CD69 on the surface than eYFP^int or eYFP^neg cells, suggesting a higher level of acute activation.

View larger version (49K): [in this window] [in a new window]   FIGURE 3. Surface phenotype of T lymphocytes in correlation with eYFP fluorescence. Yeti mice were infected i.n. with influenza virus. BAL and medLN were analyzed by flow cytometry 9 days later. The depicted dot plots were gated on CD4+ or CD8+ cells. Shown are eYFP fluorescence vs CD44 (A) or CD69 (B). The depicted plots are representative of three individual mice. Similar results were obtained in three independent experiments.

IFN- production correlates with eYFP fluorescence, but is only induced after stimulation

As described above, we observed remarkable fluorescence heterogeneity in eYFP⁺ CD4⁺ and CD8⁺ T cells (Figs. 1–3). We next asked whether the brightness of reporter expression correlated with the secretion of IFN- protein. Yeti mice and wt controls were infected, and the secretion of IFN- was analyzed 9 days later by cytokine secretion assay (26). As shown in Fig. 4A, the vast majority of eYFP⁺, but not eYFP^–, cells secreted IFN- upon short-term stimulation. The secretion of IFN- by CD4⁺, total CD8⁺, and Ag-specific CD8⁺ T cells correlated directly with the eYFP fluorescence. In fact, eYFP^high cells secreted the maximum amount of IFN- that can be measured by this assay, as determined by the addition of rIFN- as a High Control (26) (data not shown). However, the frequency of IFN--secreting cells was very low when the same cells were cultured in the absence of stimulation with plate-bound anti-CD3 despite robust eYFP fluorescence. Similar results were obtained by intracellular cytokine staining and Ag-specific stimulation with the NP_366–374 peptide (data not shown).

View larger version (34K): [in this window] [in a new window]   FIGURE 4. Ex vivo IFN- production by T cells in correlation with eYFP fluorescence. Yeti and wt mice were infected i.n. with influenza virus and were analyzed 9 days later. A, Splenocytes were cultured for 4 h in the presence or the absence of PMA plus ionomycin and were analyzed by IFN- secretion assay. The depicted dot plots were gated on CD4+, total CD8+, or PA224–233-specific T cells. The spleens from three mice per group were pooled. Similar results were obtained in two independent experiments. B, Single cell suspensions were prepared from lungs (BAL+lung) and medLN. CD4+ and CD8+ T lymphocytes with a CD62Llow phenotype were separated by cell sorting according to fluorescence intensity into eYFPneg, eYFPint, and eYFPhigh populations as defined in Fig. 2 by the quadrant and the vertical dashed line. Purified populations were cultured in the absence (–) or the presence (-CD3) of plate-bound anti-CD3. Culture supernatants were analyzed for IFN- using a cytokine bead array. Depicted are the mean ± SD. Asterisks indicate statistically significant differences (p <0.05, by Student’s t test). Certain cell populations were not obtained in sufficient numbers, and IFN- production could not be determined (ND). Similar results were obtained in two independent experiments.

eYFP fluorescence correlates directly with the production of additional effector cytokines and chemokines

We speculated that increased reporter fluorescence might correlate not only with the secretion of IFN-, but also with the enhanced secretion of additional effector cytokines and chemokines. To test this hypothesis, we analyzed the culture supernatants from cells sorted by different levels of eYFP fluorescence, as described in Fig. 4B for additional effector cytokines and chemokines. Indeed, the secretion of the type 1 effector cytokines, TNF- and GM-CSF (Fig. 5A), and the proinflammatory chemokines, CCL5 (RANTES) and CCL4 (MIP-1; Fig. 5B), was largely restricted to eYFP⁺ cells and correlated positively with the eYFP brightness of CD4⁺ and CD8⁺ from medLN or BAL/lung. In contrast, the secretion of IL-2 (Fig. 5A) and the chemokine CXCL12 (SDF-1; Fig. 5B) was not restricted to eYFP⁺ cells and did not correlate with eYFP fluorescence intensity. As seen for IFN-, tissue-derived CD4⁺ T lymphocytes secreted greater amounts of these soluble effector molecules than cells from the lymph node. In fact, among the CD4⁺ T population, GM-CSF was only detectable in the supernatants of stimulated cells derived from BAL/lung. As seen for the secretion of IFN-, the secretion of these cytokines and chemokines was also largely dependent on activation of the CD3 complex.

