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.

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 α and β 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 αβ 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 α and β 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 αβ 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 αβ pairs does not require Ii chain coexpression, but in the exceptional case of Aαbb molecules, Ii chain expression has been shown to facilitate production or maintenance of αβ 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 × 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 Aαbb 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 Aαbb 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 × 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 Aαdd 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.

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]AβbN5(B6.Aβo) mice were purchased from Taconic Farms (Germantown, NY). In all experiments comparisons were made between age- and, whenever possible, sex-matched animals.

Hybridomas used in the present study include MKD6 specific for a private Aβd epitope (37, 38), 25-9-17 (39) specific for Aβd/b, BP107 (40) specific for Aβd/b, M5/114 (41) specific for (Aβ + Eβ), 14-4-4 (42) specific for Eαd, 10-2-16 (43) specific for Aβk, Y3P (44) specific for Ab(α + β), K24-199 (45) specific for Aαd, 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 λ repressor cI peptide P12–26 (YLEDARRLKAIYEKKK), and HEL46–61 (NTDGSTDYGILQINSR) peptides were purchased from Quality Controlled Biochemicals (Hopkinton, MA).

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.

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/λ 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 × 104/well) with spleen cells (2 × 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 × 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 × 104/well) were incubated with irradiated spleen cells (5 × 105/well) in 96-well microtiter plates. For mixed lymphocyte reactions, nylon-purified T cells (4 × 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.

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 × 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.

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 Aαdd 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. 1 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 (Aβd-specific) mAb (37, 38), previously shown to react with Ii chain-independent epitope(s) (52, 60, 61). Similar results were obtained using BP107 (Aβ-specific) mAb (40) previously used to detect DM dependent conformational determinant(s) (47, 63, 64), 25-9-17 (Aβ-specific) mAb (39) reactive with peptide-dependent epitope(s) (65), M5/114 mAb (41) specific for (Aβ + Eβ) determinants, K24–199 (45) (Aαd-specific), and 14-4-4 (42) (Eαd-specific) mAb. Thus, in contrast to Aαdd-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).

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.

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.

Close modal

Biochemical and functional experiments strongly suggest that floppy Aαbb 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. 2 a, 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, λP12–26, and IgG2ab. Thus we conclude that, as for Aαbb, functionally empty Aαdd molecules are produced in the absence of Ii chain expression.

FIGURE 2.

Ag presentation capabilities. Splenocytes from +/+ (•) or Ii chain mutants (○) 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.

FIGURE 2.

Ag presentation capabilities. Splenocytes from +/+ (•) or Ii chain mutants (○) 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.

Close modal

As expected, BALB/c mutant spleen cells appear generally ineffective for presentation of intact protein Ags (Fig. 2,b). 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. 2 c, 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.

Ii chain mutants on a (C57BL/6 × 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. 3). 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).

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.

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.

Close modal

Ii chain mutants on a (C57BL/6 × 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 Aαb gene (47), but in contrast, class II mutants created by targeting the Aβb 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. 4). 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.

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.

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.

Close modal

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. 5). Despite increased surface expression observed for dendritic cells analyzed after overnight culture (Fig. 5,b) in comparison to results obtained using freshly isolated splenocytes (Fig. 5 a), 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.

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.

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.

Close modal

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. 6, 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. 6,b). Similarly as shown in Fig. 6 , 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).

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 (○) 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.

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 (○) 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.

Close modal

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 Aαdd 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 Aαdd 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 (Aβd-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 Aαdd molecules via an Ii chain-independent pathway(s).

