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The Journal of Immunology, 1999, 163: 232-241.
Copyright © 1999 by The American Association of Immunologists

BALB/c Invariant Chain Mutant Mice Display Relatively Efficient Maturation of CD4+ T Cells in the Periphery and Secondary Proliferative Responses Elicited upon Peptide Challenge1

George Kenty and Elizabeth K. Bikoff2

Department of Molecular and Cellular Biology, Biological Laboratories, Harvard University, Cambridge, MA 02138


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Allelic differences are known to influence many important aspects of class II biosynthesis, including subunit assembly, Ii chain associations, and DM-mediated peptide loading. Mutant mouse strains lacking Ii chain expression have been previously studied on mixed genetic backgrounds. The present experiments describe cellular and functional characteristics of congenic BALB/c Ii chain mutants. As expected, class II surface expression was markedly decreased, but in contrast to I-Ad-transfected cell lines, serological analysis of BALB/c Ii chain-deficient spleen cells gave no evidence for discordant expression of class II conformational epitopes. Thus, we conclude that properly folded class II molecules are exported via the Ii chain-independent pathway. Functional assays demonstrate consistently superior peptide-loading capabilities, suggesting that these I-Ad molecules are empty or occupied by an easily displaced peptide(s). Defective B cell development was observed for three mutant strains established on diverse genetic backgrounds. Ii chain function is also essential for optimal class II surface expression by mature splenic dendritic cells. Surprisingly, we observe in BALB/c Ii chain mutants, relatively efficient maturation of CD4+ T cells in the periphery and secondary proliferative responses elicited upon peptide challenge. The milder phenotype displayed by BALB/c Ii chain mutants in comparison with class II functional defects previously described for mouse strains lacking Ii chain is likely to have an effect on disease susceptibility.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The MHC class II membrane glycoproteins selectively expressed by B cells, dendritic cells, macrophages, and thymic epithelial cells guide CD4+ T cell responses toward diverse foreign pathogens and promote selection of a broad receptor repertoire during thymic development. Polymorphic residues contributed by both {alpha} and ß subunits are found in the three-dimensional structure to create the peptide binding cleft that accommodates peptides 15–20 aa in length lacking precisely defined termini. The highly conserved Ii3 chain acts as the functional equivalent of peptide during early stages of class II biosynthesis to prevent irreversible misfolding or aggregation of the subunits and protect the empty groove from association with molecular chaperones such as BiP and calnexin that are responsible for ER quality control (1, 2, 3, 4). The Ii chain facilitates export of correctly folded {alpha}ß dimers past the cis-Golgi complex and targets immature class II to peripheral endocytic compartment(s), where selective Ii chain degradation subsequently permits occupancy of the groove by specific peptide fragments (5). Peptide elution experiments (6, 7, 8, 9), transfection assays (10, 11), and x-ray crystal studies (12) all demonstrate class II associates with Ii chain via its CLIP sequence. On intact Ii chain, this region comprises a highly disordered flexible domain accessible to proteases (13). In contrast, CLIP bound to class II has an extended rigid structure and, as for conventional peptide, extensively interacts with both {alpha} and ß chain residues (12). The nonconventional class II product DM selectively acts inside endocytic compartments to cause CLIP dissociation in exchange for tightly bound peptide ligand(s) (14). Recent studies demonstrate that DM also associates with empty class II molecules to prevent misfolding in endocytic compartments and, acting in this manner, may function as a peptide editor serving to increase the overall affinities of peptide/class II complexes (15, 16, 17, 18, 19).

Class II allelic diversity influences the intrinsic stability of {alpha}ß dimers (20) and many important aspects of Ii chain and DM interactions (21, 22, 23, 24, 25, 26, 27, 28, 29, 30). As a general rule, assembly of allelically matched {alpha}ß pairs does not require Ii chain coexpression, but in the exceptional case of A{alpha}bAßb molecules, Ii chain expression has been shown to facilitate production or maintenance of {alpha}ß dimers (22). Allelic differences affect the kinetics and specificity of CLIP associations (23, 24, 27). Recent studies demonstrate that promiscuous CLIP binding to structurally diverse class II grooves is mediated via allele-specific contacts (23, 27). Similarly, allelic variants differ with respect to Ii chain degradation intermediates (21, 28, 29) and the requirement for DM activities during Ag presentation and peptide loading (25, 26). Moreover, allele-specific functional disturbances were recently described for DM mutant mice (30).

