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The Journal of Immunology, 1999, 163: 1162-1169.
Copyright © 1999 by The American Association of Immunologists

Th Cells and Th2 Responses Can Develop in the Absence of MHC Class II-CD4 Interactions1

Andreas Wack, Paola Corbella2, Nicola Harker, Kathleen Roderick, Trisha Norton, Keith Williams, Owen Williams and Dimitris Kioussis3

Division of Molecular Immunology, National Institute for Medical Research, Mill Hill, London, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In this paper, we address the question whether CD4 and MHC class II expression are necessary for the development of the T helper lineage during thymocyte maturation and for activation-induced Th2 responses. To bypass the CD4-MHC class II interaction requirements for positive selection and activation, we used mice that are doubly transgenic for CD8 and for the MHC class I-restricted TCR F5. This transgene combination leads to MHC class I-dependent maturation of CD4 lineage cells. Upon activation, these CD4 lineage T cells secrete IL-4 and give help to B cells but show no cytotoxic activity. Remarkably, neither MHC class II nor CD4 expression are necessary for the generation and helper functions of these cells. This suggests that under normal conditions, coreceptor-MHC interactions are necessary to ensure the canonical combinations of coreceptor and function in developing thymocytes, but that they do not determine functional commitment. Our results also imply that expression of the CD4 gene does not influence, but is merely associated with the decision to establish the T helper program. In addition, we show that activation through TCR-MHC class I interactions can induce Th2 responses independently of CD4 and MHC class II expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Thymocyte development takes place in a sequence of maturation steps that can be followed by the expression of the coreceptors CD4 and CD8. Thus, immature CD4-CD8- double negative cells develop into CD4+CD8+ double positive and subsequently into either CD4+CD8- or CD4-CD8+ single positive (SP)4 mature thymocytes. Normally, CD4+ T cells express an MHC class II-restricted TCR and have mainly helper function, and CD8+ T cells express an MHC class-I restricted TCR and have predominantly cytotoxic function. Thus, one of two differentiation programs is initiated in each thymocyte, resulting in two T cell lineages with coordinated functional commitment, MHC restriction, and coreceptor expression. Analysis of mice deficient for coreceptor or MHC molecules has shown their necessity for the generation of the appropriate T cell lineage. However, it is not clear whether these molecules are required for the triggering of the appropriate differentiation program, for ensuring the survival of the cells, or for both (1, 2, 3, 4).

When activated, Th cells can develop into effector cells with a range of functional profiles that can be broadly distinguished by the cytokines produced; the most polarized effector cell types are Th1 cells secreting mainly IFN-{gamma} and Th2 cells secreting mainly IL-4 and IL-5 (5). There is general agreement that the circumstances of activation greatly influence the decision of a Th cell to develop toward Th1 or Th2 profile. It has also been shown that helper T cells generated in the absence of the CD4 molecule can develop into Th1, but not Th2, cells (6, 7, 8), implying that the requirement of the CD4 molecule may be stricter for the development of Th2 effector function than for the generation of Th1 cells.

In the present study, we attempted to address the following questions. First, are CD4-MHC class II interactions necessary for thymic differentiation of mature CD4 lineage T cells? Second, is the commitment to helper function or the bias between Th1 or Th2 phenotypes of these cells influenced by the expression of MHC class II or CD4 molecules?

