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Prepublished online as a Blood First Edition Paper on December 27, 2002; DOI 10.1182/blood-2002-06-1855.

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Blood, 1 May 2003, Vol. 101, No. 9, pp. 3550-3559

IMMUNOBIOLOGY

Promoter IV of the class II transactivator gene is essential for positive selection of CD4+ T cells

Jean-Marc Waldburger, Simona Rossi, Georg A. Hollander, Hans-Reimer Rodewald, Walter Reith, and Hans Acha-Orbea

From the Institute of Biochemistry and Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, Epalinges, Switzerland; Department of Genetics and Microbiology, University of Geneva, Medical School, Geneva, Switzerland; Pediatric Immunology, Departments of Research and Clinical-biological Sciences, and the Children's Hospital, University of Basel, Switzerland; Department for Immunology, University Clinics Ulm, Germany.


    Abstract
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

Major histocompatibility complex class II (MHCII) expression is regulated by the transcriptional coactivator CIITA. Positive selection of CD4+ T cells is abrogated in mice lacking one of the promoters (pIV) of the Mhc2ta gene. This is entirely due to the absence of MHCII expression in thymic epithelia, as demonstrated by bone marrow transfer experiments between wild-type and pIV-/- mice. Medullary thymic epithelial cells (mTECs) are also MHCII- in pIV-/- mice. Bone marrow-derived, professional antigen-presenting cells (APCs) retain normal MHCII expression in pIV-/- mice, including those believed to mediate negative selection in the thymic medulla. Endogenous retroviruses thus retain their ability to sustain negative selection of the residual CD4+ thymocytes in pIV-/- mice. Interestingly, the passive acquisition of MHCII molecules by thymocytes is abrogated in pIV-/- mice. This identifies thymic epithelial cells as the source of this passive transfer. In peripheral lymphoid organs, the CD4+ T-cell population of pIV-/- mice is quantitatively and qualitatively comparable to that of MHCII-deficient mice. It comprises a high proportion of CD1-restricted natural killer T cells, which results in a bias of the Vbeta repertoire of the residual CD4+ T-cell population. We have also addressed the identity of the signal that sustains pIV expression in cortical epithelia. We found that the Jak/STAT pathways activated by the common gamma  chain (CD132) or common beta  chain (CDw131) cytokine receptors are not required for MHCII expression in thymic cortical epithelia. (Blood. 2003;101:3550-3559)

© 2003 by The American Society of Hematology.

    Introduction
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

Major histocompatibility complex class II (MHCII) molecules are crucial for the development, survival, and activation of CD4+ T cells. Recognition of self-peptide-MHCII complexes on cortical thymic epithelial cells (cTECs) determines whether or not immature CD4+ thymocytes are positively selected.1 Negative selection, mediated by MHCII+ thymic dendritic cells (DCs),2 results in the deletion of autoreactive CD4+ T cells. In the periphery, peptide-MHCII complexes expressed on antigen-presenting cells (APCs) induce the activation, proliferation, and differentiation of CD4+ T helper cells. The central importance of these functions is illustrated by the phenotype of MHCII-deficient mouse strains,3-5 by the clinical course of patients suffering from bare lymphocyte syndrome (BLS),6-10 and by CIITA and RFX5-deficient mice, 2 murine models of BLS.11-14 Patients with BLS suffer from a severe primary immunodeficiency that is entirely attributable to the nearly complete absence of MHCII expression.6-10 Patients with BLS, as well as mice deficient for MHCII, CIITA, and RFX5, exhibit impaired maturation of CD4+ T cells and are unable to mount efficient CD4+ T helper cell-dependent immune responses.

CIITA is one of the 4 genes affected in BLS.6,7 It now is widely recognized to be the master regulator of MHCII expression. In most instances, it is the expression of CIITA that dictates the tightly controlled pattern of MHCII expression. Only MHCII+ APCs (B cells, DCs, macrophages) express CIITA constitutively. The expression of CIITA, and thus of MHCII genes, can be activated in MHCII- cells by stimulation with interferon gamma  (IFNgamma ).15-23 A large and complex regulatory region containing several independent promoters controls transcription of the Mhc2ta gene. Of the 4 promoters identified in the human gene, 3 (pI, pIII, and pIV) are strongly conserved in the mouse. Promoter I is highly specific for DCs. Promoter III is used primarily in B cells but is also active in certain human DC preparations.19,24,25 Promoter IV is largely responsible for IFNgamma -induced expression.