Ii chain activities as a specific class II chaperone have been described in mutant strains created using ES cell technology (31, 32, 33). Ii chain mutant mice originally studied on a mixed (C57BL/6 x 129)F2 background display dramatically reduced surface class II, owing in large measure to decreased rates of post-ER export (31, 32, 33). The absence of Ii chain causes Ag presentation defects and markedly decreased numbers of mature CD4+ T cells in the thymus and periphery (31, 32, 33). Ii chain mutants expressing three different MHC haplotypes fail to produce mature compact class II dimers tightly occupied by peptide ligand(s) (22, 31, 32, 33). The few mature A{alpha}bAßb dimers expressed in the absence of Ii chain exhibit reduced mobilities in SDS gels and markedly enhanced peptide binding capabilities (31, 32, 33). Taken together, biochemical and functional experiments strongly suggest that these floppy A{alpha}bAßb conformers are empty or occupied by easily displaced peptide(s). Moreover, these findings argue that under physiological conditions class II peptides are predominantly loaded via an Ii chain-dependent pathway(s).

In contrast, Ii chain mutants expressing H-2k and H-2d haplotypes gave no evidence for expression of floppy conformers (22), and in T cell stimulation assays, Viville et al. (32) described indistinguishable peptide titration curves, as expected for class II molecules stably occupied by self peptide ligand(s). Considering that these experiments analyzed (C57BL/6 x 129)F2 progeny, subtle strain differences could be masked due to contributions by background genes such as the stimulatory Mlsa locus (34, 35). Alternatively, allele-specific DM activities potentially influence constitutive expression of empty class II molecules and thus the efficiency of Ii chain-independent peptide loading pathway(s). According to this way of thinking, Ii chain mutants expressing different MHC haplotypes may be expected to display strain-dependent class II functional deficiencies.

We previously described immature A{alpha}dAßd dimers efficiently assembled in the absence of Ii chain (22), but cellular and functional characteristics of our BALB/c Ii chain mutants have not yet been reported. Inbred BALB/c mice are widely used for studying genetic control of susceptibility to parasitic infections, and strain differences affecting the ratio of Th1/Th2 cytokine responses. Recent experiments suggest that Th subset differentiation occurs normally in Ii chain-deficient BALB/c backcross progeny (36), but genetic background contributions to Ii chain requirements per se were not examined. The present report describes class II functional activities of congenic BALB/c mice lacking Ii chain expression. Surprisingly, we observed relatively efficient maturation of CD4+ T cells in the periphery and secondary proliferative responses elicited upon peptide challenge. This milder phenotype displayed by BALB/c Ii chain mutants, in contrast to the striking class II functional defects previously described on mixed genetic backgrounds, predicts relatively uncompromised in vivo responses directed toward foreign pathogens.


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

Inbred 129/Sv/Ev Ii chain mutants were produced by mating the ES cell-derived germline male chimeras described previously (31) to 129/Sv/Ev females. The generation of Ii chain-deficient mice expressing three independent MHC haplotypes by backcrossing the targeted allele onto BALB/cAn (H-2d), B10.BR/SgSn (H-2k), or B.C-9, a strain congenic with C57BL/6 but expressing the Igha allotype of BALB/c, has been described (22). A PCR genotyping assay was used to identify the mutant allele (22) during subsequent backcrosses. The congenic strains analyzed in the present report were established by intercross matings at the 10th backcross generation. BALB/cAn and B.C-9 Ii chain mutants are available from The Jackson Laboratory Induced Animal Resource (Bar Harbor, ME). C57BL/6TacfBR-[KO]AßbN5(B6.Aßo) mice were purchased from Taconic Farms (Germantown, NY). In all experiments comparisons were made between age- and, whenever possible, sex-matched animals.

Abs and peptides

Hybridomas used in the present study include MKD6 specific for a private Aßd epitope (37, 38), 25-9-17 (39) specific for Aßd/b, BP107 (40) specific for Aßd/b, M5/114 (41) specific for (Aß + Eß), 14-4-4 (42) specific for E{alpha}d, 10-2-16 (43) specific for Aßk, Y3P (44) specific for Ab({alpha} + ß), K24-199 (45) specific for A{alpha}d, and anti-mouse CD11c (N418) (46). Y3P was the gift of C. A. Janeway, Jr. (Yale University Medical School, New Haven, CT), K24-199 was provided by G. Hammerling (German Cancer Research Center, Heidelberg, Germany), and other cell lines were obtained from the American Type Culture Collection (Manassas, VA). The chain specificities of class II mAbs and Ii chain influences affecting expression of conformational epitopes, have been extensively discussed (22, 47). The OVA323–339 (ISQAVHAAHAEINEAGR), IgG2ab435–451 (YFMYSKLRVQKSTWERG), bacteriophage {lambda} repressor cI peptide P12–26 (YLEDARRLKAIYEKKK), and HEL46–61 (NTDGSTDYGILQINSR) peptides were purchased from Quality Controlled Biochemicals (Hopkinton, MA).