For this purpose, we have used the F5 transgenic mice expressing an MHC class I-restricted TCR (9). When crossed to the recombination-activating gene (Rag)-1- background, F5 mice contain no CD4+ SP T cells in the thymus or the periphery. Expression of a CD8 transgene (CD8tg) on all T cells in F5/Rag-1- mice leads to maturation of CD4+ T cells, which show helper function in the periphery (10). As these CD4+ T cells have been selected on an MHC class I-restricted TCR, they appear to have made the lineage decision and their functional commitment, regardless of TCR restriction. In addition, these CD4+ cells can be activated by H-2Db presenting the cognate Ag peptide NP68. Thus, both in thymic selection and during peripheral activation, it appears that TCR-CD8-MHC class I interactions have replaced TCR-CD4-MHC class II interactions. Under these circumstances, we wanted to determine whether the CD4 and MHC class II molecules play any role in the establishment of the functional program leading to the helper lineage and in the decision between a Th1 and a Th2 response taken during activation of Th cells. For this purpose, we crossed F5/Rag-1-/CD8tg mice to mouse strains that are deficient in the expression of CD4 or MHC class II and analyzed generation and function of cells that develop in the CD4 helper lineage.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Mice transgenic for the F5 TCR (9) were generated in our laboratory. Mice transgenic for the mouse CD8{alpha}/Lyt2.1 and CD8 {beta}-chain (11) or deficient in the expression of Rag-1 (12), MHC class II (13), CD4 (14), or TCR {alpha}-chain (15) were kindly provided by Drs. Ellen Robey (University of California, Berkeley, CA), Eugenia Spanopoulou (Mount Sinai School of Medicine, New York, NY), Diane Mathis (Institut de Genetique et de Biologie Moleculaire et Cellulaire, Strasburg, France), Dan Littman (Skirball Institute of Biomolecular Medicine, New York, NY), and Mike Owen (Imperial Cancer Research Fund, London, U.K.), respectively. All strains are backcrossed to C57BL/10 mice, and, therefore, are of MHC H-2b haplotype and express endogenously the CD8{alpha}/Lyt2.2 allele. All mice shown in this study are on the Rag-1- background and heterozygous for the transgenic F5 TCR and, where appropriate, for the CD8 transgene. The mice were kept in a conventional animal colony free of pathogens and were analyzed at 6–8 wk of age.

Mouse treatment and reagents

For dexamethasone treatment, mice were injected i.p. twice at 24-h intervals with 2 mg of water soluble dexamethasone (Sigma, St. Louis, MO) dissolved in PBS. Mice were sacrificed 24 h after the second injection. For ex vivo T cell responses, mice were injected i.p. twice at 24-h intervals with 50 nmol of the NP68 peptide from the nucleoprotein of influenza virus A/NT/60/68 (ASNENMDAM) dissolved in PBS. Mice were killed and organs removed 24 h after the second injection.

Flow cytometry

For flow cytometric analysis, 106 cells were stained with the following mAbs and second layer reagents: PE-conjugated anti CD4 (Sigma), Tricolor-conjugated anti CD4 (Caltag, Burlingame, CA), biotin-conjugated KT-11 (anti V{beta}11; Ref. 16), biotin- or FITC-conjugated 2.43 (anti CD8{alpha}/Lyt2.2; Ref. 17), PE-conjugated anti-Thy1 (PharMingen, San Diego, CA), PE-conjugated anti-B220 (Sigma), biotin-conjugated anti-CD69 (PharMingen), Tricolor-conjugated anti-panCD8{alpha} (Caltag), and streptavidin-conjugated RED 670 (Life Technologies, Paisley, U.K.). For the determination of DNA content, cells were stained for expression of surface markers, permeabilized in PBS/2% FCS/0.3% Saponin (Sigma), and subsequently stained with 4 mg/ml 7-aminoactinomycin D (7AAD; Sigma). Samples were analyzed on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA) using CellQuest software (Becton Dickinson).

Intracellular cytokine staining

Intracellular IFN-{gamma} staining was performed as described previously (18). Briefly, spleen and lymph node cells from uninjected or NP68-injected mice were incubated at 106 cells/ml for 4 h at 37°C, 5% CO2 in RPMI 1640 (Life Technologies, Paisley, U.K.) supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, penicillin, streptomycin, and 50 µM 2-ME (complete medium) with 10 ng/ml phorbol dibutyrate (Sigma), 500 ng/ml ionomycin (Sigma), and 10 mg/ml Brefeldin A (Epicentre Technologies, Madison, WI). Subsequently, cells were stained for surface markers, fixed with 4% formaldehyde in hypertonic PBS, permeabilized in PBS/1% BSA/0.5% saponin (Sigma), and then stained with FITC-conjugated anti-IFN-{gamma} (clone XMG1.2, Rat IgG1; PharMingen) or the FITC-conjugated isotype-matched control Ab R3-34 (PharMingen).

Cell purification

For in vitro assays, pooled spleen and lymph node cells were purified by depletion with sheep anti-rat IgG Dynabeads M-450 (Dynal, Oslo, Norway) coupled to 2.43 (anti CD8{alpha}/Lyt2.2; Ref. 17), GK1.5 (anti CD4; Ref. 19), or to YTS 169.4 (anti panCD8{alpha}; Ref. 20) mAbs, according to manufacturer’s instructions. The final population was stained and checked by flow cytometry. Contamination by the depleted cells was <5%. The purified populations were used in the assays described below.