As for floppy Aαbb conformers, we also found here in functional assays that Aαdd 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 Aαbb migrates in SDS-PAGE as compact dimers, this population represents a much smaller percentage of mature Aαkk and Aαdd 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 Aαdd-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 β-chain bulge, affecting subunit contacts and imposing spatial restrictions for peptide interactions. Specific peptide only partially fills the relatively shallow Aαdd groove and potentially accounts for the decreased representation of compact Aαdd 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 Aαb gene (47, 97), but in striking contrast, class II mutants created by targeting the Aβb 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 × 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 × 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. 4. 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 α-chain, but not β-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-Aβ transgenes (100, 101, 102). Higher order β-chain aggregates accumulate in Ii chain mutant spleen cells (3, 33, 103). Free β-chain appears especially prone to misfolding and aggregation, probably due to the formation of inappropriate disulfide bonds during early folding of the β1 domain. In contrast, the α1 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 αβ 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.

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.

1

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

3

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

1
Schaiff, W. T., K. A. Hruska, Jr, D. W. McCourt, M. Green, B. D. Schwartz.
1992
. HLA-DR associates with specific stress proteins and is retained in the endoplasmic reticulum in invariant chain negative cells.
J. Exp. Med.
176
:
657
2
Anderson, K. S., P. Cresswell.
1994
. A role for calnexin IP90 in the assembly of class II MHC molecules.
EMBO J.
13
:
675
3
Bonnerot, C., M. S. Marks, P. Cosson, E. J. Robertson, E. K. Bikoff, R. N. Germain, J. S. Bonifacino.
1994
. Association with BiP and aggregation of class II MHC molecules synthesized in the absence of invariant chain.
EMBO J.
13
:
934
4
Nijenhuis, M., J. Neefjes.
1994
. Early events in the assembly of major histocompatibility complex class II heterotrimers from their free subunits.
Eur. J. Immunol.
24
:
247
5
Cresswell, P..
1996
. Invariant chain structure and MHC class II function.
Cell
84
:
505
6
Rudensky, A. Y., P. Preston-Hurlburt, S. C. Hong, A. Barlow, C. A. Janeway, Jr.
1991
. Sequence analysis of peptides bound to MHC class II molecules.
Nature
353
:
622
7
Chicz, R. M., R. G. Urban, W. S. Lane, J. C. Gorga, L. J. Stern, D. A. A. Vignali, J. L. Strominger.
1992
. Predominant naturally processed peptides bound to HLA-DR1 are derived from MHC-related molecules and are heterogeneous in size.
Nature
358
:
764
8
Hunt, D. F., H. Michel, T. A. Dickinson, J. Shabanowitz, A. L. Cox, K. Sakaguchi, E. Appella, H. M. Grey, A. Sette.
1992
. Peptides presented to the immune system by the murine class II histocompatibility complex molecule I-Ad.
Science
256
:
1817
9
Chicz, R. M., R. G. Urban, J. C. Gorga, D. A. A. Vignali, W. S. Lane, J. L. Strominger.
1993