Immunofluorescence analysis

For single-color analysis, spleen cell suspensions depleted of erythrocytes by ammonium chloride-Tris treatment were incubated on ice with saturating amounts of biotin-conjugated Abs followed by FITC-labeled avidin D. Fluorescence was analyzed using a FACScan flow cytometer (Becton Dickinson, Mountain View, CA), and the data are displayed as cell number vs log fluorescence. Dead cells were eliminated from the analysis by appropriate gating. For double-staining experiments analyzing B cell subsets, spleen or lymph node cells were incubated with PE-conjugated goat F(ab')2 anti-mouse IgM (u) (Caltag, San Francisco, CA; catalogue no. M31604) as a pan-B cell marker used in combination with FITC-labeled Abs directed against the IgE Fc receptor CD23 (PharMingen, San Diego, CA; catalogue no. 01234D) or surface IgD (PharMingen catalogue no. 02214D). For T cell subset analysis, suspensions of thymocytes, lymph node, or spleen cells were incubated on ice with anti-CD8-FITC, anti-CD4 PE, biotinylated anti-TCR (PharMingen catalogue no. 01044D, 01065B, and 01302D, respectively) followed by streptavidin red 670 (Life Technologies, Gaithersburg, MD). CD4 vs CD8 dot plots are shown.

For experiments analyzing dendritic cell class II expression, cell suspensions were incubated with anti-mouse CD11c (N418) culture supernatants followed by PE-conjugated goat anti-hamster IgG (H+L) (Caltag, San Francisco, CA; catalogue no. HA6004) as a dendritic cell marker in combination with biotin-labeled class II mAbs and FITC-conjugated avidin D as described above. Freshly isolated splenic dendritic cells were prepared using spleens perfused and then gently teased into RPMI 1640 medium containing 10% FCS and collagenase (Worthington Biochemical, Lakewood, NJ; catalogue no. CLSS-4, 100 U/ml), and subsequently treated for 30 min at 37°C with 400 U/ml collagenase before passage through nylon gauze to obtain single cell suspensions. This population was cultured on plastic petri dishes for 2 h at 37°C and gently rinsed to remove nonadherent cells, and the loosely adherent population harvested after overnight culture.

Ag presentation assays

T cell hybridomas used in this study include DO11.10 specific for I-Ad/OVA323–339 (48) and AODH7.1 specific for I-Ed/HGG (49) provided by Philippa Marrack (Howard Hughes Medical Institute, National Jewish Center, Denver, CO), and 7B7.3 specific for I-Ad/{lambda} P12–26 (50) given to us by Malcolm Gefter (Massachusetts Institute of Technology, Cambridge, MA). The BALB/c I-Ad-restricted hybridoma C5–46 and the cloned T cell line C1–15, both specific for IgG2a of the b allotype, have been described (51). Transfection assays originally demonstrated their fine specificity for determinants encoded by the IgG2a CH3b-coding region (51, 52). Consistent with mapping studies reported by Bartnes and Hannestad (53, 54), the C1–15 clone recognizes the IgG2ab peptide comprised of residues 435–451 (55). The BALB/c keyhole limpet hemocyanin-specific polyclonal T cell line was isolated from Ag-primed lymph node cells using the same protocol as that described for generation of C1–15 through the use of repeated Ag stimulation followed by short periods of rest in the presence of irradiated spleen cells.

IL-2 production by T cell hybridomas was assessed by incubating T cells (5 x 104/well) with spleen cells (2 x 105/well) in 200 µl of complete RPMI 1640 supplemented with 15% FCS, 10% NCTC109, 100 U/ml penicillin, 100 µg/ml streptomycin, 1 mM sodium pyruvate, 15 mM HEPES (pH 7.2), 0.1 mM nonessential amino acids, 5 x 10-5 M 2-ME, 2 mM glutamine, and increasing concentrations of Ag. Supernatants were collected after 20 h and assayed for IL-2 content in a secondary culture with CTLL indicator cells in the presence of 50% primary supernatants. Responsiveness of normal T cells was analyzed using a proliferation assay (51). Briefly rested T cells (2 x 104/well) were incubated with irradiated spleen cells (5 x 105/well) in 96-well microtiter plates. For mixed lymphocyte reactions, nylon-purified T cells (4 x 105/well) and increasing numbers of irradiated spleen cells were cultured for 72 h. The degree of stimulation was measured by a 16- to 18-h exposure to 1 µCi of [3H]thymidine. All results are expressed as the mean counts per minute of triplicate cultures.

