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Divisions of
*
Molecular Immunology and
Molecular Neuroendocrinology, The National Institute for Medical Research, London, United Kingdom; and
Institute for Molecular Biology and Biotechnology-Foundation for Research and Technology, Hellas, Heraklion, Greece
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have used a double-transgenic model in which monoclonal CD8 T cells are chronically exposed to self Ag in the periphery, but are not affected during thymic development. Chronic exposure of CD8 T cells to their cognate Ag rendered them unable to proliferate or produce cytokines such as IL-2 and IFN-
in response to antigenic stimulation in vitro. However, the cells still retained some killer function in vivo. Dendritic cells (DCs)2 expressing high levels of nucleoprotein (NP) were absent in double-transgenic mice, but detectable in lymphocyte-deficient Rag-/- mice expressing NP. In addition, when crossed with mice expressing Ag in the anterior pituitary gland (triple-transgenic mice) (13), F5 T cells migrated to this site and killed growth hormone producing somatotrophs. The anergic state was reversible upon transfer into Ag-free recipients, resulting in full recovery of in vitro responsiveness to Ag. Anergic CD8 T cells express higher levels of CD5, a negative regulator of T cell signaling (14), whereas after transfer and residence in Ag-free hosts, CD5 levels returned to normal, suggesting that chronic stimulation by Ag may result in induction of negative T cell regulators that prevent full functionality.
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Materials and Methods |
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Rag1-/- mice expressing a fragment encoding influenza A/NT/60/68 NP under control of the MHC class I H-2Kb promoter (termed NP47) (15) were crossed to F5 Rag1-/- mice harboring CD8 T cells with specificity for peptide 366–374 encoded by the NP transgene. The double-transgenic mice were termed NP47F5. Rag1-/- F5 mice expressing green fluorescent protein (GFP) under control of the CD2 promoter in all T cells (GFPF5) (16) were used to distinguish CD8 T cells from single-transgenic mice from those of double-transgenic mice. Syngeneic Rag1-/- H-2b mice were used as adoptive hosts. Mice expressing influenza NP in the anterior pituitary gland, termed strain 48 Rag-/- (13) were crossed to NP47F5 mice to obtain triple-transgenic mice, termed NP47F5 x 48 (all Rag-/-). All mice were bred in the animal facilities of the National Institute for Medical Research in accordance with established guidelines.
Determination of absolute CD8 T cell numbers
Absolute numbers of peripheral T cells were calculated as the number of CD8 T cells in the spleen plus two times the number of CD8 T cells in the mesenteric and inguinal lymph nodes which represent about one-half of the lymph nodes (17).
Flow cytometry and cell sorting
Cells were stained with fluorescent or biotin-labeled mAbs (all from BD PharMingen, San Diego, CA): anti-CD4PE (H129.19), anti CD8
-APC (53-6.7), anti-V
11-Bio (RR3-15), anti-CD44-PE and biotin (IM7), anti-CD62-L-biotin (MEL-14), anti-CD5-PE and FITC (53-7.3), and anti-HSA-FITC (M1/69). CyChrome 7-PE-streptavidin (Caltag Laboratories, Burlingame, CA) was used as a secondary reagent for biotin. Single-cell suspensions were preincubated with unlabeled mAb to FcRII/III (2.4G2) to minimize unspecific staining. Stainings with individual reagents were performed for 30 min on ice in FACS buffer (PBS, 2% FCS, 0.1% azide) and washed with FACS buffer. Mature CD8 single thymocytes were isolated for in vitro activation assays in two steps. First, NP47F5 or F5 thymus preparations were depleted of CD4,CD8 double-positive thymocytes using CD4 Dynabeads (Dynal, Oslo, Norway). Second, the remaining thymocytes were FACS sorted from CD4-negative-HSAlow cells using anti-CD4-PE and anti HSA-FITC mAbs. Four-color cytometry was done on a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA) and the results were analyzed with CellQuest software. Cell sorting was done on a MoFlo cell sorter (Cytomation, Fort Collins, CO).
