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Blood, 15 April 2004, Vol. 103, No. 8, pp. 3102-3110. Prepublished online as a Blood First Edition Paper on December 24, 2003; DOI 10.1182/blood-2003-09-3311.
IMMUNOBIOLOGY Do thymically and strictly extrathymically developing T cells generate similar immune responses?From the Guy-Bernier Research Center, Maisonneuve-Rosemont Hospital, Montreal, QC, Canada; and Institute for Experimental Immunology, University Hospital, Zürich, Switzerland.
If present in sufficient numbers, could extrathymic T cells substitute for thymus-derived T cells? To address this issue, we studied extrathymic T cells that develop in athymic mice under the influence of oncostatin M (OM). In this model, extensive T-cell development is probably due to amplification of a minor pathway of T-cell differentiation taking place only in the lymph nodes. Extrathymic CD4 T cells expanded poorly and were deficient in providing B-cell help after infection with vesicular stomatitis virus (VSV) and lymphocytic choriomeningitis virus (LCMV). Compared with classic T cells, stimulated extrathymic CD8 T cells produced copious amounts of interferon  (IFN-), and their expansion was precocious but of limited amplitude because of a high apoptosis rate. Consequently, although extrathymic cytotoxic T lymphocytes (CTLs) responded to LCMV infection, as evidenced by the expansion of GP33-41 tetramer-positive CD8 T cells, they were unable to eradicate the virus. Our data indicate that the site of development impinges on T-cell quality and function and that extrathymic T cells functionally cannot substitute for classical thymic T cells. (Blood. 2004;103:3102-3110)
One hallmark of the adaptive immune system is that the thymus has been conserved as the primary T-lymphoid organ during 450 million years of evolution.1 This level of conservation is remarkable when one considers that about 10 different organs have been used as primary sites of hematopoiesis in jawed vertebrates.1 Thymic epithelial cells (TECs) derived from the third pharyngeal pouch are the main constituent of the thymic environment.2 TECs provide 2 types of signals to thymocytes: T-cell receptor (TCR)–dependent and TCR-independent.3-5 In conjunction with mesenchymal cells, TECs have a unique ability to provide TCR-independent interactions that are essential for several thymocyte developmental events but whose nature is still elusive.2,6-9 Indeed, studies in wild-type and TCR-transgenic euthymic and nude mice indicated that the efficiency of generating mature T cells was 100 to 1000 times less in nude compared to euthymic mice.5 In contrast, TCR-mediated signals, dictating which TCR clonotypes are positively selected, can be supported by major histocompatibility complex (MHC)–peptide complexes displayed by other cell types. Thus, studies involving hematopoietic chimeras and thymus grafts have shown that hematopoietic cells can mediate positive selection of CD8 T cells in vivo.5,10,11 In line with this, studies in tetraparental aggregation chimeras have demonstrated that the MHC of TECs is not required for efficient positive selection of MHC Ia– and MHC II–restricted T cells.12 Moreover, under normal circumstances, preferential or exclusive positive selection on hematopoietic cells appears to be a general characteristic shared by many (if not all) MHC class Ib-restricted T cells.13-15 These data indicate that the nonredundant role of TECs is to provide TCR-independent signals to thymocytes, but that TCR signals can be provided by other cells in the thymus and the periphery.5,16 These considerations raise the question of whether the canonical influence of TECs on T-cell development is essential for survival. Would lymphocytes developing in a TEC-free milieu be functional and reach sufficient numbers to eradicate pathogens? Oncostatin M (OM)–transgenic mice represent a unique model to directly address this issue. Remarkably, chronic exposure to OM transforms the lymph node (LN) into a "primary" lymphoid organ whose ability to support T-cell development and to seed secondary lymphoid organs is similar to that of a normal thymus.17,18 The lymphopoietic pathway modulated by OM is truly thymus-independent and takes place only in the LNs.17,18 The proportions of double-negative, double-positive, and single-positive T cells in the OM+ LNs reproduce those found in a thymus, and the TCR repertoire of the single-positive cells is diversified. The effect of OM on extrathymic T-cell development in the LNs is probably due to some amplification of a cryptic pathway that is operative in conditions of defective thymopoiesis and was nicely characterized in nude mice.19 Like normal LNs, OM-transgenic LNs contain no reticular epithelial cells.20 Studies in hematopoietic chimeras have demonstrated that MHC class I expression strictly on hematopoietic cells was sufficient to support the development of a diversified repertoire of CD8 T cells in the OM+ LNs.21 To directly evaluate the functionality of T cells generated in an environment devoid of TECs, we therefore sought to characterize in vitro and in vivo the function of extrathymic T cells that develop in the OM-conditioned LNs of athymic mice. More specifically, we addressed 2 questions: Do extrathymic T cells proliferate normally following TCR engagement and are they able to generate protective immune responses against model infections, such as lymphocytic choriomeningitis virus (LCMV) and vesicular stomatitis virus (VSV) infection?