. Specificity and promiscuity among naturally processed peptides bound to HLA-DR alleles.
J. Exp. Med.
178
:
27
10
Bijlmakers, M. J. E., P. Benaroch, H. L. Ploegh.
1994
. Mapping functional regions in the lumenal domain of the class II-associated invariant chain.
J. Exp. Med.
180
:
623
11
Romagnoli, P., R. N. Germain.
1994
. The CLIP region of invariant chain plays a critical role in regulating major histocompatibility complex class II folding, transport, and peptide occupancy.
J. Exp. Med.
180
:
1107
12
Ghosh, P., M. Amaya, E. Mellins, D. C. Wiley.
1995
. The structure of an intermediate in class II MHC maturation: CLIP bound to HLA-DR3.
Nature
378
:
457
13
Park, S. J., S. Sadegh-Nasseri, D. C. Wiley.
1995
. Invariant chain made in Escherichia coli has an exposed N-terminal segment that blocks antigen binding to HLA-DR1 and a trimeric C-terminal segment that binds empty HLA-DR1.
Proc. Natl. Acad. Sci. USA
92
:
11289
14
Busch, R., E. D. Mellins.
1996
. Developing and shedding inhibitions: how MHC class II molecules reach maturity.
Curr. Opin. Immunol.
8
:
51
15
Denzin, L. K., C. Hammond, P. Cresswell.
1996
. HLA-DM interactions with intermediates in HLA-DR maturation and a role for HLA-DM in stabilizing empty HLA-DR molecules.
J. Exp. Med.
184
:
2153
16
Sanderson, F., C. Thomas, J. Neefjes, J. Trowsdale.
1996
. Association between HLA-DM and HLA-DR in vivo.
Immunity
4
:
87
17
Kropshofer, H., A. B. Vogt, G. Moldenhauer, J. Hammer, J. S. Blum, G. J. Hammerling.
1996
. Editing of the HLA-DR-peptide repertoire by HLA-DM.
EMBO J.
15
:
6144
18
van Ham, S. M., U. Gruneberg, G. Malcherek, I. Broker, A. Melms, J. Trowsdale.
1996
. Human histocompatibility leukocyte antigen (HLA)-DM edits peptides presented by HLA-DR according to their ligand binding motifs.
J. Exp. Med.
184
:
2019
19
Kropshofer, H., S. O. Arndt, G. Moldenhauer, G. J. Hammerling, A. B. Vogt.
1997
. HLA-DM acts as a molecular chaperone and rescues empty HLA-DR molecules at lysosomal pH.
Immunity
6
:
293
20
Kozono, H., J. White, J. Clements, P. Marrack, J. Kappler.
1994
. Production of soluble MHC class II proteins with covalently bound single peptides.
Nature
369
:
151
21
Avva, R. R., P. Cresswell.
1994
. In vivo and in vitro formation and dissociation of HLA-DR complexes with invariant chain-derived peptides.
Immunity
1
:
763
22
Bikoff, E. K., R. N. Germain, E. J. Robertson.
1995
. Allelic differences affecting invariant chain dependency of MHC class II subunit assembly.
Immunity
2
:
301
23
Malcherek, G., V. Gnau, G. Jung, H. G. Rammensee, A. Melms.
1995
. Supermotifs enable natural invariant chain-derived peptides to interact with many major histocompatibility complex-class II molecules.
J. Exp. Med.
181
:
527
24
Sette, A., S. Southwood, J. Miller, E. Appella.
1995
. Binding of major histocompatibility complex class II to the invariant chain derived peptide, CLIP, is regulated by allelic polymorphism in class II.
J. Exp. Med.
181
:
677
25
Stebbins, C. C., G. E. Loss, Jr, C. G. Elias, A. Chervonsky, A. J. Sant.
1995
. The requirement for DM in class II-restricted antigen presentation and SDS-stable dimer formation is allele and species dependent.
J. Exp. Med.
181
:
223
26
Stebbins, C. C., M. E. Peterson, W. M. Suh, A. J. Sant.
1996
. DM-mediated release of a naturally occurring invariant chain degradation intermediate from MHC class II molecules.