T-dependent proliferative responses

Eight- to 10-wk-old animals were immunized s.c. at the base of the tail with intact Ags or peptide (100 µg) in CFA. Seven days later, inguinal and para-aortic lymph nodes were gently teased, and cell suspensions (5 x 105 in 200 µl) were cultured in complete RPMI 1640 medium supplemented as described above. Proliferation was assessed after 2 days by a 16-h exposure to 1 µCi of [3H]thymidine.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Conformational analysis of surface class II

In transfected cells, the Ii chain acts as a chaperone to enhance class II export (56, 57, 58, 59) and promote mature class II conformation (52, 60, 61, 62). Thus, surface A{alpha}dAßd molecules produced in the absence of Ii chain exhibited a distinctive serological profile (52, 60, 61). To examine Ii chain contributions to mature class II conformation in professional APCs under physiological conditions, surface expression by BALB/c Ii chain-deficient and control wild-type spleen cells was tested using a panel of mAbs. As shown in Fig. 1Go in the absence of Ii chain function, there was no evidence for conformational changes affecting class II reactivity patterns. Rather, BALB/c Ii chain mutant spleen cells were weakly stained with MKD6 (Aßd-specific) mAb (37, 38), previously shown to react with Ii chain-independent epitope(s) (52, 60, 61). Similar results were obtained using BP107 (Aß-specific) mAb (40) previously used to detect DM dependent conformational determinant(s) (47, 63, 64), 25-9-17 (Aß-specific) mAb (39) reactive with peptide-dependent epitope(s) (65), M5/114 mAb (41) specific for (Aß + Eß) determinants, K24–199 (45) (A{alpha}d-specific), and 14-4-4 (42) (E{alpha}d-specific) mAb. Thus, in contrast to A{alpha}dAßd-transfected cells, BALB/c Ii chain-deficient spleen cells gave no evidence for selective expression of class II conformational epitopes. These findings demonstrate that mature properly folded class II molecules are exported via an Ii chain-independent pathway(s).



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  		FIGURE 1. Surface expression of Ii chain-dependent conformational epitopes. Splenocytes from +/+ (thick line) or Ii- (thin line) BALB/c mice were stained with biotin-conjugated mAbs followed by FITC-conjugated avidin.

 
Superior peptide-loading capabilities and partially defective presentation of intact protein Ags

Biochemical and functional experiments strongly suggest that floppy A{alpha}bAßb conformers produced in the absence of Ii chain are empty, or occupied by easily displaced peptide(s). In contrast, Ii chain mutants expressing the k haplotype gave indistinguishable peptide titration curves, as expected for class II molecules stably occupied by self peptide ligand(s) (32). These results suggest allelic differences influence constitutive pathways for self peptide capture. To examine this possibility, we tested BALB/c Ii chain mutant spleen cells for their abilities to stimulate IL-2 production by T cell hybridomas and proliferative responses of long-term T cell lines. As shown in Fig. 2Goa, BALB/c mutant spleen cells consistently display markedly enhanced peptide-loading capabilities. Similar results were obtained using T cell clones specific for three different peptides, namely OVA323–339, {lambda}P12–26, and IgG2ab. Thus we conclude that, as for A{alpha}bAßb, functionally empty A{alpha}dAßd molecules are produced in the absence of Ii chain expression.



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  		FIGURE 2. Ag presentation capabilities. Splenocytes from +/+ (•) or Ii chain mutants ({circ}) were tested for their ability to present peptides (a), intact proteins (b), or alloantigens (c). IL-2 production by T cell hybridomas or proliferative responses of long-term T cell lines as indicated (a and b), and stimulation of allogeneic T cells (c) were measured by a 16-h exposure to 1 µCi of [3H]thymidine. All results are expressed as the mean counts per minute of triplicate cultures.