IL-4 bioassays

A total of 2 x 106 purified cells from untreated mice was incubated in the presence of 105 irradiated splenocytes from Rag-1-/H-2b mice as APCs at 37°C, 5% CO2 in 24-well plates in 2 ml complete medium with or without 1 µM NP68. After 48 h, supernatants were collected, and IL-4 production was assessed in a bioassay using the IL-4-dependent cell line CT4S (21). A total of 100 µl of supernatants was plated out in 96-well plates, and anti-IL-2 mAb (PharMingen) alone or anti-IL-2 + anti-IL-4 (PharMingen) were added. CT4S cells were washed three times, and 5–10 x 103 cells/well were distributed. After 24 h, 1 µCi of [3H]thymidine was added to each well. Then,16 h later, cells were harvested onto glass fiber filters (Wallac, Milton Keynes, U.K.) and counts analyzed on a 1205 Betaplate counter (Wallac). Supernatant from an IL-4-transfected cell line (22), containing known concentrations of IL-4, was used for the standard curve in each individual experiment and to convert the data into pg/ml of IL-4 produced.

B cell-T cell coculture assays

Small resting B cells were obtained from the spleens of TCR{alpha}- mice by centrifugation through a percoll (Pharmacia, Uppsala, Sweden) gradient. B cells were loaded with carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes, Eugene, OR) by incubating them at 5 x 107 cells/ml for 15 min at 37°C in PBS + 5 µM CFSE. Cells were washed extensively, resuspended in complete medium, and placed in 96-well plates at 105 cells/well. A total of 2 x 105 purified T cells was added, and triplicate cocultures were incubated for 4 days at 37°C, 5% CO2 with or without 1 µM NP68. At the end of the culture, CFSE content of B cells was analyzed by flow cytometry.

CTL assays

Purified T cell subsets from peptide-treated mice were plated out in serial dilutions in complete medium into 96-well plates. EL-4 target cells were labeled with 50 µCi of 51Cr-sodium chromate in complete medium for 1 h at 37°C, 5% CO2 with or without 50 µM NP68. Cells were washed three times in warm medium, and 5 x 103 cells were added to each well. After brief centrifugation, plates were incubated at 37°C, 5% CO2 for 5 h. Subsequently, 25 µl of the supernatant were removed, spotted onto glass fiber filtermats, and analyzed in a 1205 Betaplate counter. Percent specific lysis was calculated as follows: % specific lysis = [(experimental release - spontaneous release)/(maximum release - spontaneous release)] x 100. In all experiments, spontaneous release was <15%.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Thymocyte differentiation toward the CD4 lineage in F5/CD8tg mice does not require MHC class II

Thymocyte differentiation in F5/Rag-1- mice leads to the generation of mature CD8+ SP cells, but not of CD4+ SP cells (Fig. 1GoA). As described previously (10), the combination of the F5 TCR with a constitutively expressed CD8 transgene allows maturation of thymocytes into both the CD8 and the CD4 lineage. Thus, staining of cells from F5/Rag-1-/CD8tg mice for CD4 and endogenous CD8 (CD8end; Fig. 1GoB) reveals two mature T cell populations in the thymus and the spleen, the CD4-CD8end+ (CD8) and CD4+CD8end- (CD4) subpopulations. Both of these populations express the transgenic CD8 molecule, but we will refer to these populations as SP T cells. These mice, as all other mice in this study, are heterozygous for F5 and the CD8 transgene and are on the Rag-1- background.



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  		FIGURE 1. Maturation of CD4 and CD8 lineage cells in F5/CD8tg mice is not dependent on MHC class II. Flow cytometric analysis of thymus and spleen cells from F5 (A), F5/CD8tg (B), and F5/CD8tg/MHC-II- (C) mice. Thymocytes were stained for CD4 and CD8end molecules, and dot plots show total thymocytes. Splenocytes were triple stained for Thy1, CD4, and CD8end molecules, and the dot plots shown are gated on Thy1+ splenocytes. The percentage of cells in each quadrant is indicated in the dot plot, total thymocyte or splenocyte numbers are shown below the dot plots. In the spleens of mice with the genotypes shown, 50–65% of total splenocytes are Thy1+.