We have recently generated mice carrying a targeted deletion of promoter IV (pIV) (Figure 1). pIV-/- mice were engineered to carry a deletion of approximately 500 base pair (bp), encompassing exon IV and its associated promoter. Transcription from the remaining promoters (pI and pIII) is unaffected by the deletion of pIV.25 This ensures normal levels of basal and activated MHCII expression on B cells, macrophages, and DCs in the thymus and periphery of pIV-/- mice (Figure 1). IFNgamma -induced MHCII expression on extrahematopoietic cells is on the other hand completely abrogated in pIV-/- mice.


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Figure 1. Selective loss of MHCII expression in pIV-/- mice. Mice with a targeted deletion of CIITA pIV lack IFNgamma -induced MHCII expression on nonhematopoietic cells and constitutive MHCII expression on cortical thymic epithelial cells (cTECs) and mTECs. Professional APCs retain IFNgamma -induced CIITA expression via pI (microglia, macrophages) and constitutive expression via pI (DCs) and pIII (B cells, DCs).

Surprisingly, pIV-/- mice also lack MHCII expression in the thymic cortex.25 This results in a defect in cTEC-mediated positive selection and in drastically reduced numbers of CD4+ T cells in the thymus and periphery. The periphery of pIV-/- mice contains ample numbers of MHCII+ DCs, B cells, and macrophages. Interactions between the T-cell receptor (TCR) of CD4+ T cells and MHCII molecules on peripheral APCs are believed to be crucial for the survival of CD4+ T cells.26,27 CD4+ T cells should thus encounter a favorable environment for their survival in pIV-/- mice. These mice therefore represent an unprecedented model in which to study the fate of the residual CD4+ T cells generated in the absence of the "classical" positive selection pathway.

In this study we characterized the residual population of CD4+ T cells in pIV-/- mice. We further defined MHCII expression in the thymus of pIV-/- mice and analyzed negative selection by endogenous retroviruses. To address the question on the transcription factors required for constitutive MHCII expression on cTECs, we analyzed positive selection of CD4+ T cells in mice lacking stromal expression of interleukin-7 receptor (IL-7R) or the common gamma chain.


    Materials and methods
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

Mice and generation of bone marrow chimeras

Mice carrying a deletion of pIV of the Mhc2ta gene were generated previously in our laboratory.25 I-Aalpha -/- mice were a generous gift from H. Bluethmann5 (Hoffman La Roche, Basel, Switzerland). IL-7R-deficient mice were previously described.28 To introduce a functional I-E allele in the original Sv129-C57Bl/6 background, pIV-/- mice were backcrossed to B10BR animals. F2 offspring were genotyped by polymerase chain reaction (PCR) to distinguish between the wild-type (primers F1 + r1) and deleted (primers F2 + r2) alleles of pIV: F1, 5'-CCTAGGAGCCACGGAGCTG-3'; r1, 5'-TCCAGAGTCAGAGGTGGTC-3'; f 2, 5'-CAGACTATCCTGAAA-TGCC-3'; r2, 5'-CGAGATCTAGATATCGATAAGCTTG-3'. pIV-/- F2 progeny were tested for I-E expression by fluorescence-activated cell-sorter scanner (FACS). All other experiments were performed with CIITA pIV-/- and pIV+/- littermates on a mixed Sv129-C57Bl/6 background. Mice 8 to 12 weeks of age were used to generate bone marrow chimeras. A total of 107 cells depleted of mature CD4+ cells (by negative selection using monoclonal anti-CD4 antibodies and magnetic beads [Dynal, Oslo, Norway] according to the manufacturer's instructions) were transferred per recipient (irradiated with 3 Gy). Mixed bone marrow chimeras were reconstituted with an equal number of bone marrow cells derived from CD45.2 pIV-/- mice and CD45.1 congenic C57BL/6 mice.29 All bone marrow transfer experiments involving pIV-/- mice (Table 1; Figures 3 and 6) were performed using the congenic markers Ly5.1 and Ly5.2. Reconstitution of the chimeras was allowed for 2 months. The analysis of reconstituted IL-7R-deficient mice did not require the use of congenic markers, because few endogenous CD4+ T cells are produced. The same applies to nude mice reconstituted with c-kit, gamma-c-deficient thymuses (Figure 8). c-Kit, gamma c-deficient thymuses30 were transplanted under the kidney capsule of nude mice. The thymocytes were analyzed 8 weeks later for CD4 and CD8 expression. Animals were housed either under specific pathogen-free conditions at Research and Consulting Company (RCC), Fullinsdorf, Switzerland, or under standard conditions in a conventional mouse facility.