In vitro T cells activation assays
T cell activation assays were either performed with FACS-sorted T cells or total splenocytes adjusted to the same percentage of T cells as in F5 spleens by adding Rag-/- H-2b splenocytes. A total of 1 x 103 CD8 T cells were cultured with 1 x 104 DCs, derived from BM cultures of C57B/10 mice with GM-CSF as described (18), in 96-well round bottom plates with different concentrations of NP peptide. Triplicate cultures were plated of each T cell population tested. Cells were incubated at 37°C in 5% CO2 for 48 h. Proliferation of the responding cells was measured as incorporation of [3H]thymidine, given for the last 16 h. IL-2 production was assessed on day 2 by transferring culture supernatants to fresh wells with 5 x 103 IL-2-dependent CTLL cells and proliferation was assessed by uptake of [3H]thymidine or by fluorescence measurement of the dye Alamar blue (19) (Biosource, Nivelles, Belgium) (19). IFN- production was assayed on day 2 using sandwich ELISA with anti-IFN- mAb AN-18 and biotinylated R4-6A2. All assays were set up in triplicates and data are represented as mean absorbance values with SD.
In vivo cytotoxicity assay
Target cells were prepared for in vivo evaluation of cytotoxic activity as previously described (20). Briefly, cells from C57B/10 spleen suspensions were pulsed with 1 µM NP peptide for 90 min at 37°C, washed, and labeled with CFSE (21) at a density of 107 cells/ml in a final concentration of 5 µM for 10 min. at 37°C. Uncoated control target cells were labeled at a lower CFSE concentration of 0.5 µM. For i.v. injection 5 x 106 cells of each population were mixed in 200 µl PBS. Specific in vivo cytotoxicity was determined by collecting spleen and lymph node cells from mice 19 h after injection of the target cells. Detection of the differentially labeled fluorescent target cell population was analyzed by flow cytometry. The ratio R between the numbers of uncoated vs NP coated (CFSElow/CFSEhigh) was calculated to obtain a numerical value of cytotoxicity.
In vitro cytotoxicity assay
Spleen cells from NP47F5, F5 and F5
NP47 donors (2.5 x 106/ml) were cultured for 4 days in vitro in the presence of syngeneic bone marrow culture-derived DC (2.5 x 105/ml). They were then tested for cytotoxic activity in the Jam test (22), using Con A-activated B10 spleen cells pulsed with NP peptide or unpulsed as target cells.
Generation of BM chimeras
Two-month-old GFP F5 or NP47F5 mice were treated with 1.0 Gy of ionizing radiation (60Co), and then were reconstituted with 10 x 106 BM cells from NP47F5 or GFPF5 mice, respectively. After 8 wk the degree of chimerism was analyzed by FACS for the presence of endogenous or transferred T cells that can be differentiated by the expression or not of GFP. At this time point most of the peripheral T cells were of donor origin (>95%) for the NP47F5GFPF5 chimeras and (>80%) for the GFPF5NP47F5 chimeras.
Generation of memory F5 CD8 T cells
A total of 5 x 106 T cells from F5 Rag-/- hosts were transferred with 5 x 106 influenza virus-pulsed bone marrow culture-derived DC into syngeneic Rag-/- hosts. Resting memory T cells were obtained from 4 wk after transfer.
In vitro Ag presentation assays
Isolation of splenic APCs for in vitro Ag presentation assays was performed by enzyme digestion of whole spleens with 1.6 mg/ml collagenase type IV (Sigma-Aldrich, St. Louis, MO) and 0.1% deoxyribonuclease (DNase-I, fraction IX, Sigma-Aldrich) in Iscove’s modified Dulbecco medium (Sigma-Aldrich) for 45 min. Subsequently, DCs were positively selected by AutoMACS using anti-mouse CD11-c MicroBeads (Miltenyi Biotec, Auburn, CA). The negative fraction was stained with biotinylated Ab F4/80 and macrophages were positively selected using streptavidin MicroBeads (Miltenyi Biotec). A total of 1 x 105 F5 lymph node cells per well were cultured in triplicates in round bottom 96-well plates with a mixture of CD11-c- and F4/80-positive spleen cells isolated from either NP47, NP47F5, or Rag-/- H-2b mice. After 6 days of culture, IL-2 production in the culture supernatant was assessed with an Alamar blue-based CTLL assay. At the same time point (day 6) cultured cells were stained with anti CD8-APC, CD44-BIO, CD69 FITC and CD25-PE mAbs and analyzed by FACS for the expression of activation markers on F5 CD8 T cells.