 
To examine whether MHC class II interactions are necessary for the maturation of CD8 and CD4 SP thymocytes in F5/CD8tg mice, these mice were crossed with MHC class II-deficient mice (MHC-II-; Ref. 13). Fig. 1GoC shows that F5/CD8tg/MHC-II- mice contain distinct populations of CD8 and CD4 SP thymocytes and splenocytes, in percentages and absolute numbers similar to those found in F5/CD8tg mice. We conclude, therefore, that MHC class II does not contribute to the efficiency of CD4 SP T cell generation in these mice.

Commitment to the CD4 lineage in F5/CD8tg mice is independent of CD4

To test whether or not F5/CD8tg mice develop CD4 lineage T cells in the absence of the CD4 molecule itself, these mice were crossed to mice deficient in CD4 (14). Thymus and spleen cells from the resulting mice were stained for TCR (by anti-V{beta}11 mAbs), Thy1, and CD8end. Fig. 2Go shows that, in thymus and spleen from F5 mice, all TCRhigh cells are also CD8end+, whereas F5/CD8tg mice contain two different populations of mature T cells: one that consists of TCRhighCD8end+ cells and one of TCRhighCD8end- cells. The latter population is also positive for CD4 (data not shown). These two populations of TCRhighCD8end+ cells and TCRhighCD8end- cells are also seen in F5/CD8tg/CD4- mice, suggesting that, in these mice, thymocyte differentiation leads to the generation of both CD8 and "CD4 lineage" T cells, the latter being identified by the absence of CD8end.



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  		FIGURE 2. Both CD4 and CD8 lineage cells can mature in F5/CD8tg/CD4- mice. Flow cytometric analysis of thymus and spleen cells from F5 (A), F5/CD8tg (B), or F5/CD8tg/CD4- (C) mice. Thymi were taken from untreated or dexamethasone treated mice as indicated, thymocytes were stained for V{beta}11 and CD8end molecules, and dot plots show total thymocytes. Splenocytes were triple stained for Thy1, V{beta}11, and CD8end molecules, and the dot plots shown are gated on Thy1+ splenocytes. The percentage of cells in each quadrant is indicated in the dot plot, total thymocyte or splenocyte numbers are shown below the dot plots. In the spleens of mice with the genotypes shown, 50–65% of total splenocytes are Thy1+.

 
To confirm that the thymic TCRhigh populations consist of mature cells, F5, F5/CD8tg and F5/CD8tg/CD4- mice were treated with dexamethasone, which eliminates immature, but spares mature thymocytes (23). After treatment, F5 thymi contain only TCRhighCD8end+ mature cells, but both F5/CD8tg and F5/CD8tg/CD4- thymi contain TCRhighCD8end+ and TCRhighCD8end- populations (Fig. 2Go), consistent with the hypothesis of thymocyte differentiation into mature CD8 (retaining expression of endogenous CD8) and CD4 lineage cells (with extinguished expression of endogenous CD8). In the spleen, the percentages of Thy1+TCRhighCD8end- T cells and the ratio of CD8end- to CD8end+ T cells are comparable in F5/CD8tg and F5/CD8tg/CD4- mice (Fig. 2Go). These Thy1+CD8end+ and Thy1+CD8end- cells do not belong to the NKT lineage, as judged by the absence of NK1.1 staining and their resting phenotype (data not shown). This suggests that the expression of the CD4 molecule does not affect the efficiency of generating CD4 lineage T cells in this mouse model.

CD8end- T cells generated in the absence of MHC class II or CD4 can be activated through the F5 TCR and show a Th2 type cytokine profile

To test whether MHC class II or CD4 is necessary for the functional commitment to the helper lineage, we compared the functional capabilities of CD4 and CD8 lineage T cells from F5/CD8tg with those from F5/CD8tg/MHC-II- or F5/CD8tg/CD4- mice in a number of assays.