                              
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Table 1. Characterization of bone marrow chimeras

Cytofluorimetric analysis

Single-cell suspensions from peripheral lymph nodes, thymus, or spleen were prepared by crushing the tissues between 2 frosted glass slides. Peripheral blood lymphocytes (PBLs) were harvested by tail blood sampling and isolated by centrifugation on a Ficoll gradient. Ice-cooled single-cell suspensions were pretreated with Fc-block (anti-CD16/CD32; PharMingen, San Diego, CA) and then incubated with specific antibodies (PharMingen) directed against CD4 (RM4-5), CD8 (53-6.7), I-Ab (AF6-120.1) I-Ed (14-4-4S), Ly5.1 (A20), Ly5.2 (104), NK-1.1 (PK136), CD62L (MEL-14), CD44H (TM-1), CD54 (3E2), TCRbeta (H57-597), CD3 (17A2), Vbeta 3 (KJ25), Vbeta 4 (KT4), Vbeta 5.1 and 5.2 (MR9-4), Vbeta 6 (RR4-7), Vbeta 7 (TR310), Vbeta 8 (F23.1), Vbeta 9 (MR10-2), Vbeta 10b (B21.5), Vbeta 11 (RR3-15), Vbeta 12 (MR11-1), Vbeta 13 (MR12-3), Vbeta 14 (14-2). Then 104 (primary cultures) to 105 (suspensions from fresh organs) cells were analyzed using a FACSCalibur (Becton Dickinson). alpha -Galactosylceramide (alpha -GalCer)-loaded tetramers were a generous gift from M. Kronenberg, San Diego, CA. Detection of CD1-restricted natural killer T (NKT) cells with mCD1 tetramers loaded with alpha -GalCer were performed as described previously.31 Briefly, cells were pretreated with Fc-block (anti-CD16/CD32; PharMingen) and neutravidin (Molecular Probes, Eugene, OR) at 4°C and then stained for 20 minutes at 23°C with anti-NK1.1 Ab, anti-CD4 Ab, anti-TCRbeta Ab and alpha -GalCer-loaded mCD1 (tetramerized with neutravidin phycoerythrin).

Immunohistochemistry

Sections (8-µm) from frozen adult organs and whole newborn mice were air dried, fixed in ice-cold acetone, blocked in phosphate-buffered saline (PBS) containing 0.6% H2O2, 5% goat serum and 0.1% NaN3, and stained with digoxygenin-conjugated antibody directed against MHCII (M5/114), F4/80 (Serotec no. MCAP497), CD11c (HL3; Pharmingen), and keratin (antipankeratin serum provided by E. Reichmann, Zurich, Switzerland). Staining was revealed using antidigoxygenin peroxidase and 3-amino-9-ethyl carbazole (Sigma-Aldrich) to give a red precipitate. Sections were counterstained with methylene blue.

Immunofluorescence

For detection of MHCII expression by medullary epithelial cells, thymuses were isolated and embedded in cryoembedding media (Tissue-Tek; Sakura Finetek Europe BV, Zoeterwoude, The Netherlands). Frozen samples were cut into sections 6 mm thick, fixed with 4% paraformaldehyde/PBS, and exposed to PBS with 1% fetal calf serum 0.1% Tween at pH 7.3 for 10 minutes before incubation with primary antibody for 1 hour at room temperature. Washing was repeated before incubation with the secondary antibody (20 minutes at room temperature). Isotype controls were used in all experiments. Thymic sections were stained for MTS10 (Pharmingen) and costained against I-Abeta (AF6-120.1; Pharmingen).32 Medullary areas were microscopically localized and analyzed by 2-color immunofluorescence with a confocal microscope (Carl-Zeiss AG, Feldbach, Switzerland).