J. Immunol.
157
:
4892
27
Gautam, A. M., M. Yang, P. J. Milburn, R. Baker, A. Bhatnagar, J. McCluskey, T. Boston.
1997
. Identification of residues in the class II-associated Ii peptide (CLIP) region of invariant chain that affect efficiency of MHC class II-mediated antigen presentation in an allele-dependent manner.
J. Immunol.
159
:
2782
28
Villadangos, J. A., R. J. Riese, C. Peters, H. A. Chapman, H. L. Ploegh.
1997
. Degradation of mouse invariant chain: roles of cathepsins S and D and the influence of major histocompatibility complex polymorphism.
J. Exp. Med.
186
:
549
29
Takaesu, N. T., J. A. Lower, D. Yelon, E. J. Robertson, E. K. Bikoff.
1997
. In vivo functions mediated by the p41 isoform of the MHC class II-associated invariant chain.
J. Immunol.
158
:
187
30
Wolf, P. R., S. Tourne, T. Miyazaki, C. Benoist, D. Mathis, H. L. Ploegh.
1998
. The phenotype of H-2 M-deficient mice is dependent on the MHC class II molecules expressed.
Eur. J. Immunol.
28
:
2605
31
Bikoff, E. K., L.-Y. Huang, V. Episkopou, J. van Meerwijk, R. N. Germain, E. J. Robertson.
1993
. Defective major histocompatibility complex class II assembly, transport, peptide acquisition, and CD4+ T cell selection in mice lacking invariant chain expression.
J. Exp. Med.
177
:
1699
32
Viville, S., J. Neefjes, V. Lotteau, A. Dierich, M. Lemeur, H. Ploegh, C. Benoist, D. Mathis.
1993
. Mice lacking the MHC class II-associated invariant chain.
Cell
72
:
635
33
Elliott, E. A., J. R. Drake, S. Amigorena, J. Elsemore, P. Webster, I. Mellman, R. A. Flavell.
1994
. The invariant chain is required for intracellular transport and function of major histocompatibility complex class II molecules.
J. Exp. Med.
179
:
681
34
Takaesu, N. T., J. A. Lower, E. J. Robertson, E. K. Bikoff.
1995
. Major histocompatibility class II peptide occupancy, antigen presentation and CD4+ T cell function in mice lacking the p41 isoform of invariant chain.
Immunity
3
:
385
35
Janeway, C. A., Jr, P. J. Conrad, J. Tite, B. Jones, D. B. Murphy.
1983
. Efficiency of antigen presentation differs in mice differing at the Mls locus.
Nature
306
:
80
36
Brown, D. R., K. Swier, N. H. Moskowitz, M. F. Naujokas, R. M. Locksley, S. L. Reiner.
1997
. T helper subset differentiation in the absence of invariant chain.
J. Exp. Med.
185
:
31
37
Kappler, J. W., B. Skidmore, J. White, P. Marrack.
1981
. Antigen-inducible, H-2 restricted, interleukin-2-producing T cell hybridomas: lack of independent antigen and H-2 recognition.
J. Exp. Med.
153
:
1198
38
Braunstein, N. S., R. N. Germain.
1987
. Allele-specific control of Ia molecule surface expression and conformation: implications for a general model of Ia structure-function relationships.
Proc. Natl. Acad. Sci. USA
84
:
2921
39
Ozato, K., D. H. Sachs.
1981
. Monoclonal antibodies to mouse MHC antigens: hybridoma antibodies reacting to antigens of the H-2b haplotype reveal genetic control of isotype expression.
J. Immunol.
126
:
317
40
Symington, F. W., J. Sprent.
1981
. A monoclonal antibody detecting an Ia specificity mapping in the I-A or I-E subregion.
Immunogenetics
14
:
53
41
Bhattacharya, A., M. E. Dorf, T. A. Springer.
1981
. A shared alloantigenic determinant on Ia antigens encoded by the I-A and I-E subregions: evidence for I region gene duplication.