Depending on the genotype of the mice, T cell subsets were identified on the FACS by different stainings: F5/CD8tg and F5/CD8tg/MHC-II- mice were stained with anti-CD8end and anti-CD4 mAbs; thus, CD8 and CD4 lineage cells could be distinguished as CD4-CD8end+ and CD4+CD8end-, respectively. In contrast, F5/CD8tg/CD4- mice were stained with anti-Thy1 and anti-CD8end mAbs, and Thy1+CD8end+ cells were considered CD8 lineage cells, whereas Thy1+CD8end- cells were considered CD4 lineage cells. First, these mice were injected i.p. with 50 nmol of the cognate peptide NP68 twice at 24 h intervals and sacrificed 24 h after the second injection. Both CD8 and CD4 lineage T cells respond in a similar manner to the cognate stimulus by becoming blasts and up-regulating the activation markers CD69 (Fig. 3Go), CD25, and CD44 (data not shown). In addition, when stained by 7AAD at the same time point, 40–50% of both CD8 and CD4 lineage T cells show hyperdiploid DNA content indicative of proliferation (Fig. 3Go). The response of the CD4 lineage population to the peptide is similar in F5/CD8tg, F5/CD8tg/MHC-II-, and F5/CD8tg/CD4- mice, indicating that the combination of F5 TCR and transgenic CD8 ensures responsiveness to the Ag and that this responsiveness is not dependent on the presence of MHC class II or CD4.



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  		FIGURE 3. Both CD4 and CD8 lineage cells are activated by the cognate peptide NP68. Splenocytes from untreated (shaded plot) or peptide-treated (bold solid line) mice with the indicated genotypes were surface stained with anti-CD8end plus either anti-CD4 or anti-Thy1, and subsequently either with anti-CD69 (left column) or with 7AAD for DNA content (right column). Histograms show CD69 and 7AAD staining of CD4 and CD8 lineage cells, gated as Thy1+CD8end- (CD4 lineage) and Thy1+CD8end+ (CD8 lineage) for F5, F5/CD8tg, and F5/CD8tg/CD4- mice, or as CD4+CD8end- (CD4 lineage) and CD4-CD8end+ (CD8 lineage) for F5/CD8tg/MHC-II- mice. Splenocytes from F5/CD8tg/MHC-II- mice were stained on a different day, explaining the different levels of CD69 and 7AAD staining compared with all other mice. However, staining levels were indentical to those on cells from control mice stained on the same day.

 
To assess functional commitment in the absence of CD4 or MHC class II, we examined the production of IFN-{gamma} and IL-4, cytokines normally associated with the cytotoxic/Th1 and Th2 lineages, respectively. Given that the kinetics of all activation parameters are similar for CD4 and CD8 SP T cells (see above), we decided to perform the functional assays at the same time point (48 h postactivation). To compare IFN-{gamma} production by T cells from F5/CD8tg, F5/CD8tg/MHC-II-, and F5/CD8tg/CD4- mice, animals were injected with Ag or left untreated and spleens removed after 48 h. Splenocytes were cultured for 4 h in the presence of phorbol dibutyrate, ionomycin, and Brefeldin A, surface-stained with anti-CD4 and anti-CD8end or with anti-Thy1 and anti-CD8end, as described above, followed by staining for intracellular IFN-{gamma}. As shown in Fig. 4GoA, ~50% of the CD8 T cells from F5/CD8tg mice produce IFN-{gamma} after peptide injection (bold line), a percentage comparable to that of CD8+ cells from peptide-injected F5 mice (data not shown). In contrast, no significant fraction of the CD4 lineage cells was IFN-{gamma} positive in F5/CD8tg mice using this assay. However, a CD8end+-depleted T cell population from untreated F5/CD8tg mice was able to produce detectable amounts of IFN-{gamma} upon in vitro stimulation by peptide using an ELISA assay (data not shown). Similar results were obtained for F5/CD8tg/MHC-II- and F5/CD8tg/CD4- mice: after peptide injection the majority of CD8end+ cells, but only a small proportion of CD8end- cells, are positive for IFN-{gamma}.



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  		FIGURE 4. CD4 lineage T cells show low IFN-{gamma} but high IL-4 production. A, Splenocytes from untreated (thin solid line) or peptide-treated (bold solid line and shaded plot) mice with the indicated genotypes were stimulated for 4 h in vitro, as described in Materials and Methods, surface-stained with anti-CD8end plus either anti-CD4 or anti-Thy1, and subsequently with either anti-IFN-{gamma} (solid lines) or control (shaded plot) mAbs. Histograms show IFN-{gamma} staining of CD4 and CD8 lineage cells, gated as CD4+CD8end- (CD4 lineage) and CD4-CD8end+ (CD8 lineage) for F5/CD8tg and F5/CD8tg/MHC-II- mice, or as Thy1+CD8end- (CD4 lineage) and Thy1+CD8end+ (CD8 lineage) for F5/CD8tg/CD4- mice. B and C, Concentration of IL-4 in the supernatants of purified T cell populations from untreated mice with the indicated genotypes. T cell populations were purified as described in Results. Supernatants were collected after 48 h of culture in the presence of 1 µM NP68. Specificity was tested by adding anti-IL-4 mAbs. All results are representative of at least three experiments.