    Results
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

Positive selection of CD4+ T cells is abrogated in pIV-/- mice

To our surprise, a severe depletion of CD4+ T cells was observed when we examined thymocytes and mature peripheral T cells in pIV-/- mice.25 The single-positive CD4+ T-cell numbers and percentages in pIV-/- thymuses did not exceed those found in MHCII-deficient mice (Figure 2A). In contrast, CD8+ and double-positive CD4+ CD8+ thymocytes are present in normal or slightly increased numbers. In the peripheral lymphoid organs, the percentage of CD4+ T cells is reduced to 1%-5% of total lymphocyte numbers (Figure 2B-C), while that of CD8+ T cells is markedly increased. The extent of the CD4+ T-cell deficiency in pIV-/- mice was surprising because MHCII expression is normal on B cells, macrophages, and DCs in all organs, including the thymus.25 Since peripheral MHC/peptide engagements have been shown to be necessary for the survival and homeostatic expansion of CD4+ T cells,26,27 the peripheral MHCII+ environment of pIV-/- mice should permit the survival and accumulation of any positively selected CD4+ T cells. In spite of this, CD4+ T-cell counts are as low as in mice that have a complete lack of MHCII expression, such as CIITA, RFX5, and MHCII-deficient mutants.


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Figure 2. The depletion of CD4+ T cells found in pIV-/- mice is comparable to that observed in Aalpha -/- mice. CD4+ and CD8+ T-cell populations were analyzed by FACS. Numbers indicate the percentage of CD4+, CD8+, and CD4+CD8+ cells. (A) Representative FACS analysis of thymocytes from pIV+/-, pIV-/- and I-Aalpha -/- mice. (B) % of CD4+ and CD8+ T-cell populations from the lymph nodes, spleen, and PBLs were analyzed for pIV-/- mice and pIV+/- control littermates. (C) Representative FACS analysis showing the CD4+ and CD8+ T-cell populations in lymph nodes from control pIV+/-, pIV-/-, and I-Aalpha -/- mice.

There are 2 explanations that could account for the loss of CD4+ T cells. First, pIV could be essential for MHCII expression in the thymic compartment that drives positive selection of CD4+ T cells. The lack of MCHII expression in pIV-/- thymuses would have to be very tight because MHCII+ DCs, B cells, and macrophages in the periphery of pIV-/- mice should ensure the survival of any positively selected CD4+ T cells. The second possibility is that pIV performs a function that is intrinsically important for T-cell development. For instance, CIITA has been suggested to be involved in the differentiation of peripheral CD4+ T cells into Th2 cells.33 To discriminate between these possibilities we produced radiation bone marrow chimeras with various combinations of recipient and donor phenotypes (Table 1). The development of T cells was examined 2 months after engraftment. The origin of the lymphocytes was assessed by using the congenic markers Ly 5.1 and Ly 5.2.29 Most CD3-negative lymphocytes were of donor origin. In all chimeras, a small fraction of recipient cells persisted in the CD3-positive subset.

When both the recipient and donor phenotypes are wild type, all lymphocyte populations are reconstituted to normal levels (Table 1, group A). This is also true when wild-type mice are reconstituted with pIV-/- donor cells (Table 1, group B). In contrast, pIV-/- mice reconstituted with either wild-type (Table 1, group C) or pIV-/- donor cells (Table 1, group F) had very low CD4+ T-cell counts but normal levels of CD8+ T cells. We also mixed wild-type with pIV-/- donor cells and reconstituted wild-type or pIV-/- mice (Table 1, groups D and E). Again, CD4+ T cells were generated normally if the recipient was wild type (Table 1, group D), whereas pIV-/- recipients were unable to produce normal numbers of CD4+ T cells (Table 1, group E). Taken together these results exclude the possibility that the lack of CD4+ T cells in pIV-/- mice could be due to an intrinsic defect in bone marrow progenitors.