 
As expected, BALB/c mutant spleen cells appear generally ineffective for presentation of intact protein Ags (Fig. 2Gob). We also observe in the presence of selected T cell clones (for example, C1–15), vigorous Ii chain-independent responses. Thus, the ability of Ii chain to enhance Ag presentation seems to depend on the particular T cell epitope. As shown in Fig. 2Goc, Ii chain mutant spleen cells efficiently function as stimulators for allogeneic T cells. Strong mixed lymphocyte responses were directed toward Ii chain mutant spleen cells expressing three independent MHC haplotypes. These results strongly suggest that class II molecules produced in the absence of Ii chain efficiently present via constitutive pathway(s) a broad spectrum of diverse peptides. Moreover, this degree of occupancy appears sufficient for initiating CD4+ T cell responses.

Mature CD4+ T cells in the periphery

Ii chain mutants on a (C57BL/6 x 129)F2 mixed background have greatly reduced numbers of mature CD4+ T cells in the thymus and periphery (31, 32, 33). To examine the extent of CD4+ T cell development in BALB/c mice lacking Ii chain function, we analyzed T cell subpopulations using three-color flow cytometry. Consistent with previous results, mutant thymi display roughly 3-fold fewer mature CD4+ T cells (Fig. 3Go). Surprisingly, we found that BALB/c mutants contain substantial numbers of peripheral CD4+ T cells. Thus, spleen and lymph node CD4+ T cell percentages decreased at most 2-fold. The less severe CD4+ maturation defect observed here for BALB/c mice lacking Ii chain does not simply reflect changes in animal health status over time, because our H-2b Ii chain mutants analyzed in the same experiments consistently display a more striking phenotype (data not shown). Thus, in the context of the BALB/c background, Ii chain plays a critical role during thymic selection, but peripheral expansion of CD4+ T cells is partially rescued via an Ii chain-independent pathway(s).



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  		FIGURE 3. T cell subsets. Thymus, spleen, and lymph node cell suspensions were stained for CD4 and CD8 expression and analyzed by flow cytometry. The numbers refer to the percentages of total cells within the indicated gates. Representative data from one of seven identical experiments with similar results are shown.

 
Impaired selection of mature B cells

Ii chain mutants on a (C57BL/6 x 129)F2 mixed background display cellular disturbances affecting B cell development (47, 66). Similar changes affecting the representation of B cell subsets are caused by disruption of the A{alpha}b gene (47), but in contrast, class II mutants created by targeting the Aßb locus were found to contain normal B cell populations (66, 67, 68). These studies leave open the question whether Ii chain functions during B cell maturation are restricted to class II chaperone activities. One simple scenario is these functional discrepancies reflect strain differences contributed by unlinked loci. To test this possibility, we examined B cell maturation in congenic Ii chain mutant strains established on three diverse genetic backgrounds, namely BALB/cAn, B10.BR/SgSnJ, and 129/Sv/Ev. As a marker for mature B cells, we analyzed surface expression of CD23, the low affinity IgE Fc receptor (69, 70). Spleen and lymph node IgM+ B cells were also tested for coexpression of surface IgD. In wild-type mice, the predominant population of mature IgM+ B cells coexpresses both IgD and CD23 surface markers (Fig. 4Go). In contrast, Ii chain mutants contain increased percentages of immature B cells lacking surface IgD and CD23 expression. The loss of Ii chain function also causes a striking depletion of IgM+ B cells in lymph node populations. Thus, Ii chain mutants expressing diverse genetic backgrounds exhibit defective B cell maturation. Interestingly, wild-type 129/Sv/Ev mice consistently have decreased percentages of mature B cells, suggesting that background loci also influence B cell development.



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  		FIGURE 4. Defective B cell maturation. Spleen and lymph node cell suspensions from 12- to 15-wk-old animals were stained using PE-conjugated anti-IgM in combination with FITC-conjugated anti-IgD or anti-CD23 and were analyzed by flow cytometry. The numbers refer to the percentages of total cells within the indicated gates. Representative data from one of five identical experiments with similar results are shown.

 
Ii chain necessary for optimal class II surface expression by dendritic cells

Recent experiments suggest that dendritic cells selectively capture peptides via Ii chain-independent pathway(s) (71), and allelic differences were also reported to influence dendritic cell class II activities. Thus, dendritic cells derived from B10.BR Ii chain mutants were found to strongly express surface I-Ak and stimulate I-Ak-restricted T cells, whereas dendritic cells isolated from H-2b mice were reported to display Ii chain chaperone requirements (71). To examine this important issue, we decided to assess class II surface expression by splenic dendritic cells, comparing our Ii chain mutant strains carrying three independent genetic backgrounds. Freshly isolated collagenase-treated splenocytes or loosely adherent cell populations recovered after overnight culture were doubly stained using anti-CD11c (N418 mAb) (46) to identify dendritic cells and MKD6 (37), 10-2-16 (43), or Y3P (44) mAb for detection of class II surface expression. As judged by the staining profiles of gated N418+ dendritic cell populations, the mutants exhibit markedly decreased levels of class II surface Ags (Fig. 5Go). Despite increased surface expression observed for dendritic cells analyzed after overnight culture (Fig. 5Gob) in comparison to results obtained using freshly isolated splenocytes (Fig. 5Goa), we consistently found that Ii chain mutant dendritic cells display reduced fluorescence intensities. Thus, we conclude that the Ii chain is necessary for optimal class II surface expression by splenic dendritic cells.