View larger version (53K): [in this window] [in a new window]   FIGURE 5. Ex vivo cytokine and chemokine production by T lymphocytes. Yeti mice were infected with influenza virus, and the indicated populations were isolated 9 days later as described in Fig. 4. Subsequently, eYFPneg, eYFPint, and eYFPhigh populations were cultured in the absence (–) or the presence (-CD3) of plate-bound anti-CD3. Culture supernatants were analyzed for the cytokines TNF-, GM-CSF, and IL-2 (A) and the chemokines RANTES/CCL5, MIP-1/CCL4, and SDF-1/CXCL12 (B) using a cytokine bead array. Depicted are the mean ± SD. Detection limits for the cytokines or chemokines are indicated in the respective graphs. Asterisks indicate statistically significant differences (p < 0.05, by Student’s t test). Certain cell populations were not obtained in sufficient numbers, and protein production could not be determined (ND). Similar results were obtained in two independent experiments.

Anatomical restriction of eYFP^high cells, not their heterogeneity, depends on the pathogen

Finally we wanted to study whether the heterogeneity of eYFP fluorescence or the anatomical restriction of eYFP^high cells is specific for a given pathogen. To this end, Yeti mice were infected orally with the protozoan parasite T. gondii, and various organs were analyzed 1 wk later by FACS (Fig. 6). T. gondii-infected mice revealed a similar heterogeneity of eYFP fluorescence of CD4⁺ and CD8⁺ T cells as influenza-infected animals. In contrast to influenza-infected mice, however, eYFP^high cells were restricted to other organs, such as mesLN or blood. In fact, blood-borne CD4⁺ T cells from T. gondii-infected animals had substantially higher MFI_eYFP values than cells in any organ of influenza virus-infected animals (Figs. 2 and 6). Consistent with this observation, T. gondii-infected mice have high serum IFN- concentrations during acute infection (13, 23) (data not shown). Thus, the heterogeneous expression of IFN- by CD4⁺ and CD8⁺ T cells and the selective accumulation of eYFP^high cells in certain tissues are not limited to viral infection. In contrast, in which tissue highly differentiated cells accumulate is dependent on the pathogen despite the wide dissemination of eYFP⁺ cells.

View larger version (80K): [in this window] [in a new window]   FIGURE 6. Expression of the bicistronic IFN--eYFP reporter in T. gondii-infected Yeti mice. Yeti mice were infected orally with T. gondii, and CD4+ and CD8+ T cells in the indicated organs were analyzed after 1 wk for their eYFP fluorescence. The vertical dashed line demarcates eYFPint and eYFPhigh cells. Data are representative of two independent experiments with a minimum of three individual mice or three mice per group.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

The expression of IFN- was broadly heterogeneous in both CD4⁺ and CD8⁺ T cells and occurred before cell division (Fig. 1). In contrast to CD4⁺ T cells, which expressed IFN- only under Th1-polarizing conditions, CD8⁺ T cells activated the Ifn gene more rapidly and regardless of a polarizing cytokine milieu. However, the expression levels of IFN- were substantially regulated by exogenous factors, such as the presence of polarizing cytokine conditions (Fig. 1). This mechanism might be important for the local modulation of CD8⁺ effector functions in vivo. Indeed, a similar heterogeneity of IFN- expression by CD4⁺ and CD8⁺ T cells was observed after infection with the respiratory influenza virus. This heterogeneity represents a distinct functional adaptation of CD4⁺ and CD8⁺ T cells, because innate IFN- producers, such as NK and NK T cells, which are constitutively eYFP⁺ (20, 21), do not reveal a similar heterogeneity in their reporter fluorescence in naive or infected mice (K. D. Mayer and M. Mohrs, unpublished observations). Despite the systemic dissemination of T cells with heterogeneous eYFP fluorescence, eYFP^high cells were present only in the influenza-infected lung airways and parenchyma (Figs. 2 and 3). The broad spectrum of eYFP fluorescence was paralleled by the heterogeneous surface expression of acute activation markers, such as CD25, CD69, and CD122, indicating recent antigenic stimulation (28, 32, 33, 34). Consistent with this observation, the frequency of cells presenting MHC class I- and class II-restricted antigenic peptides is indeed highest in the infected lung (S. R. Crowe and D. L. Woodland, unpublished observations) (22, 35, 36). It is also conceivable that the highly inflammatory environment in the infected lung enhances IFN- expression, potentially synergizing with increased antigenic stimulation (37, 38, 39).

The fluorescence heterogeneity and anatomical restriction of eYFP^high cells are not due to differences in Ag specificity, because identical patterns were observed in NP_366–374- and PA_224–233-specific T cells (data not shown). Moreover, recruitment into tertiary sites alone does not result in high levels of eYFP expression, because eYFP^high cells were not present in the pleural cavity despite similar recruitment and frequency of Ag-specific cell as the infected lung (Fig. 2C). Because increased eYFP fluorescence correlates directly with the abundance of IFN- transcripts and the potential to secret larger amounts of IFN- (Fig. 4), the accumulation of eYFP^high cells has a striking functional significance.