J. Immunol.
127
:
2488
42
Ozato, K., N. Mayer, D. H. Sachs.
1980
. Hybridoma cell lines secreting monoclonal antibodies to mouse H-2 and Ia antigens.
J. Immunol.
124
:
533
43
Oi, V. T., P. P. Jones, J. W. Goding, L. A. Herzenberg, L. A. Herzenberg.
1978
. Properties of monoclonal antibodies to mouse Ig allotype, H2, and Ia antigens.
Curr. Top. Microbiol. Immunol.
81
:
115
44
Janeway, C. A., Jr, P. J. Conrad, E. A. Lerner, J. Babich, P. Wettstein, D. B. Murphy.
1984
. Monoclonal antibodies specific for Ia glycoproteins raised by immunization with activated T cells: possible role of T cellbound Ia antigens as targets of immunoregulatory T cells.
J. Immunol.
132
:
662
45
Koch, N., G. J. Hammerling, N. Tada, S. Kimura, U. Hammerling.
1982
. Cross-blocking studies with monoclonal antibodies against I-A molecules of haplotype b, d, and k.
Eur. J. Immunol.
12
:
909
46
Metlay, J. P., M. D. Witmer-Pack, R. Agger, M. T. Crowley, D. Lawless, R. M. Steinman.
1990
. The distinct leukocyte integrins of mouse spleen dendritic cells as identified with new hamster monoclonal antibodies.
J. Exp. Med.
171
:
1753
47
Kenty, G., W. D. Martin, L. Van Kaer, E. K. Bikoff.
1998
. MHC class II expression in double mutant mice lacking invariant chain and DM functions.
J. Immunol.
160
:
606
48
Shimonkevitz, R., J. Kappler, P. Marrack, H. Grey.
1983
. Antigen recognition by H-2 restricted T cells. I. Cell-free antigen processing.
J. Exp. Med.
158
:
303
49
Roehm, N. W., P. Marrack, J. W. Kappler.
1982
. Antigen-specific, H-2 restricted helper T cell hybridomas.
J. Exp. Med.
156
:
191
50
Guillet, J.-G., M.-Z. Lai, T. J. Briner, J. A. Smith, M. L. Gefter.
1986
. Interaction of peptide antigens and class II major histocompatibility complex antigens.
Nature
324
:
260
51
Bikoff, E. K., L. A. Eckhardt.
1989
. Presentation of IgG2a antigens to class II-restricted T cells by stably transfected B lymphoma cells.
Eur. J. Immunol.
19
:
1903
52
Bikoff, E. K..
1992
. Formation of complexes between self peptides and MHC class II molecules in cells defective for presentation of exogenous protein antigens.
J. Immunol.
149
:
1
53
Bartnes, K., K. Hannestad.
1991
. Igh-1b-specific CD4+CD8 T cell clones of the Th1 subset selectively suppress the Igh-1b allotype in vivo.
Eur. J. Immunol.
21
:
2365
54
Bartnes, K., Ø. Rekdal, J.-P. Briand, K. Hannestad.
1993
. Th1 clones that suppress IgG2ab specifically recognize an allopeptide determinant comprising residues 435–451 of γ2ab.
Eur. J. Immunol.
23
:
2655
55
Avery, A. C., Z.-S. Zhao, A. Rodrigues, E. K. Bikoff, M. Soheilian, C. S. Foster, H. Cantor.
1995
. Resistance to herpes stromal keratitis conferred by an IgG2a-derived peptide.
Nature
376
:
431
56
Layet, C., R. N. Germain.
1991
. Invariant chain promotes egress of poorly expressed, haplotype-mismatched class II major histocompatibility complex AαAβ dimers from the endoplasmic reticulum/cis-Golgi compartment.
Proc. Natl. Acad. Sci. USA
88
:
2346
57
Sant, A. J., L. R. Hendrix, J. E. Coligan, W. L. Maloy, R. N. Germain.
1991
. Defective intracellular transport as a common mechanism limiting expression of inappropriately paired class II major histocompatibility complex α/β chains.
J. Exp. Med.
174
:
799
58
Anderson, M. S., J. Miller.
1992
. Invariant chain can function as a chaperone protein for class II major histocompatibility complex molecules.