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  		FIGURE 5. Reduced class II surface expression by splenic dendritic cells. Collagenase-treated splenocytes (a) or loosely adherent cell populations recovered after overnight culture (b) were stained with anti-CD11c (N418 mAb) and MKD6 (anti-I-Ad), 10-2-16 (anti-I-Ak), or Y3P (anti-I-Ab) mAb for detection of class II surface expression. The histograms show class II staining profiles of gated N418+ cell populations. The mean fluorescence values for +/+ (thick line) or Ii chain mutant (thin line) double-positive cell populations are indicated by the numbers. Data are representative of three independent experiments.

 
Efficient secondary proliferative responses elicited upon peptide challenge

The experiments above demonstrate enhanced peptide-loading capabilities and efficient presentation of alloantigens by BALB/c Ii chain mutant spleen cells. The mutants also contain substantial numbers of mature CD4+ T cells in the periphery. To test whether the Ii chain is required for activation of class II-restricted CD4+ T cells in vivo under physiological conditions, we analyzed secondary proliferative responses of local draining lymph node populations. As shown in Fig. 6Go, the BALB/c mutant mice gave decreased responses directed toward intact protein Ags. Interestingly, we observe indistinguishable dose-response curves upon secondary challenge with OVA323–339 peptide, in BALB/c mice immunized with intact OVA (Fig. 6Gob). Similarly as shown in Fig. 6Go, d–e, B10.BR mutants fail to respond to intact HEL, but do generate vigorous proliferative responses toward the immunodominant HEL46–61 peptide. In contrast, H-2b mutant mice gave barely detectable responses following peptide immunization (Ref. 72 and our unpublished observations).



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  		FIGURE 6. T-dependent proliferative responses. Draining lymph node cell populations isolated from BALB/c (a–c) or B10. BR (d and e) wild-type (•) or congenic Ii chain mutants ({circ}) primed with intact OVA (a and b), keyhole limpet hemocyanin (c), HEL (d), or HEL46–61 peptide (e) were challenged in vitro with whole proteins (a, c, and d) or peptides (b and e). After 2 days, proliferation was assessed by a 16-h exposure to 1 µCi of [3H]thymidine. Results are expressed as the mean counts per minute of triplicate cultures. Data are representative of three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Here we describe for the first time cellular and functional characteristics of Ii chain mutants expressing the H-2d haplotype. We examined class II activities of BALB/c Ii chain mutants at the 10th backcross generation, greatly increasing the likelihood that phenotypic difference(s) solely reflect Ii chain influences. Surprisingly, we found that BALB/c Ii chain mutant mice appear relatively immunocompetent in comparison with the striking class II defects previously attributed to Ii chain loss of function mutations (31, 32, 33, 72). These congenic BALB/c Ii chain mutants should prove useful for dissecting contributions made by Ii chain to host disease in the absence of other genetic influences.

Although early transfection experiments demonstrated substantial surface expression of A{alpha}dAßd molecules in the absence of Ii chain (73), subsequent studies describe Ii chain abilities to enhance class II export and promote conformational maturation (52, 56, 57, 58, 59, 60, 61, 62). Thus, surface A{alpha}dAßd molecules expressed by transfected cells lacking Ii chain exhibit a distinctive serological profile (52, 60, 61). This discordant representation of class II conformational epitopes has been taken as evidence for Ii chain influences affecting the structure of mature class II expressed on the cell surface. Here we observe that the absence of Ii chain function causes markedly reduced amounts of total class II surface expression, but in striking contrast to results obtained using transfected cell lines (52, 60, 61), the present experiments gave no evidence for discordant representation of class II conformational epitopes. Rather, several mAbs directed against distinct sites, including MKD6 (Aßd-specific) mAb, previously used to detect Ii chain-independent epitope(s) (52, 60, 61), all weakly stained surface class II expressed by BALB/c mutant spleen cells. Such discrepancies in the literature probably reflect suboptimal Ii chain expression levels, possibly the absence of DM peptide-editing functions, or other essential components of the class II maturation pathway lacking in transfection recipient cell lines. In contrast, here comparisons were made using spleen cell populations identical in every respect except for Ii chain expression. The present data clearly demonstrate that professional APCs can export properly folded A{alpha}dAßd molecules via an Ii chain-independent pathway(s).