pIV-/- mice specifically lack MHCII expression on thymic epithelial cells

The thymus of pIV-/- mice is characterized by an abnormal pattern of MHCII expression (Figure 3A). In wild-type thymuses, the cortex shows a fine reticular pattern of MHCII expression that is characteristic of the epithelial cell matrix. In the medulla, the staining is more diffuse and is attributable to DCs, B cells, macrophages, and medullary epithelial cells. In pIV-/- mice, the cortical reticular staining is absent, indicating that the cortical thymic epithelial cells (cTECs) lack MHCII expression. Only patchy areas of MHCII expression remain in the cortex. In contrast, the diffuse staining of the medulla is similar to wild-type controls. By staining of adjacent sections, the patchy MHCII expression in the cortex was found to colocalize with F4/80 positive macrophages. CD11c-positive cells, on the other hand, were restricted to the medulla and corticomedullary junction, in accordance with previous studies.34 Finally, the fine reticular stain obtained with an antikeratin antibody is consistent with a conserved architecture of the thymic stroma. Medullary thymic epithelial cells (mTECs) were analyzed by immunofluorescence for MHCII expression (Figure 3B). In control pIV+/- thymic medulla, the majority of MTS10+ cells (major mTECs) express MHCII. Strikingly, in pIV-/- mice MTS10+ mTECs do not express MHCII. The absence of MHCII expression by mTECs has been confirmed by FACS experiments (data not shown).


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Figure 3. pIV-/- mice lack MHCII expression on thymic epithelial cells, and passive transfer of MHCII molecules to T cells is abrogated. (A) Thymic sections were stained (brown) for MHCII, F4/80, CD11c, and epithelial cells (keratin). The counterstain is methylene blue. The strong reduction in MHCII expression in pIV-/- thymuses is restricted to the cortex (compare upper 2 panels). In pIV-/- thymuses, the residual patchy MHCII expression in the cortex overlaps with F4/80+ thymic macrophages (compare middle 2 panels). CD11c-positive DCs are restricted to the medulla (bottom left panel). The keratin stain indicates a normal architecture of the stroma in both the cortex and the medulla (bottom right panel). m indicates medulla; c, cortex. Original magnification, × 200 for all images in panels A and B. (B) Immunohistology of thymic medullary areas. MTS10+ and MHCII+ medullary cells appear blue and green, respectively. Double-positive cells appear cyan in the thymic medulla from the pIV+/- control. No costaining is observed in the pIV-/- medulla. (C-E) Passive transfer of MHCII molecules to thymocytes does not occur in pIV-/- mice. (C) MHCII expression was analyzed by FACS on double-positive CD4+CD8+ thymocytes from pIV-/- (gray profile) and pIV+/- mice (open profile). (D) Double-positive CD4+CD8+ thymocytes derived from pIV-/- bone marrow progenitors acquire MHCII molecules by passive transfer if they develop in the thymus of an irradiated wild-type recipient (open profile). On the other hand, wild-type thymocytes developing in a pIV-/- recipient do not acquire MHCII molecules (gray profile). pIV-/- donor cells (open profile) were identified by gating on Ly5.2+ cells. Wild-type donor cells (gray profile) were selected within the Ly5.1+ gate. (E) MHCII expression on double-positive CD4+CD8+ thymocytes is compared between pIV-/- mice (gray profile) and I-Aalpha -/- mice (open profile).

Thymocytes acquire MHCII molecules by passive transfer from TECs

Mouse T cells do not express significant amounts of CIITA and are consequently devoid of MHCII molecules. However, experiments with radiation chimeras35 have indicated that mouse thymocytes can acquire MHCII molecules in a cell contact-dependent fashion. Thymocytes acquire these MHCII molecules by passive transfer from an unidentified thymic stromal cell.36 Strikingly, the passive transfer of MHCII to CD4+CD8+ thymocytes is abrogated in pIV-/- mice (Figure 3C). In irradiated recipients reconstituted with pIV-/- donor cells, the passive MHCII transfer to thymocytes was corrected only if the host thymus is wild type (Figure 3D and data not shown). In contrast, wild-type thymocytes grafted into irradiated pIV-/- hosts remain MHCII-. The lack of MHCII transfer in pIV-/- mice is complete, as shown by the negative control (I-Aalpha -/- mice) in Figure 3E. These observations confirm that the presence of MHCII molecules at the surface of thymocytes results from a passive transfer from cTECs or mTECs.