Proc. Natl. Acad. Sci. USA
89
:
2282
59
Romagnoli, P., C. Layet, J. Yewdell, O. Bakke, R. N. Germain.
1993
. Relationship between invariant chain expression and major histocompatibility complex class II transport into early and late endocytic compartments.
J. Exp. Med.
177
:
583
60
Peterson, M., J. Miller.
1990
. Invariant chain influences the immunological recognition of MHC class II molecules.
Nature
345
:
172
61
Peterson, M., J. Miller.
1992
. Antigen presentation enhanced by the alternatively spliced invariant chain gene product p41.
Nature
357
:
596
62
Rath, S., R.-H. Lin, A. Rudensky, C. A. Janeway, Jr.
1992
. T and B cell receptors discriminate major histocompatibility complex class II conformations influenced by the invariant chain.
J. Immunol.
22
:
2121
63
Fung-Leung, W. P., C. D. Surh, M. Liljedahl, J. Pang, D. Leturcq, P. A. Peterson, S. R. Webb, L. Karlsson.
1996
. Antigen presentation and T cell development in H2-M-deficient mice.
Science
271
:
1278
64
Miyazaki, T., P. Wolf, S. Tourne, C. Waltziner, A. Dierich, N. Barois, H. Ploegh, C. Benoist, D. Mathis.
1996
. Mice lacking H2-M complexes, enigmatic elements of the MHC class II peptide-loading pathway.
Cell
84
:
531
65
Chervonsky, A. V., R. M. Medzhitov, L. K. Denzin, A. K. Barlow, A. Y. Rudensky, C. A. J. Janeway.
1998
. Subtle conformational changes induced in major histocompatibility complex class II molecules by binding peptides.
Proc. Natl. Acad. Sci. USA
95
:
10094
66
Shachar, I., R. A. Flavell.
1996
. Requirement for invariant chain in B cell maturation and function.
Science
274
:
106
67
Cosgrove, D., D. Gray, A. Dierich, J. Kaufman, M. Lemeur, C. Benoist, D. Mathis.
1991
. Mice lacking MHC class II molecules.
Cell
66
:
1051
68
Markowitz, J. S., P. R. Rogers, M. J. Grusby, D. C. Parker, L. H. Glimcher.
1993
. B lymphocyte development and activation independent of MHC class II expression.
J. Immunol.
150
:
1223
69
Rao, M., W. T. Lee, D. H. Conrad.
1987
. Characterization of a monoclonal antibody directed against the murine B lymphocyte receptor for IgE.
J. Immunol.
138
:
1845
70
Waldschmidt, T. J., D. H. Conrad, R. G. Lynch.
1988
. The expression of B cell surface receptors I. The ontogeny and distribution of the murine B cell IgE Fc receptor.
J. Immunol.
140
:
2148
71
Rovere, P., V. S. Zimmermann, F. Forquet, D. Demandolx, J. Trucy, P. Ricciardi-Castagnoli, J. Davoust.
1998
. Dendritic cell maturation and antigen presentation in the absence of invariant chain.
Proc. Natl. Acad. Sci. USA
95
:
1067
72
Wong, P., A. Y. Rudensky.
1996
. Phenotype and function of CD4+ T cells in mice lacking invariant chain.
J. Immunol.
156
:
2133
73
Miller, J., R. N. Germain.
1986
. Efficient cell surface expression of class II MHC molecules in the absence of associated invariant chain.
J. Exp. Med.
164
:
1478
74
Zhong, G., C. Reis e Sousa, R. N. Germain.
1997
. Antigen-unspecific B cells and lymphoid dendritic cells both show extensive surface expression of processed antigen-major histocompatibility complex class II complexes after soluble protein exposure in vivo or in vitro.
J. Exp. Med.
186
:
673
75
Schumacher, T. N. M., H. L. Ploegh.
1994
. Are MHC-bound peptides a nuisance for positive selection?.
Immunity
1
:
721
76
Katz, J. F., C. Stebbins, E. Appella, A. J. Sant.