As for floppy A{alpha}bAßb conformers, we also found here in functional assays that A{alpha}dAßd molecules expressed by BALB/c mutant spleen cells display markedly enhanced peptide-loading capabilities. Thus, an Ii chain-dependent pathway(s) is essential for self peptide capture under physiological conditions. The equivalent dose-response curves previously observed for Ii chain mutant spleen cells suggestive of class II molecules stably occupied by self peptide ligand(s) (32) probably reflect background influences contributed by unlinked loci. Consistent with this idea, C4H3 mAb specific for I-Ak/HEL46–61 complexes detects increased binding of exogenous peptide administered in vivo, as expected for congenic B10.BR Ii chain mutant spleen cells with superior peptide-loading capabilities (74). Thus, as a general rule, Ii chain mutant spleen cells are quite competent for presentation of already processed peptide ligands.

Ii chain-deficient spleen cells function effectively as stimulators for allogeneic T cells. Moreover, we observed vigorous mixed lymphocyte responses elicited by mutant spleen cells expressing three independent MHC haplotypes. Thus, class II molecules exported to the cell surface via an Ii chain-independent pathways(s) efficiently engage a broad repertoire of polyclonal TCR. At first glance, Ii chain-independent presentation of alloantigens appears somewhat at odds with the present observations, suggesting that functionally empty class II molecules are produced in the absence of Ii chain. On the other hand, the idea that allogeneic responses are directed toward polymorphic residues on empty class II molecules rather than specific peptide/class II complexes has considerable merit (75). However, recent evidence strongly argues that this is not the case. Thus, alloreactive T cell clones have the ability to distinguish diverse peptides as expected if specific peptide promotes TCR associations (65, 76, 77). Biochemical studies also demonstrate that binding site occupancy is necessary for class II export through the secretory pathway (78). Recent x-ray crystal studies strongly suggest that CD4+ T cells specific for I-Ad peptide complexes have reactivity toward peptides that only partially fill the class II groove (79). Perhaps in cells lacking the Ii chain, class II transiently associates with signal peptides (80, 81) or intact polypeptides available in the ER (82, 83). Moreover, recent evidence suggests that short-lived class II peptide complexes such as those extensively studied in vitro (84, 85, 86, 87, 88, 89) may allow escape from tolerance induction in vivo (90, 91, 92). Surface class II expression is essential for peripheral CD4+ T cell survival (93, 94). The present findings strongly suggest that low affinity peptide/class II complexes expressed in BALB/c Ii chain mutants can initiate TCR cross-linking and promote peripheral expansion of CD4+ T cells.

Although >90% of A{alpha}bAßb migrates in SDS-PAGE as compact dimers, this population represents a much smaller percentage of mature A{alpha}kAßk and A{alpha}dAßd molecules in the steady state (22, 95). These allelic differences may reflect polymorphic influences affecting interchain contacts and/or selection of self peptides. The peptides bound to I-Ad appear to lack a clearly distinct nine-residue sequence motif (8, 96). Recent x-ray crystal studies of A{alpha}dAßd-peptide complexes demonstrate that high affinity interactions are achieved without insertion of large anchor residues into deep binding pockets (79). The floor of its peptide-binding groove contains an unusual ß-chain bulge, affecting subunit contacts and imposing spatial restrictions for peptide interactions. Specific peptide only partially fills the relatively shallow A{alpha}dAßd groove and potentially accounts for the decreased representation of compact A{alpha}dAßd dimers.

Recent experiments suggest that the Ii chain on its own plays a critical role during B cell maturation. Thus, Ii chain mutants exhibit defective B cell development (47, 66). Similar changes affecting representation of B cell subsets are caused by disruption of the A{alpha}b gene (47, 97), but in striking contrast, class II mutants created by targeting the Aßb locus contain normal B cell populations (66, 67, 68, 98). Similarly, B cell development appears unperturbed in CIITA-deficient mice (66), suggesting that B cell defects are not caused by the loss of class II per se. Because mutant mouse strains were independently established and separately maintained in different laboratories, functional discrepancies potentially reflect strain differences contributed by unlinked loci. Consistent with this suggestion, we found that inbred 129 mice consistently have decreased percentages of mature B cells compared with other strains. This contribution is likely to influence B cell characteristics observed for mutant strains randomly established on a mixed (C57BL/6 x 129)F2 genetic background.