Negative selection by endogenous mtv superantigens is conserved in pIV-/- mice

The data obtained from radiation chimeras and the histologic findings in pIV-/- thymuses strongly argue that the CD4+ T-cell deficiency in pIV-/- mice is entirely attributable to the lack of MHCII expression by cortical thymic epithelial cells (cTECs). In contrast to other MHCII-deficient mutants, pIV-/- mice retain normal MHCII expression on B cells, DCs, and macrophages in both the thymus and the periphery.25 This provided a unique opportunity to test whether MHCII+ APCs can perform negative selection in the absence of positive selection of CD4+ T cells. With this aim, we examined clonal deletion of T cells in pIV-/- mice. T cells expressing Vbeta 5 and Vbeta 11 TCRs are deleted in the thymus of mouse strains that express I-E and retroviral superantigens encoded by mtv-8 and -9.37 Because pIV-/- mice were generated in a mixed B6/129 background (b haplotype), they lack a functional I-Ealpha gene. To obtain efficient, superantigen-mediated deletion we therefore introduced a functional I-E allele by crossing the pIV-/- mice with B10.BR mice. Figure 4A compares the frequency of CD4+ T cells expressing Vbeta 5, Vbeta 11, and Vbeta 8 in the presence and absence of the I-Ealpha gene. In wild-type mice and in pIV-/- mice, introduction of the I-Ealpha gene has no effect on the control Vbeta 8 family. In wild-type mice, introduction of the I-Ealpha gene leads to efficient deletion of the susceptible Vbeta 5 and Vbeta 11 families. A deletion of the Vbeta 11 family and, to a lesser extent, of the Vbeta 5 subset was also observed in the residual CD4+ T-cell population of pIV-/- mice. The fact that deletion in the CD4+ Vbeta 5 subset is only partial in pIV-/- mice may be explained by the recently described MHCI- and MHCII-independent population of CD4+ T cells.38 This population displays a 6-fold increase in Vbeta 5 expression, which becomes apparent in the residual CD4+ T-cell compartment of pIV-/- mice. In the CD8+ T-cell compartment, the I-E dependent, superantigen-mediated deletion of the Vbeta 5 and Vbeta 11 families was, as expected, similar in pIV-/- and control littermates (Figure 4B). There was again no change in the percentage of Vbeta 8+ CD4+ T cells after introduction of the I-Ealpha gene. Taken together, these results establish that superantigen-mediated negative selection of CD4+ T cells occurs normally in pIV-/- mice. The residual CD4+ thymocytes in pIV-/- mice are negatively selected despite the defect in their positive selection. This defect in positive selection is as strong in the B10BR background as it is in the mixed Sv129-C57Bl/6 background (compare Figure 4C to Figure 2).


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Figure 4. Negative selection occurs normally in pIV-/- mice. Vbeta 5, Vbeta 11, and Vbeta 8 subsets of CD4+ and CD8+ T cells were analyzed by FACS of splenocytes extracted from pIV-/- mice and control pIV+/- littermates. The numbers indicated alongside the bar graphs represent the percentages of the indicated Vbeta family relative to the total CD4+ (A) or CD8+ (B) T-cell populations. White and filled bars represent the mean value (2 to 4 mice) for a nondeleting (I-E-) and a deleting (I-E+) background, respectively. (C) Representative FACS analysis showing the CD4 and CD8 populations in B10BR thymuses and lymph nodes. B10BR controls (I-E+ I-A+ pIV+/-, on the left) are compared with B10BR pIV-/- mice (I-E+ I-A+ pIV-/- , on the right).

Thymocytes and peripheral CD4+ T cells exhibit the same activated phenotype in pIV-/- and MHCII-deficient mice

CD4+ T cells in MHCII-deficient mice present typical anomalies in the expression of several cell surface markers. Figure 5A shows that CD4+ thymocytes of pIV-/- mice present the same anomalies as their counterparts isolated from animals fully deficient in MHCII molecules. CD4+ thymocytes have reduced TCR levels in both I-Aalpha -/- and pIV-/- mice. MHCII deficiency also has been reported to have an impact upstream of the CD4+ single-positive stage, leading to elevated CD4 and TCR levels on double-positive CD4+CD8+ thymocytes.39 This phenotype is again reproduced in the pIV-/- animals.