1996
. Invariant chain and DM edit self-peptide presentation by major histocompatibility complex (MHC) class II molecules.
J. Exp. Med.
184
:
1747
77
Lightstone, L., R. Hargreaves, G. Bobek, M. Peterson, G. Aichinger, G. Lombardi, R. Lechler.
1997
. In the absence of the invariant chain, HLA-DR molecules display a distinct array of peptides which is influenced by the presence or absence of HLA-DM.
Proc. Natl. Acad. Sci. USA
94
:
5772
78
Zhong, G., F. Castellino, P. Romagnoli, R. N. Germain.
1996
. Evidence that binding site occupancy is necessary and sufficient for effective major histocompatibility complex (MHC) class II transport through the secretory pathway redefines the primary function of class II-associated invariant chain peptides (CLIP).
J. Exp. Med.
184
:
2061
79
Scott, C. A., P. A. Peterson, L. Teyton, I. A. Wilson.
1998
. Crystal structures of two I-Ad-peptide complexes reveal that high affinity can be achieved without large anchor residues.
Immunity
8
:
319
80
Henderson, R. A., H. Michel, K. Sakaguchi, J. Shabanowitz, E. Appella, D. F. Hunt, V. H. Engelhard.
1992
. HLA-A2. 1-associated peptides from a mutant cell line: a second pathway of antigen presentation.
Science
255
:
1264
81
Wei, M. L., P. Cresswell.
1992
. HLA-A2 molecules in an antigen-processing mutant cell contain signal sequence-derived peptides.
Nature
356
:
443
82
Busch, R., I. Y. Vturina, J. Drexler, F. Momburg, G. J. Hammerling.
1995
. Poor loading of major histocompatibility complex class II molecules with endogenously synthesized short peptides in the absence of invariant chain.
Eur. J. Immunol.
25
:
48
83
Busch, R., I. Cloutier, R.-P. Sekaly, G. J. Hammerling.
1996
. Invariant chain protects class II histocompatibility antigens from binding intact polypeptides in the endoplasmic reticulum.
EMBO J.
15
:
418
84
Sadegh-Nasseri, S., H. M. McConnell.
1989
. A kinetic intermediate in the reaction of antigenic peptide and I-Ek.
Nature
337
:
274
85
Mason, K., H. M. McConnell.
1994
. Short-lived complexes between myelin basic protein peptides and I-Ak.
Proc. Natl. Acad. Sci. USA
91
:
12463
86
Sadegh-Nasseri, S., L. J. Stern, D. C. Wiley, R. N. Germain.
1994
. MHC class II function preserved by low-affinity peptide interactions preceding stable binding.
Nature
370
:
647
87
Witt, S. N., and H. M. McConnell. 1994. Formation and dissociation of short-lived class II MHC-peptide complexes. Biochemistry:1861.
88
Lee, C., M. N. Liang, K. M. Tate, J. D. Rabinowitz, C. Beeson, P. P. Jones, H. M. McConnell.
1998
. Evidence that autoimmune antigen myelin basic protein (MBP) Ac1–9 binds towards one end of the major histocompatibility complex (MHC) cleft.
J. Exp. Med.
187
:
1505
89
Rabinowitz, J. D., M. Vrljic, P. M. Kasson, M. N. Liang, R. Busch, J. J. Boniface, M. M. Davis, H. M. McConnell.
1998
. Formation of a highly peptide-receptive state of class II MHC.
Immunity
9
:
699
90
Fairchild, P. J., R. Wildgoose, E. Atherton, S. Webb, D. C. Wraith.
1993
. An autoantigenic T cell epitope forms unstable complexes with class II MHC: a novel route for escape from tolerance induction.
Int. Immunol.
5
:
1151
91
Liu, G. Y., P. J. Fairchild, R. M. Smith, J. R. Prowle, D. Kioussis, D. C. Wraith.
1995
. Low avidity recognition of self-antigen by T cells permits escape from central tolerance.