The recent report by Zimmerman et al. (104) suggests that the Ii chain is not required for B cell maturation in H-2k mice. In contrast, we found that the Ii chain loss of function mutation causes defective B cell maturation on three diverse backgrounds. There are several likely reasons why our findings differ. Firstly, these investigators used genetically mixed (B10.BR x CBA/J)F2 animals for their analysis, whereas, in contrast, we examined congenic B10.BR Ii chain mutants. Secondly, their single-color FACS profiles do not offer the same high degree of sensitivity as our two-color analysis shown in Fig. 4Go. Additionally, these investigators assessed only IgD and not CD23 as a B cell differentiation marker. Finally, these investigators studied only splenic B cells, whereas we found that lymph node cell populations consistently display more severe B cell defects. Intrinsic B cell defects associated with the absence of Ii chain and class II {alpha}-chain, but not ß-chain, expression is probably coincident with the onset of class II surface expression (99). Interestingly, similar B cell abnormalities are associated with high copy number I-Aß transgenes (100, 101, 102). Higher order ß-chain aggregates accumulate in Ii chain mutant spleen cells (3, 33, 103). Free ß-chain appears especially prone to misfolding and aggregation, probably due to the formation of inappropriate disulfide bonds during early folding of the ß1 domain. In contrast, the {alpha}1 domain does not contain paired cysteines. Additional work is needed to describe the downstream mechanism(s) compromising B cell viability under these circumstances.

Isolated dendritic cells recovered from B10.BR Ii chain mutant mice were reported to express surface I-Ak and stimulate I-Ak-restricted T cells at levels comparable to wild-type (71) and, in contrast, here we observe defective class II surface expression by freshly isolated splenic dendritic cells. Similar conclusions were reached assessing Ii chain mutants on three diverse genetic backgrounds, namely B.C-9, BALB/c, and B10.BR expressing H-2b, H-2d, and H-2k haplotypes, respectively. Moreover, our recent studies demonstrate Ii chain-dependent I-Ek surface expression on the three APC populations in the spleen, B cells, marcophages, and dendritic cells (105). Interestingly, we found that loosely nonadherent cell populations after overnight culture exhibit less striking Ii chain influences. Class II surface expression by enriched populations of dendritic cells purified on density gradients or following long-term in vitro stimulation and freshly isolated splenic dendritic cells examined immediately after collagenase treatment is strongly upregulated. The present findings demonstrate that the Ii chain functions as a class II chaperone to promote optimal surface expression by mature splenic dendritic cells, consistent with the idea that the Ii chain is required for self peptide capture under physiological conditions in the intact animal. On the other hand, it is of course possible that selected dendritic cell subsets, particularly those studied in growth factor-dependent long-term cultures, may preferentially use Ii chain-independent class II peptide-loading pathways.

Allelic diversity influences the intrinsic stability of {alpha}ß dimers (20) and many important aspects of Ii chain and DM functions (21, 22, 23, 24, 25, 26, 27, 28, 29, 30). Class II polymorphic differences also have an impact on CD4 interactions (106). The present results demonstrate strain-specific Ii chain functional requirements. Recent experiments document Ii chain-independent protective hosts responses to the intracellular parasite Leishmania (36) and selected viruses (107, 108). These complex responses directed toward pathogenic organisms are probably influenced by multiple loci, as is polygenic control of autoimmune disease. Congenic BALB/c mutant mice described in the present report should prove useful for studying Ii chain contributions to host protection and disease susceptibility.


   Acknowledgments
 
We thank Debbie Pelusi for valuable assistance screening mutant progeny, Patti Lewko and Joe Rocca for careful maintenance of the mouse colony, Jessica Wapner for secretarial assistance, and Renate Hellmiss for preparing the figures.


   Footnotes
 
1 This work was supported by Grant AI-19047 from the National Institutes of Health. Back

2 Address correspondence and reprint requests to Dr. Elizabeth K. Bikoff, Department of Molecular and Cellular Biology, Biological Laboratories, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138. E-mail address: Back

3 Abbreviations used in this paper: Ii, invariant; ER, endoplasmic reticulum; CLIP, class II-associated Ii chain-derived peptide; ES, embryonic stem. Back

Received for publication December 31, 1998. Accepted for publication April 23, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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