Mice C57BL/6J (B6) mice were obtained from the Jackson Laboratory (Bar Harbor, ME) and the Institute of Laboratory Animal Science (University of Zürich, Switzerland). B6.SJL-PtprcaPep3b/BoyJ (Ly5a) (B6.SJL; Ly 5.1+) and B6.129S7-Rag1tm1/Mom (RAG1-/-) mice were purchased from the Jackson Laboratory. LckOM-transgenic mice on a B6 background have been previously described.18,22 Unless stated otherwise, differences between groups were tested using the Mann-Whitney test. Thymectomy and fetal liver transplantation Thymectomy was performed as previously described.18 Hematopoietic chimeras were created by intravenously injecting 2 x 106 LckOM fetal liver cells, collected on day 13 after coitus, into irradiated (10 Gy) thymectomized RAG1-/- or B6.SJL recipients. Functional studies in hematopoietic chimeras were initiated 80 to 120 days after transplantation. In vitro T-cell stimulation T-cell isolation. Splenocytes were depleted of B lymphocytes using microbeads coated with anti-B220 antibody (Dynal, Lake Success, NY), and adherent cells (macrophages) were removed. Fluorescent labeling of T cells with carboxy-fluorescein diacetate succinimidyl ester (CFSE; Molecular Probes, Eugene, OR) was performed as described.23 Culture conditions. CFSE-labeled cells were plated at 1 x 105 cells/well in round-bottom 96-well microtiter plates. T-cell activation was achieved by coated anti-CD3 (145-2C11; PharMingen, Mississauga, ON, Canada) with or without soluble anti-CD28 (1 µg/mL; 37.51; PharMingen) or control hamster immunoglobulin (1 µg/mL; Ha4/8, PharMingen). At the time of harvest, CFSE-labeled cells were counted, washed in RPMI 1640, and stained with a combination of antibodies before analysis. Division peaks (as determined by CFSE intensity) were labeled from 0 to n. Since a single T cell dividing n times will generate 2n daughter cells, if the total number of T cells that have divided 3 times (n = 3) is 8, then exactly one precursor had to divide 3 times to generate these 8 cells (23 = 8). Making use of this mathematical relationship, the number of T cells that have divided was extrapolated from the number of daughters under each division peak, and the total number of mitotic events was calculated as described.24 The proliferative burst size (number of daughter cells generated by a dividing T-cell "precursor") was obtained by dividing the total number of mitoses by the number of precursors that had divided.24 For intracellular cytokine staining, cells were restimulated with phorbol myristate acetate (PMA; 20 ng/mL; Sigma, St Louis, MO) and ionomycin (750 ng/mL; Sigma) 4 hours prior to cell harvesting and monensin (PharMingen) was added 1 hour later. LCMV and VSV infection VSV Indiana (VSV-IND; Mudd-Summers isolate) was grown on BHK21 cells. LCMV-WE and LCMV-Armstrong (ARM) were propagated on L929 fibroblast cells. Mice were infected by intravenous injection of LCMV-ARM (200 pfu), LCMV-WE (200 pfu), or VSV-IND (2 x 106 pfu).25-27 LCMV titers in different organs were determined as described with the immunologic focus assay.28 LCMV-NP (nucleoprotein)–specific IgG antibodies were measured by enzyme-linked immunosorbent assay (ELISA),27 and the VSV neutralization assay was performed as previously described.29,30 In general, experiments with VSV and LCMV-WE were done in Zürich and experiments using LCMV-ARM in Montréal. Flow cytometric analyses
Analyses were performed with a FACSCalibur flow cytometer using CellQuest software (Becton Dickinson, Oakville, ON, Canada) using the following monoclonal antibodies (PharMingen): peridinin chlorophyll protein (PerCP)– or allophycocyanin (APC)–conjugated anti-CD4 (RM4-5), APC-conjugated anti-CD8 (53-6.7), biotin-conjugated anti-Ly5.1 and anti-Ly5.2, and phycoerythrin (PE)– or PerCP-conjugated streptavidin. H2Db/LCMV GP33-41 tetramers and H2Db/B6dom1 tetramers31,32 were obtained from the National Institute of Allergy and Infectious Disease (NIAID) MHC Tetramer Core Facility (Atlanta, GA) and Canadian Network for Vaccines and Immunotherapies (CANVAC) Tetramer Core Facility (Montreal, QC, Canada), respectively. Intracellular cytokine staining for interleukin 2 (IL-2) and interferon Immunization with DCs