Radioimmunoassays
Mouse growth hormone in pituitary extracts was assayed by RIA, as previously described for the rat (23), using mouse reagents kindly provided by the National Institute for Diabetes and Digestive and Kidney Diseases.
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Results |
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Although the NP transgene under control of a MHC class I promoter would be expected to be expressed ubiquitously on all cells in the body, line NP47 failed to express NP in a functional manner in the thymus. As a consequence, F5 T cells specific for NP undergo thymic development in double-transgenic NP47F5 mice indistinguishable from that of normal F5 mice, as indicated by the absolute number (Table I) and percentage of single-positive CD8 T cells (Fig. 1A), their level of TCR, and low expression of the activation marker CD44 (Fig. 1B). Furthermore, purified single-positive CD8 thymocytes from double-transgenic mice proliferated in response to cognate NP peptide in vitro to the same degree as F5 CD8 single-positive thymocytes (Fig. 1C). In contrast, peripheral CD8 T cells from double-transgenic mice, while present in similar numbers as in F5 control mice (Table I), express lower levels of TCR (Fig. 1D) and have up-regulated the activation marker CD44 compared with peripheral F5 T cells (Fig. 1E). In addition, NP47F5 T cells failed to proliferate, secrete IL-2, or IFN- in response to NP peptide in vitro, resembling an anergic phenotype (Fig. 1F). Nevertheless NP47F5 T cells had comparable cytotoxic activity in vitro to F5 T cells. It should be emphasized that all mice used in this study were crossed onto a Rag1-/- background excluding any T cells of other specificities.
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To establish whether the functional impairment of F5 T cells in NP47F5 mice is the result of interactions with nonprofessional, e.g., epithelial cells or with bone-marrow-derived DCs and macrophages, bone marrow chimeras were generated. NP47F5 mice were lethally irradiated, followed by injection of bone marrow from GFPF5 mice. In the resulting chimeras Ag expression is restricted to radioresistant, non-bone marrow-derived cells. As shown in Fig. 2A (group I), in such mice GFPF5 T cells developed normally, did not up-regulate CD44, and proliferated, as well as secreted cytokines in response to NP peptide in vitro (Fig. 2B). In contrast, bone marrow chimeras in which Ag expression was restricted to bone marrow-derived cells after lethal irradiation of GFPF5 mice followed by reconstitution with bone marrow from NP47F5 (Fig. 2A, group II), showed the phenotype and impaired function (Fig. 2B) observed in NP47F5 T cells.
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The phenotype of peripheral T cells from NP47F5 mice indicated a degree of heterogeneity with some cells retaining high levels of TCR and low levels of CD44. This could be due to the continuous thymic output of naive T cells in F5 mice that do not undergo significant thymic involution (24). Recent thymic emigrants may be Ag responsive for a period of time before they are tolerized. To obtain a more homogenous population of "anergic" T cells, F5 T cells were transferred into NP47 (Rag1-/-) hosts and analyzed 20 days after transfer. At early points after transfer, F5 T cells expanded and then underwent a contraction phase (not shown), but by 20 days after transfer the number of recovered cells stabilized and they exhibited a homogenous CD44high phenotype (Fig. 3A). Their function was compromised as seen with NP47F5 T cells and likewise they exhibited cytotoxic activity in vitro (Fig. 3B). To be able to attribute functional behavior to a homogenous cell population, some of the experiments were conducted with NP47 mice that had received transfer of F5 mice at least 20 days before. These were termed F5NP47 to distinguish them from T cells isolated directly from double-transgenic NP47F5 mice.