 
Next, we assessed the ability of purified T cell populations to produce IL-4 in vitro. Pooled lymph node and spleen cells from untreated F5, F5/CD8tg, or F5/CD8tg/MHC-II- mice were either left unseparated or depleted with magnetic beads coupled to anti-CD8end mAbs or anti-CD4 mAbs. Cells were cultured in the presence of 1 µM NP68 peptide for 48 h, and supernatants were collected and tested using the IL-4-dependent cell line CT4S. The proliferation of CT4S cells could be blocked by anti-IL-4 Abs, confirming the specificity of the assay. As shown in Fig. 4GoB, only activated CD4, but not activated CD8 populations produce significant amounts of IL-4. The unseparated T cells show relatively low IL-4 production compared with the CD4 population, probably due to dilution of CD4 T cells by CD8 T cells, which take over the culture under these conditions, or to excess IFN-{gamma} produced by CD8 T cells, which blocks Th2 type responses (IL-4) by the CD4 T cells. No IL-4 above background was produced by any cell population from F5 mice. For illustration purposes, the bar charts shown here represent the lowest dilution of supernatant in a titration curve (data not shown).

In F5/CD8tg/CD4- mice, CD4 cannot be used for depletion, and, therefore, we compared unseparated cells with populations depleted by anti-CD8end mAbs and, therefore, enriched in CD8end- T cells or panCD8-depleted populations that lack T cells altogether. As shown in Fig. 4GoC, the CD8end--enriched activated populations produce more IL-4 than unseparated activated cells. T cell-depleted activated populations do not produce measurable amounts of IL-4, suggesting that the stimulated CD4 lineage T cells are responsible for IL-4 production in these mice. Cell subsets from F5 mice that underwent the same depletion procedures do not produce detectable levels of IL-4. As the capacity of CD4 lineage T cells to produce IL-4 appears to be similar in F5/CD8tg, F5/CD8tg/MHC-II-, and F5/CD8tg/CD4- mice, we conclude that neither MHC class II nor CD4 is required for the development of this functional property, which is characteristic of Th2-type responses.

Helper and cytotoxic characteristics of T cells from F5/CD8tg/MHC-II- and F5/CD8tg/CD4- mice

To test more directly the helper capacities of T cell subsets from F5/CD8tg/MHC-II- and F5/CD8tg/CD4- mice, T cell-B cell coculture assays were performed. Purified T cell subsets were prepared as described in the previous section and cultured with or without 1 µM NP68 in coculture with CFSE-loaded B cells from TCR{alpha}- mice. After 4 days of coculture, cells were stained for B220 and panCD8 and FACS-analyzed. Since the amount of the fluorescent compound CFSE per cell is halved with every cell division (24), B cell proliferation can be assayed as percentage of B220+panCD8- cells with CFSE fluorescence lower than that of untreated B cells. The result of this experiment is shown in Fig. 5Go, A and B. Both for F5/CD8tg and F5/CD8tg/MHC-II- mice, B cell proliferation, as measured by loss of CFSE staining, is observed after coculture with activated CD4 T cells, but not after coculture with CD8 T cells or T cell-depleted cells (Fig. 5GoA). The unseparated T cell populations do not cause significant B cell proliferation, and, as mentioned above, in the case of IL-4 production, this is probably a combined effect of dilution of CD4 T cells and/or suppression of Th2 differentiation by high amounts of IFN-{gamma} produced by the CD8 T cells.



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  		FIGURE 5. CD4 but not CD8 lineage cells give help to B cells. B cell division after coculture with purified T cell populations from mice with the indicated genotype in the presence (hatched bars) or absence (black bars) of NP68. T cell populations were purified as described in Results. After 4 days, cocultures were stained for B220 and CD8 expression, and B cell proliferation was determined as percentage of CFSEdull cells within B220+CD8- cells. All experiments were repeated three times with similar outcome.