and in vivo CTL assays Enrichment for splenic dendritic cells (DCs) was performed as described.33 Spleen DCs were resuspended in medium containing GP33-41 at a concentration of 10-6 M, incubated for 60 minutes at 37°C, washed 3 times with RPMI 1640, and the proportion of CD11c+MHCII+ DCs was assessed by flow cytometry. The cell concentration was adjusted so that the cell suspension used for intravenous immunization contained 2 x 106 CD11c+MHCII+ DCs/mL. In vivo cytotoxic T-lymphocyte (CTL) assay was performed as described by Coles et al.34 Briefly, B6 splenocytes pulsed with 10-6 M GP33-41 peptide or not were labeled with a high (250 nM) and low (25 nM) concentration of CFSE, respectively. Equal numbers of cells from each population (107 cells) were mixed, then injected intravenously. On day 0, CFSE-labeled cells were injected into recipients that were naive or had been previously injected with peptide-pulsed DCs on days -21 and -7. Recipients were killed 4 hours later. The following formula was used to calculate specific lysis: ratio = (percentage CFSElow/percentage CFSEhigh), and percentage specific lysis = [1 - (ratio unprimed/ratio primed)] x 100.
In vitro function of T lymphocytes of thymic versus extrathymic origin Proliferation. T cells of extrathymic origin, which developed in OM+ LNs, were tested for proliferative behavior and compared to T cells of thymic origin by stimulation with anti-CD3 with or without anti-CD28. Spleen T cells from B6 mice (thymic T cells) and from irradiated (10 Gy) adult-thymectomized B6.SJL (Ly5.1) hosts reconstituted with LckOM fetal liver cells (Ly5.2; extrathymic T cells) were labeled with CFSE and cultured in CD3-coated microtiter plates in the presence or not of soluble anti-CD28. After culturing for 24 to 96 hours, cells were labeled with 7-amino-actinomycin D (7-AAD) to exclude necrotic cells and stained with antibodies against CD4, CD8, Ly5.1, and Ly5.2. At all time points after stimulation more than 99% of harvested T lymphocytes were Ly5.1-Ly5.2+ (data not shown). The absolute numbers of CD4 and CD8 T cells recovered after stimulation for 24 to 96 hours and their CFSE content (which decreases by 50% after each cell division) are shown in Figure 1. The numbers of CD4 and CD8 T cells harvested at 48 to 96 hours were expressed as a function of X, which represents the number of cells/well at 24 hours (before the beginning of cell division). Calculations of the proliferative burst size and the doubling time were based on the results of 5 consecutive experiments (Table 1). The proliferative burst size corresponds to the number of daughter cells generated by a dividing T-cell "precursor," and the doubling time represents the time required for the average responding T cell to achieve a single cell division.24 As expected, proliferation of thymic T cells was more rapid in the presence of costimulation with anti-CD28 than when stimulated with anti-CD3 alone. Furthermore, expansion of thymic CD8 T cells was more rapid and extensive than for CD4 T cells. When compared to their thymic counterparts, extrathymic CD4 T cells showed an increased burst size when stimulated with anti-CD3 alone but not when anti-CD28 was added (Table 1). Of note, in the presence of anti-CD3 with or without anti-CD28, total accumulation of extrathymic CD4 T cells at 72 hours was decreased by about 50% relative to thymic T cells. When stimulated with anti-CD3 with or without anti-CD28, extrathymic CD8 T cells proliferated more rapidly and showed a greater burst size than thymic T cells. However, although extrathymic T cells accumulated to higher levels than thymic CD8 T cells when stimulated with anti-CD3 alone, their expansion, in contrast to thymic T cells, was barely augmented by costimulation with anti-CD28. Thus, altogether, extrathymic T cells divided more rapidly and extensively than thymic T cells. However, a great discrepancy emerged between the doubling time and burst size on one side and total cell accumulation of extrathymic T cells on the other side. This discrepancy suggested that accumulation of extrathymic T cells following TCR engagement might possibly be curtailed by an increased apoptosis rate.