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Figure 5. Thymocytes and CD4+ T cells from pIV-/- and I-Aalpha -/- mice display a similar phenotype. (A) Double-positive CD4+CD8+ thymocytes from pIV-/- mice express higher levels of TCR and CD4 than controls (top 2 histograms). CD8 levels are similar to controls (data not shown). Single-positive CD4+ T cells display lower TCR levels in pIV-/- and I-Aalpha -/- thymuses compared to control littermates (middle 2 histograms). This is not observed for CD8+ T cells (bottom 2 histograms). (B) CD4+ splenocytes were analyzed by FACS for CD62L, CD54, and TCR expression. CD62L and TCR levels are lower in I-Aalpha -/- and pIV-/- CD4+ T cells than in controls. CD54 is increased in both mutants. (C) CD8+ T cells from the spleens of I-Aalpha -/- , pIV-/-, and control littermates are similar with respect to their expression levels of CD62L, CD54, and TCR.

The similarity between pIV-/- and MHCII-deficient mice extends to the peripheral T-cell compartments. The residual CD4+ T cells in the periphery of pIV-/- mice have the same activated phenotype as the CD4+ T cells of MHCII-deficient mice (Figure 5B). They display a down-regulation of CD62L with a concomitant increase in CD54. Moreover, their TCR levels are markedly reduced. In contrast, CD8+ T cells do not present these anomalies in either the pIV-/- or MHCII-deficient mice (Figure 5C). We conclude that normal MHCII expression in the periphery of pIV-/- mice does not prevent the residual CD4+ T cells from adopting an activated phenotype.

There are 2 explanations that could account for the unusual activated phenotype of the residual CD4+ T cells in the pIV-/- mice. First, the absence of pIV expression in the CD4+ T cells could itself induce an abnormal phenotype. Alternatively, the atypical phenotype could result not from an intrinsic defect in CD4+ T cells but from the development of these cells in the absence of MHCII expression on TEC. The analysis of CD4+ T cells in the radiation chimeras described above (Table 1) is consistent with the latter hypothesis. CD4+ T cells that developed in wild-type hosts display wild-type levels of cell-surface markers even if they are derived from pIV-/- donor cells. On the other hand, wild-type CD4+ T cells that developed in pIV-/- recipients exhibit the same characteristic alterations seen in the pIV-/- and MHCII-deficient mutants (Figure 6). These results rule out the possibility that pIV expression in bone marrow-derived cells is required to obtain a normal phenotype in CD4+ T cells.


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Figure 6. The characteristic cell surface phenotype of thymocytes and peripheral CD4+ T cells in pIV-/- mice is reproduced in bone marrow chimera experiments only if the irradiated recipient is a pIV-/- mouse. Filled profiles represent T cells derived from pIV-/- progenitors grafted into a wild-type host. Open profiles represent T cells derived from wild-type progenitors grafted into a pIV-/- host. T cells were identified as donor-derived by gating with the appropriate congenic marker (Ly5.1 or Ly5.2). Groups B, C, D, and E refer to the groups of bone marrow chimeras listed in Table 1. The 6 histograms in the upper panel reveal that thymocytes derived from wild-type bone marrow progenitors present alterations identical to those described in pIV-/- and I-Aalpha -/- mice if they develop in a pIV-/- host. The 4 histograms in the lower panel represent a similar analysis of splenic CD4+ T cells. Peripheral pIV-/- CD4+ T cells display a normal surface phenotype when they develop in wild-type hosts, whereas wild-type CD4+ T cells that develop in pIV-/- recipients up-regulate CD44 and express lower levels of TCR.

Taken together, our results show that the activated phenotype of CD4+ T cells is a consequence of deficient MHCII expression on cTECs and mTECs. This is consistent with the finding that the residual CD4+ T cells in pIV-/- and MHCII-/- mice share the same atypical phenotype. Moreover, the fact that these 2 mutants exhibit the same phenotype implies that it must arise independently of whether or not peripheral APCs express MHCII molecules.