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The results from the bone chimera experiments suggested that F5 T cells are chronically exposed to Ag presented by bone marrow-derived DCs, and macrophages which are responsible for the reduced in vitro functionality of these T cells. Nevertheless, their cytotoxic activity in vitro suggests that they might still retain some functionality. To determine whether F5 T cells chronically exposed to Ag have cytotoxic activity in vivo, a sensitive in vivo killing assay was used. Spleen cells from H-2b mice were labeled with two different concentrations of CFSE, resulting in two distinct CFSE-positive populations. One of these was pulsed with NP peptide, whereas the other one was left untreated and both populations were coinjected into either naive F5 mice (Fig. 4A, top left panel), previously immunized F5 (Fig. 4A, bottom left panel), tolerized F5NP47 (Fig. 4A, top right panel), or NP47F5 mice (Fig. 4A, bottom right panel). The ratio of CFSElow (unpulsed):CFSE high (NP pulsed) target cells was assessed 19 h after injection of the target cells. While in naive F5 recipients no killing of NP-pulsed target cells had taken place, indicated by approximately equal numbers of unlabeled:labeled targets recovered (ratio 1.5); effector cells generated in immunized F5 killed NP-pulsed targets, resulting in a much higher ratio of 10. Interestingly, tolerized F5 T cells also killed NP-pulsed targets (ratio 6.1 and 2.6) indicating that these cells still had cytotoxic capacity in vivo. Similar in vivo killing activity had been reported with anergic CD4 T cells (25).
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Additional evidence that some cytotoxic function is preserved in NP47F5 T cells came from a further cross to mice expressing NP in the anterior pituitary gland in somatotrophs that are producing growth hormone (48 strain). While F5 mice crossed with this strain develop as dwarfs due to early and excessive destruction of growth hormone producing cells (13), triple-transgenic NP47F5 x 48 mice do not develop a dwarf phenotype. This is probably due to reduced numbers of T cells with full killing activity, but the mice nevertheless have drastically reduced levels of growth hormone in their pituitary gland (Fig. 4C).
Peripheral T cells in NP47F5 mice express high levels of CD5
It is thought that the developmentally controlled expression of CD5 is important for setting the TCR response threshold for p:MHC complexes encountered during thymic development and the strength of signaling through the TCR (26, 27). More recently it has been suggested that some degree of sensory adaptation of CD5 levels is continued in the periphery during T cell interaction with p:MHC survival ligands (28). We analyzed whether CD5 levels in T cells from NP47F5 mice differ from those in F5 mice that develop in the absence of cognate Ag or memory T cells that had encountered a transient antigenic stimulus. Staining of single-positive CD8 thymocytes cells from NP47F5 and F5 mice revealed similar levels of CD5 expression. However, peripheral T cells from NP47F5 mice continued to express the high levels of CD5 found on thymocytes, whereas peripheral F5 T cells had down-regulated levels of CD5 (Fig. 5A). Fig. 5B gives a comparison of CD5 levels on naive F5 T cell, anergized NP47F5 or F5NP47 T cells and memory F5 T cells.
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NP47 T cells into Ag-free recipients restores functional activity and resets CD5 levels
It has been described previously that the "anergic’ phenotype of in vivo-tolerized T cells is dependent on continuous contact with Ag, but can be reversed upon transfer into Ag-free hosts. We transferred F5NP47 T cells as well as F5 control T cells into Ag-free syngeneic Rag-/- hosts and assessed functional activity of the T cells 3 and 6 wk after transfer. While 3 wk after transfer F5NP47 T cells still showed impaired proliferation and cytokine production compared with F5 T cells transferred into syngeneic Rag-/- hosts (Fig. 6A), functional activity was restored 6 wk after transfer (Fig. 6B). Fig. 6C shows that CD5 levels on F5NP47 T cells 2 days after transfer into Ag-free recipients are higher than those of transferred naive F5 T cells (left panel), similar to what was observed in peripheral T cells of the original mice (see Fig. 5). Little change of CD5 levels was observed after 3 wk (Fig. 6C, middle panel). However, 6 wk after transfer T cells from F5NP47 mice had down-regulated CD5 levels so that both groups expressed similar levels of CD5 (Fig. 6C, right panel).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. The residual functional activity seen in NP47F5 mice could be due to recent thymic emigrants that have not yet been tolerized, especially since F5 mice do not undergo significant thymic involution (24) and continue to export thymocytes even in old age. Nevertheless, the functional impairment of peripheral T cells is apparent early on as indicated for instance by the reduced autoimmune phenotype in triple-transgenic mice that also express Ag in the pituitary gland. Mice that contain normal F5 T cells and only express the Ag in the pituitary gland develop as dwarfs due to the presence of T cells in the pituitary gland as early as 2 wk after birth, before the onset of growth hormone-dependent growth (13). NP47F5 T cells in contrast do not influence the growth rate of mice containing NP in the pituitary gland, although they eventually destroy growth hormone producing somatotrophs as indicated by the drastically reduced levels of growth hormone found in pituitary glands of triple-transgenic mice.