Apoptosis. Annexin V labeling showed that after stimulation with anti-CD3 with or without anti-CD28, the apoptosis rate was greater for extrathymic than for thymic CD4 and CD8 T cells (Figure 2A). Analysis of annexin V staining of T cells as a function of their CFSE content revealed that the difference between thymic and extrathymic T cells prevailed irrespective of whether they had gone through 0 to more than 6 cell divisions after stimulation (Figure 2B). Therefore, the higher apoptosis rate of extrathymic CD4 and CD8 T cells was a constant finding and probably was not due to their more rapid posttriggering proliferation rate.
Cytokine production. We next asked whether the brisk proliferative response of extrathymic T cells was coupled with a more rapid cytokine production. Production of IL-2 and IFN- In vivo responses against viruses
We assessed in vivo responses of extrathymic T cells against 2 model infections, LCMV and VSV. Initial control of LCMV, a noncytopathic virus, is mainly dependent on CD8 CTLs,35,36 whereas long-term virus control requires B cells and CD4 T cells.25,37 VSV, a cytolytic virus, is eliminated by early produced neutralizing IgM (T-independent) followed by IgG (CD4-dependent) antibodies against VSV glycoprotein.38,39 To study animals in which 100% of T cells were of extrathymic origin, we transplanted LckOM fetal liver cells into thymectomized-irradiated RAG1-/- mice (OM
CD8 response against LCMV-ARM. Following infection with LCMV-ARM (200 pfu intravenously), the kinetics of anti-LCMV CD8 response were assessed with H2Db/GP33-41 tetramers and its efficacy in terms of virus clearance was estimated with the immunologic focus assay.28 The percentage of tetramer-positive CD8 splenocytes increased more rapidly in OM
In OM
Antigen driven T-cell expansion is not synonymous with protective immunity. In fact, some antigen-specific CD8 T cells may expand considerably in vivo yet show defective effector activity.42,43 LCMV-ARM titers were similar in the spleen and lung of OM
CD8 response against LCMV-WE. A correlation was observed between resistance to IFN-
CTL response against LCMV GP33-41–coated DCs. To evaluate the response of extrathymic CTLs against a nonreplicative antigen, we immunized OM
Extrathymic CD8 T cells present an intrinsic defect. The LNs of OM-transgenic mice show a disturbed architecture because they are dual primary and secondary lymphoid organs where mature T cells are not apparently segregated from immature T lymphocytes.20 We therefore performed adoptive transfer experiments to evaluate whether extrathymic T cells could eradicate LCMV-ARM in recipients whose LNs were unencumbered with immature T cells. RAG1-/- mice were transfused with 30 x 106 splenocytes from B6 mice (thymic T cells) or OM
Antibody production against LCMV and VSV. To evaluate the functionality of CD4 extrathymic T cells, we assessed their ability to help B cells for production of antibody against LCMV-NP (ELISA) and VSV (neutralizing antibody; Figure 7). Compared to B6 controls, both OM
Athymic mice reconstituted with OM-transgenic hematopoietic stem cells provide a unique model for analysis of T cells generated extrathymically in an environment devoid of TECs. A main conclusion of this work is that OM+ extrathymic T cells do not behave like classic thymic T cells following antigen stimulation. Extrathymic CD4 T cells responded by proliferation and intracellular IFN- production (Figures 1-2) to in vitro stimulation with anti-CD3 with or without CD28. However, because of their high apoptosis rate, OM+ CD4 T cells failed to accumulate to normal levels in vitro despite their normal doubling time and burst size (Table 1; Figure 2). In line with this, they did not provide adequate help to B cells when mice were infected with VSV or LCMV. Thus, extrathymic CD4 T cells are functionally deficient. The functionality of extrathymic CD8 T cells was superior to that of their CD4 counterpart. In vitro they proliferated extensively and produced greater quantities of IFN- than thymus-derived T cells (Table 1; Figure 2). Under these conditions, their accumulation in 72-hour culture assays was nevertheless not commensurate with their proliferative activity because it was curtailed by a high apoptosis rate. Consistent with the high apoptosis rate observed in vitro (Figure 2A-B), GP33-41 tetramer-positive extrathymic CD8 T cells were unduly