The potential for IFN- secretion is tightly linked to clonal expansion of Ag-specific CD8⁺ lymphocytes, because all NP_366–374- or PA_224–233-specific T cells were eYFP⁺ in all tissues (Fig. 2C and data not shown). This observation implies that both Ifn alleles are frequently expressed, because otherwise a substantial fraction of the cells would be eYFP^neg in the heterozygous reporter knockin mice that were used throughout the experiments. Expression of the Ifn gene in CD4⁺ T cells was also biallelic, because >95% of these cells in the lung airways and parenchyma were eYFP⁺ (Figs. 1B and 3A). Thus, the bicistronic IFN- reporter marks cells competent for rapid cytokine production, whereas the secretion of this effector cytokine is dependent on antigenic simulation. In fact, the rapid on/off cycling of IFN- production by CD4⁺ and CD8⁺ T cells has been demonstrated (40, 41, 42). As we show in this study, the amount of IFN- that can be secreted upon stimulation correlates directly with the expression of the bicistronic eYFP reporter. The accessible Ifn gene locus and the constitutive presence of transcripts may allow accelerated cytokine production (43), whereas the requirement for Ag-specific stimulation may limit collateral damage. The greatly enhanced secretion of cytokines upon TCR-mediated stimulation might also be important to focus Ag-specific Th functions to MHC class II-bearing cells (44, 45, 46). The translational silencing of IFN- has recently been demonstrated in NK and NK T cells derived from wt and Yeti mice (20, 21). Moreover, CD8⁺ memory T cells have been shown to contain high levels of IFN- and RANTES (CCL5) transcripts in the absence of detectable protein production (47, 48, 49). Our present in vivo study with bicistronic reporter mice suggests that this mechanism is also operational in conventional CD4⁺ and CD8⁺ effector T cells in a situation of acute infection. However, we cannot rule out that the half-life of the fluorescent protein (50) exceeds the half-lives of IFN- transcripts.

In addition to IFN-, eYFP fluorescence of CD4⁺ and CD8⁺ T cells correlates positively with the enhanced secretion of a select set of cytokines and chemokines (Fig. 5), suggesting a generally more differentiated effector status of eYFP^high cells. The association of IFN- and TNF- with the immune response to viral infections is well established (10, 11, 12, 51). The synergistic function of GM-CSF and TNF- is supported by data showing that the combined exposure of APCs in the lung to both cytokines is required for optimal Ag presentation and Th1 priming (52, 53, 54). The chemokine receptors for the inflammatory chemokines RANTES/CCL5 and MIP-1/CCL4 (CCR1, -3, and -5) are expressed on a variety of cell types, including effector and memory T cells and macrophages (55, 56, 57). The increased secretion of soluble effector molecules was selective and therefore not simply due to a generally enhanced secretory capacity. For example, the production of IL-2 was not limited to eYFP⁺ cells, and the secreted amounts did not positively correlate with eYFP fluorescence intensity (Fig. 5A). In fact, eYFP^high CD4⁺ and CD8⁺ cells produced less IL-2 than eYFP^int cells, and reduced IL-2 production has been associated with progressive Th1 effector polarization (58, 59, 60).

Finally, oral infection with the protozoan parasite T. gondii also resulted in functional heterogeneity, and highly fluorescent cells were similarly restricted to certain tissues despite the widespread dissemination of this pathogen (Fig. 6). Thus, the functional heterogeneity and anatomical restriction of highly differentiated effector T cells are general immunological mechanisms and are not limited to viral infections of the lung. Understanding the mechanisms that result in highly differentiated effector T cells in certain tissues will be important to develop efficient vaccination strategies.

	Acknowledgments

 
We thank Pamela (Scottie) Adams and the Trudeau Institute Molecular Biology Core Facility for generation of MHC class I reagents, and Simon Monard and Brandon Sells for cell sorting. We thank Drs. Marcia Blackman, Troy Randall, Steve Smiley, and Iain Scott for critical review of the manuscript.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by National Institutes of Health Grants AI45666 (to M.M.), AI0465530 (to M.M.), ML63925 (to D.L.W.), AI55154 (to D.L.W.), and AI61587 (to L.L.J.) and funds from the Trudeau Institute.

² Address correspondence and reprint requests to Dr. Markus Mohrs, Trudeau Institute, 154 Algonquin Avenue, Saranac Lake, NY 12983. E-mail address: mmohrs{at}trudeauinstitute.org

³ Abbreviations used in this paper: eYFP, enhanced yellow fluorescent protein; BAL, bronchoalveolar lavage; cRPMI, complete RPMI 1640; i.n., intranasal; p.i., postinfection; medLN, mediastinal lymph node; MFI, median fluorescence intensity; NP, nucleoprotein; PA, acid polymerase protein; SDF, stromal cell-derived factor; wt, wild type; Tc1, CTL producing type-1 cytokines; Tc2, CTL producing type-2 cytokines.

Received for publication January 26, 2005. Accepted for publication April 12, 2005.

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