Continuous presence of Ag has been shown to be a prerequisite for the maintenance of an "anergic" phenotype (29, 39, 40). In agreement with this we also find that NP specific CD8 T cells recover their function following transfer into an Ag-free environment, although recovery was not as rapid as observed in a system using transgenic CD4 T cells (1). One of the hallmarks of anergized CD8 T cells was their high expression of CD5. CD5 is a negative regulator of TCR signaling and is thought to be set during thymic development according to the avidity for peptide:MHC molecules encountered during positive selection (26, 27). In contrast, it was suggested that contact with peptide:MHC molecules in the periphery modulates expression of CD5 (28). Naive transgenic F5 T cells in peripheral lymphoid organs, like peripheral T cells from wildtype mice express lower levels of CD5 than thymocytes (27). However, peripheral CD8 T cells in NP47F5 mice fail to down-regulate CD5. Since we previously observed that peripheral CD4 T cells sorted on the basis of high or low expression of CD5 did not change their CD5 profile in response to homeostatic or antigenic signals (41), we assume that normal, transient interactions with cognate ligands do not influence CD5 levels which is also supported by low CD5 levels on memory F5 T cells equivalent to those on naive F5 T cells. In contrast, continuous contact with cognate Ag as experienced by thymic emigrants from NP47F5 mice might prevent down-regulation of CD5 to allow negative regulation of TCR signaling in the face of inescapable exposure to cognate Ag. This effect may be exacerbated by concomitant down-regulation of TCR expression (42). Increased levels of CD5 expression were also reported for anergic B cells and breeding of mice containing B cells anergized by chronic exposure to transgenic HEL Ag onto a CD5-/- background resulted in loss of the anergic phenotype (43). The assumption that high CD5 expression is related to the anergic phenotype of NP47F5 T cells is supported by the finding that CD5 levels are down-regulated following transfer into Ag-free adoptive hosts. Although these hosts were lymphopenic, therefore allowing homeostatic expansion of transferred cells, it is unlikely that this caused the changes in CD5 levels, as control F5 T cells also subjected to homeostatic expansion after transfer into Rag-/- hosts did not alter their CD5 levels. Thus, it seems that maintaining high levels of CD5 expression resulting in increased thresholds for TCR triggering could be a means to control self specific T cells and avoid overt autoreactivity in the continual presence of self Ag.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: DC, dendritic cell; NP, nucleoprotein; GFP, green fluorescent protein.
Received for publication March 10, 2003. Accepted for publication May 30, 2003.
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References |
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6+ T lymphocytes on immunizing Mls-1b mice with Mls-1a expressing cells. Nature 339:541.[Medline]
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) thymocytes after stimulation with NP peptide (C). Peripheral F5 (D, left panel) or NP47F5 (D, right panel) cells were stained for CD8, TCR, and CD44 mAbs. Percentages of CD8 populations with different levels of expression of TCR are given in the contour plot. Histogram overlays (E) compare expression of CD44 on F5 (gray filled histogram) and NP47F5 (black line) on peripheral CD8 cells. Proliferation, IL-2 and IFN-
) or RagNegbb (
) mice. On day 6 of culture, responder F5 CD8 T cells were stained for CD8, CD25, CD44 and CD69 mAbs and analyzed by FACS. At the same time point culture supernatant was measured for the amount of IL-2 secreted by the responder cells by CTLL assay. The response of F5 CD8 T cells is shown as percentage of F5 CD8 T cells positive for CD25, CD44, CD69, and IL-2 secretion. C, Pituitary growth hormone content in 5-mo-old male NP47F5 x 48 compared with NP47F5 age-matched male control mice.