susceptible to apoptosis following LCMV-ARM infection (Figure 4C). Moreover, extrathymic CD8 T cells specific for the GP33-41 epitope developed only low cytotoxic activity following in vivo stimulation (Figure 5), and they accumulated to lower levels than thymic T cells after LCMV-WE infection (Figure 3E). Although LCMV-ARM titers were similar in chimeras and controls up to day 15, it cannot be excluded that innate resistance levels differ and influence initial replication. It seems probable that in chimeras control of LCMV-ARM during the first 2 weeks correlated with higher IFN- production of extrathymic T cells. However, control of LCMV-ARM was only transient because viral replication became evident by day 30 (Figure 4A). Furthermore, extrathymic T cells provided no significant protection against LCMV-WE. This discrepancy between effects on LCMV-ARM versus LCMV-WE is in line with an earlier finding of the former's greater susceptibility to IFN- and lower propensity to induce T-cell exhaustion.40
Considering their ability to rapidly produce high amounts of IFN-
Because T lymphocytes have a finite lifespan (of Our demonstration that one crucial limitation of extrathymic T cells is their difficulty in dealing with viruses would be consistent with the idea that coevolution with viruses has been a driving force in the remarkable conservation of the thymus as the primary T-lymphoid organ in all jawed vertebrates. Interestingly, our work demonstrates that OM+ extrathymic T cells share the phenotype and functional properties of a minor subset of T cells found in euthymic mice, MHC Ib-restricted T cells. Similar to MHC Ib-restricted T cells,14 the behavior of OM+ T cells seems to fit somewhere between conventional T cells and their evolutionary precursors, natural killer T cells. It will be important to determine how the environment where they develop can impinge on the function of T lymphocytes. Moreover, further studies will be needed to determine whether OM+ extrathymic T cells show the same behavior as MHC Ib-restricted T cells against various pathogens. If this is the case, modulation of the OM-induced extrathymic development pathway may not substitute for classic thymic T cells but could have interesting therapeutic potential.
We are indebted to the NIAID MHC Tetramer Core Facility and CANVAC Tetramer Core Facility for providing tetramers. We thank Nathalie Labrecque for insightful comments, Pascale Blais for help with statistical analyses, and J. A. Kashul for editorial assistance. We are also grateful to Sylvie Brochu, Caroline Côté, and Edit Horvath for helpful discussions and technical assistance.
Submitted September 26, 2003; accepted December 10, 2003.
Prepublished online as Blood First Edition Paper, December 24, 2003; DOI 10.1182/blood-2003-09-3311.
Supported by grants from the Canadian Institutes for Health Research (C.P.), the Swiss National Science Foundation, and the Kanton Zürich.
M.-È.B., G.G., and M.M.M. contributed equally to this work.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Claude Perreault, Guy-Bernier Research Center, Maisonneuve-Rosemont Hospital, 5415 de l'Assomption Blvd, Montreal, QC, Canada H1T 2M4; e-mail: c.perreault{at}videotron.ca.
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Abstract
Introduction
; 
) and CD8 (
; 
) populations of thymic (
) and extrathymic (
) T cells. Three mice were in each group. For panel A, statistically significant differences between thymic and extrathymic T cells are indicated for CD4 (*) and CD8 (
) T cells. (C) Production of cytokines by thymic (shaded histograms) and extrathymic (solid boldface lines) T cells was assessed by intracellular staining with IL-2- and IFN-
RAG1-/- chimeras), and infected the mice with LCMV or VSV approximately 90 days later. Two LCMV isolates, LCMV-ARM and LCMV-WE, were used that differ with regard to susceptibility to IFN-
12% on day 3) than in B6 controls (
) were harvested on days 8 and 20 after infection. Two to 3 mice were in each group. In panels A and B, dotted lines indicate the detection limit of the immunologic focus assay. (C) Apoptosis of splenic CD8 T cells following LCMV-ARM infection. Histogram bars show the mean proportion of annexin V+ elements among GP33 tetramer-positive (+) and tetramer-negative (-) CD8 T cells in B6 mice (black and dark gray bars) and OM 
T lymphocyte lineage commitment. Immunity. 1998;9: 187-197.