 
For F5/CD8tg/CD4- mice, coculture assays were performed with unseparated cells or populations that are enriched in CD8end- T cells or populations without T cells, as described above. Significant B cell proliferation above background is detected only after coculture with the population of activated CD8end- T cells (Fig. 5GoB). None of the populations from F5 mice cause B cell proliferation above background. Comparison of CD8end- T cells from F5/CD8tg/CD4- mice to those from F5/CD8tg mice shows no significant difference in the ability to give help to B cells over three coculture experiments.

Cytotoxic function of CD4 and CD8 lineage T cells from F5, F5/CD8tg, F5/CD8tg/MHC-II-, and F5/CD8tg/CD4- mice was measured ex vivo using 51Cr release assays. Mice were injected twice with NP68 i.p., sacrificed after 48 h, pooled lymph node and spleen cells were purified as described above, and their specific cytotoxic function was tested using as targets 51Cr-loaded EL-4 cells that were incubated with or without NP68. The CD4-populations from F5/CD8tg and F5/CD8tg/MHC-II- mice show insignificant peptide-specific lysis, whereas the total and the CD8 populations lyse peptide-loaded target cells very efficiently (Fig. 6GoA). In a similar manner, the total T cell population from F5/CD8tg/CD4- mice kills targets efficiently, whereas neither the population of CD8end- T cells nor the T cell-depleted population show significant peptide-specific lysis of target cells (Fig. 6GoB).



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  		FIGURE 6. CD8 but not CD4 lineage cells show peptide-specific cytotoxicity. T cell populations from NP68-treated mice with the indicated genotypes were left unseparated or depleted as indicated. The graph shows percent specific lysis of EL-4 target cells, loaded ({circ}) or not (•) with NP68, by the purified effector populations. Results are representative of five experiments.

 
In summary, by all criteria tested, it is apparent that CD4 lineage T cells in F5/CD8tg/MHC-II- and F5/CD8tg/CD4- mice are fully functional Th cells, identical to those from F5/CD8tg mice. In addition, the production of IL-4 suggests that CD4 lineage cells can develop Th2 function. Therefore, we conclude that, in this mouse model, generation of CD4 lineage cells, their commitment to helper function, and their Th2 characteristics are independent of the MHC class II or CD4 molecules.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we have addressed the question of whether CD4-MHC class II interactions are necessary for the establishment of a helper program during thymocyte development and for the induction of Th2 function upon peripheral activation. To separate the requirements for lineage decisions from those necessary to complete maturation, we have used F5/CD8tg mice, which develop both CD8 and CD4 lineage T cells through an F5 TCR-MHC class I-mediated process. Therefore, this model allowed us to examine the requirement of MHC class II or CD4 during maturation, when a helper lineage program is established.

For this purpose, we crossed F5/CD8tg mice to mice deficient in MHC class II or CD4 expression. The results presented here show clearly that, in this mouse model, lack of MHC class II or CD4 does not affect the numbers of CD4 lineage cells generated or the Th2 function they show upon activation. These results imply that MHC class II-CD4 interactions are not necessary at any developmental stage for the generation of Th cells, as long as the completion of their development is guaranteed by appropriate transgenes.

The role of MHC or coreceptor molecules in lineage commitment has been addressed in studies of mice singly deficient for these molecules. Thus, reports using MHC class II-deficient mice have shown a dramatic decrease in the number of CD4 T cells and a lack of Th function in such mice (6, 13, 25). In these studies, it is not possible to determine whether MHC class II deficiency prevents commitment to the CD4 helper lineage or if it prevents CD4-committed cells from completing their maturation. Similarly, the TCRhigh double negative T cells with helper function that are generated in low numbers in CD4-deficient mice (6, 7, 26) may express TCRs that are CD4-independent because of very high affinity for MHC class II, which would imply that commitment and maturation of helper cells in CD4- mice are still MHC class II-dependent.

Our results show that MHC class II-CD4 interactions become obsolete for generation and function of CD4 helper cells if the latter express CD8 and an MHC class I-restricted TCR as transgenes. This is in agreement with earlier reports, which showed that on a polyclonal background, CD8 transgene expression allows generation of CD4 lineage cells in the absence of MHC class II (27, 28), and with another report, in which lineage commitment toward CD4 in the absence of MHC class II was detected by assaying coreceptor reexpression after pronase treatment of thymocytes (29).