Immunity
3
:
407
92
Akkaraju, S., W. Y. Ho, D. Leong, K. Canaan, M. M. Davis, C. C. Goodnow.
1997
. A range of CD4 T cell tolerance: partial inactivation to organ-specific antigen allows nondestructive thyroiditis of insulitis.
Immunity
7
:
255
93
Takeda, S., H.-R. Rodewald, H. Arakawa, H. Bluethmann, T. Shimizu.
1996
. MHC class II molecules are not required for survival of newly generated CD4+ T cells, but affect their long-term life span.
Immunity
5
:
217
94
Rooke, R., C. Waltzinger, C. Benoist, D. Mathis.
1997
. Targeted complementation of MHC class II deficiency by intrathymic delivery of recombinant adenoviruses.
Immunity
7
:
123
95
Germain, R. N., L. R. Hendrix.
1991
. MHC class II structure, occupancy and surface expression determined by post-endoplasmic reticulum antigen binding.
Nature
353
:
134
96
Rammensee, H.-G., T. Friede, S. Stevanovic.
1995
. MHC ligands and peptide motifs: first listing.
Immunogenetics
41
:
178
97
Kontgen, F., G. Suss, C. Stewart, M. Steinmetz, H. Bluethmann.
1993
. Targeted disruption of the MHC class II Aα gene in C57BL/6 mice.
Int. Immunol.
5
:
957
98
Grusby, M. J., R. S. Johnson, V. E. Papaioannou, L. H. Glimcher.
1991
. Depletion of CD4+ T cells in major histocompatibility complex class II-deficient mice.
Science
253
:
1417
99
Kearney, J. F., M. D. Cooper, J. Klein, E. R. Abney, R. M. E. Parkhouse, A. R. Lawton.
1977
. Ontogeny of Ia and IgD on IgM-bearing B lymphocytes in mice.
J. Exp. Med.
146
:
297
100
Gilfillan, S., S. Aiso, S. A. Michie, H. O. McDevitt.
1990
. Immune deficiency due to high copy numbers of an Aβk transgene.
Proc. Natl. Acad. Sci.USA
87
:
7319
101
Gilfillan, S., S. Aiso, D. Smilek, D. L. Woodland, E. Palmer, H. O. McDevitt.
1991
. An immune response defect due to low levels of class II cell surface expression.
J. Immunol.
147
:
4074
102
Singer, S. M., D. T. Umetsu, H. O. McDevitt.
1996
. High copy number I-Aβ transgenes induce production of IgE through an interleukin 4-dependent mechanism.
Proc. Natl. Acad. Sci. USA
93
:
2947
103
Marks, M. S., R. S. Germain, J. S. Bonifacino.
1995
. Transient aggregation of major histocompatibility complex class II chains during assembly in normal spleen cells.
J. Biol. Chem.
104
:
75
104
Zimmerman, V. S., P. Rovere, J. Trucy, K. Serre, P. Machy, F. Forquet, L. Leserman, J. Davoust.
1999
. Engagement of B cell receptor regulates the invariant chain-dependent MHC class II presentation pathway.
J. Immunol.
162
:
2495
105
Wright, R. J., E. K. Bikoff, B. Stockinger.
1998
. The Ii41 isoform of invariant chain mediates both positive and negative selection events in T-cell receptor transgenic mice.
Immunology
95
:
309
106
Fleury, S., J. Thibodeau, G. Croteau, N. Labrecque, H.-E. Aronson, C. Cantin, E. O. Long, R.-P. Sékaly.
1995
. HLA-DR polymorphism affects the interaction with CD4.
J. Exp. Med.
182
:
733
107
Battegay, M., M. F. Bachmann, C. Burhkart, S. Viville, C. Benoist, D. Mathis, H. Hengartner, R. M. Zinkernagel.
1996
. Antiviral immune responses of mice lacking MHC class II or its associated invariant chain.
Cell. Immunol.
167
:
115
108
Oxenius, A., M. F. Bachmann, D. Mathis, C. Benoist, R. M. Zinkernagel, H. Hengartner.
1997
. Functional in vivo MHC class II loading by endogenously synthesized glycoprotein during viral infection.
J. Immunol.
158
:
5717