A large proportion of the residual CD4+ T cells in pIV-/- mice are CD1-restricted NKT cells

In addition to the activated phenotype, the residual CD4+ T cells in pIV-/- mice contain a higher proportion of Vbeta 8+ and Vbeta 7+ cells (see Figure 7). These features are typical of NKT cells.40,41 This led us to suspect that the residual CD4+ T-cell population in pIV-/- mice contains a high proportion of NKT cells. We therefore performed a 4-color FACS analysis with antibodies directed against CD4, TCR-beta , and NK1.1, and with CD1 tetramers coupled to alpha GalCer (Figure 7A). The tetramers allowed us to detect NKT cells that stain negative for the NK1.1 marker.42 CD4+ T cells from pIV-/-, MHCII-deficient, and wild-type mice were found to comprise similar absolute numbers of NK1.1+ and CD1-restricted cells. In contrast, the NK1.1-CD1alpha GalCer- CD4+ T cells, which comprise the classical MHCII-restricted CD4+ T cells, are much less abundant in the MHCII-deficient and pIV-/- animals. The consequence is a considerably higher proportion of NKT cells in the pIV-/- and MHCII-deficient animals (up to half of the total number of CD4+ T cells).


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Figure 7. The proportion of CD1-restricted and NK1.1+ cells is increased in pIV-/- and I-Aalpha -/- mice. (A) The density plots show NK1.1 and CD1alpha GalCer-tetramer staining of CD4+, TCRbeta + T cells from pIV+/-, pIV-/-, and I-Aalpha -/- mice. Numbers indicate the percentage of CD4+ T cells in each quadrant relative to the total number of cells. NK1.1-, tetramer-, CD4+ T cells (lower left quadrant) are reduced 10- to 15-fold in pIV-/- and I-Aalpha -/- mice. In contrast, similar percentages of NK1.1+ and tetramer+ cells are detected in the control and mutant mice. (B) The higher proportion of NKT cells in pIV-/- and I-Aalpha -/- mice leads to an increase in the numbers of Vbeta 8+ CD4+ T cells. The bar graph shows the mean percentages of Vbeta 8+ cells for the 3 mouse strains. Total CD4+ T cells comprise a higher proportion of Vbeta 8+ cells in pIV-/- and I-Aalpha -/- mice as compared to control pIV+/- littermates (left). NK1.1+ CD4+ T cells exhibit a similar Vbeta 8 bias in all 3 mouse strains (right). (C) Representation of various Vbeta families in CD4+ and CD8+ T cells in pIV-/- mice and control pIV+/- littermates. The upper bar graph indicates the mean percentage of the CD4+ T cells belonging to the indicated Vbeta families (at least 4 mice for each data point). The higher proportion of NKT cells in the pIV-/- mice leads to an increase in the percentage of Vbeta 8 and Vbeta 7 CD4+ T cells. As a result, the representation of most other families is reduced. The distribution of Vbeta families in CD8+ T cells is identical between control and pIV-/- mice (lower bar graph).

Since NKT cells display a strong Vbeta 8 bias in their TCR repertoire,41 Vbeta 8+ cells are more frequent in the total CD4+ T-cell population of the pIV-/- and MHCII-deficient mice (Figure 7B). Within the CD4+ NK1.1+ population, the same high proportion of Vbeta 8+ cells is, of course, evident in all strains.

To complete our characterization of the T-cell population in pIV-/- mice, we performed a detailed analysis of the Vbeta repertoire of CD4+ and CD8+ splenocytes. For each Vbeta subset, 4 to 6 pIV-/- mice were compared to an equivalent number of control littermates. No change in the CD8 repertoire was evident in the pIV-/- mice (Figure 7C, lower bar graph). On the other hand, the higher proportion of NKT cells leads to an increase in the representation of Vbeta 8 and Vbeta 7 families in the CD4+ T splenocytes of pIV-/- mice (Figure 7C, upper bar graph). The percentages of CD4+ Vbeta 5+ T cells in pIV-/- mice is also marginally increased compared to control mice. This may be explained by the recently described MHCI- and MHCII-independent population of CD4+ T cells.38 This population displays a 6-fold increase in Vbeta 5 expression and is proportionally increased in pIV-/- mice, given the lack of MHCII-restricted CD4+ T cells. Most of the other Vbeta subsets in the CD4+ T-cell compartment of pIV-/- mice are reduced to approximately 50% of the wild-type levels because of the overrepresentation of Vbeta families of NKT cells.

No requirement for IL-7R signaling in MHCII expression by cTECs

The Jak/signal transducers and activators of transription (STAT) pathway is able to activate Mhc2ta transcription through promoter IV. We addressed the question whether the STAT-1,-3,-5 associated cytok