We cannot formally rule out the possibility that the CD8end- T cells found in F5/CD8tg/CD4- mice belong to an unusual double negative lineage rather than the CD4 lineage. Thus, the absence of CD4 may prevent the development of CD4 lineage cells and promote the maturation of double negative T cells, which are otherwise rare. We think this is unlikely since the CD8end- T cells found here are phenotypically and functionally identical to the corresponding CD8end-CD4+ T cell population found in F5/CD8tg mice. In addition, F5/CD4- mice do not contain a comparable population of double negative T cells. However, conclusive evidence that the CD8end- T cells belong to the CD4 lineage would require the knock in of a reporter gene into the CD4 locus, as was recently described by Chan et al. (30).

Recent publications have suggested that the CD4 molecule is required for IL-4 induction in Th cells (8, 31). In F5/CD8tg mice, we were able to test whether T cells expressing an MHC class I-restricted TCR, but committed to the helper lineage, are impaired in the development of Th2 responses. Here, we show not only that CD4 lineage cells in F5/CD8tg mice readily produce IL-4 after activation, but also that this Th2 function is unaffected by the absence of MHC class II or CD4. Thus, the activation signal mediated by F5-CD8-MHC class I interaction can efficiently replace the signal that normally induces Th2 responses in MHC class II-restricted T cells. This suggests that there is nothing qualitatively specific in the signal induced by MHC class II-CD4 interactions that dictates Th commitment.

It has been reported that a subclass of cytotoxic cells named Tc2 can produce IL-4 under specific culture conditions (32, 33). However, these cells retain their cytolytic activity. As the cells rescued in the CD4 lineage and described in this paper do not possess cytolytic function, we believe that they do not represent Tc2 type cells. Finally, we believe that the levels of CD8 present on the surface of the different populations do not play a significant role in the lineage decisions as F5/CD8tg mice deficient in CD8end (F5/CD8tg/CD8-/-) develop two cell lineages, i.e., CD4+CD8tg+ and CD4-CD8tg+, with characteristics identical to those described in all other genetic combinations presented in this paper.

The results presented here are consistent with a model that stipulates that the decision between a cytotoxic and helper program is taken either randomly or through interactions not requiring specific MHC-coreceptor ligation, for instance through interactions between notch and its ligands (34). Our findings that the lack of MHC class II or CD4 does not influence commitment to the CD4 lineage in F5/CD8tg mice are in line with this notion (10, 35). More importantly, in this paper, we extend the model to decisions concerning helper function and show that the induction of Th2 responses is equally independent of both MHC class II and CD4. In none of the mouse strains examined in this report could phenotypic differentiation (i.e., CD4 expression and/or down-regulation of endogenous CD8) be separated from functional commitment (i.e., IL-4 expression, B cell help). Thus, lineage commitment appears to entail the establishment of a differentiation program that results in the coordinate expression of a number of genes, including genes necessary for function and those encoding coreceptors. As the program for helper function can be activated even in the absence of CD4, we conclude that expression of this molecule by itself does not regulate this process, but that CD4 is merely one of the genes that is coordinately expressed within this differentiation program. In summary, our data suggest that coreceptor-MHC interactions are indispensable in the final steps of positive selection, but that they do not determine lineage commitment during early thymocyte development and do not dictate the functional profile of the T cells produced.


   Acknowledgments
 
We thank Drs. Ellen Robey, Diane Mathis, Dan Littman, and Mike Owen for the gift of mice; Drs. Caroline Johnson-Leger, Tracy Hussell, Barbara Fazekas de St. Groth, David Gray, and Mark Coles for helpful experimental advice; and Drs. Rose Zamoyska and Ellen Robey for critically reading the manuscript.


   Footnotes
 
1 A.W. was supported by a grant from the Boehringer Ingelheim Fonds and O.W. by a grant from the Leukemia Research Fund. Back

2 Current address: Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milano, Italy. Back

3 Address correspondence and reprint requests to Dr. Dimitris Kioussis, Division of Molecular Immunology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, U.K. E-mail address: Back

4 Abbreviations used in this paper: SP, single positive; CD8end, endogenous CD8; CFSE, carboxyfluorescein diacetate succinimidyl ester; NP, influenza virus nucleoprotein; Rag-1, Recombination-activating gene 1; tg, transgene; 7AAD, 7-aminoactinomycin D. Back

Received for publication February 23, 1999. Accepted for publication May 11, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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