Cell, Vol. 66, 1051-1066.

September

6, 1991, Copyright

0 1991 by Cell Press

M ice Lacking MHC Class II Molecules

Dominic Cosgrove,’ David Gray,? Andree Dierich; Jim Kaufman,? Marianne Lemeur,’ Christophe Benoist,’ and Diane Mathis* Laboratoire de Genetique Moleculaire des Eucaryotes du CNRS Unite 184 de Biologie Moleculaire de Genie Genetique de I’INSERM lnstitut de Chimie Biologique Faculte de Medecine 67085 Strasbourg Cedex France tBasel Institute for Immunology Grenzacherstrasse 487 CH-4058 Base1 Switzerland l

Summary We have produced mice that lack major histocompatibility complex class II antigens, permitting us to evaluate the role of these molecules in diverse aspects of T and 6 cell differentiation. The mutant mice show near-complete elimination of CD4+ T lymphocytes from the spleen and lymph nodes; the few remaining CD4positive cells are preferentially localized to B cell follicles. Surprisingly, substantial numbers of CD4 singlepositive cells reside in the thymus; however, these are not mature thymocytes as we currently recognize them. B lymphocytes occur in normal numbers and are capable of terminal differentiation to plasma cells. Nevertheless, several aberrations in the B cell compartment are demonstrable: a lack of germinal centers, fewer IgM+IgD+ cells in certain individuals, reduced production of serum IgGl, and complete inability to respond to T-dependent antigens. In short, the class II-negative mice have confirmed some old ideas about lymphocyte differentiation, but have provided some surprises. Introduction Major histocompatibility complex (MHC) class II molecules play a cardinal role in regulating the immune response (for reviews see Carbone and Bevan, 1989; Robinson and Kindt, 1989; Hodes, 1989). These polymorphic heterodimers are displayed on diverse immune system cell types, seeming to exert their influence by controlling cell-cell interactions. They occur on antigen-presenting cells, such as macrophages and dendritic cells, where they capture antigenic peptides and present them to T lymphocytes expressing the CD4 differentiation marker. They are also found prominently on B cells and appear to coordinate the collaboration with CD4’T helpers thought to be necessary for engendering an efficient antibody response. And finally, they occur on various thymic stromal cells, where

they govern the positive and negative selection of CD4+ T lymphocytes, crucial events in the generation of a peripheral repertoire that is both appropriate and self-tolerant. That MHC class II molecules exert important influences on antigen presentation, T-B cell collaboration, and thymocyte education has been recognized for many years. Nonetheless, the precise mechanisms involved have never been detailed to satisfaction. That class II molecules might have other influences inside or outside the immune system is accepted less generally, either because evidence for the purported function is controversial or because it has only recently been presented. Among these, one can cite the influence of class II molecules-either directly, or indirectly though their regulation of T helper cells-on the development of class l-restricted T cell responses (for review see Waldman, 1989) on the composition ofthey5Tcell repertoire(Lefrancoiset al., 1990; Matis et al., 1989; Rellahan et al., 1991) on the maturation of B cells (Fultz et al., 1982), and on the differentiation and/or targeting of sperm (Ashida and Scofield, 1987). With these thoughts in mind, we set out to produce a strain of mice lacking MHC class II molecules, an endeavor that seemed feasible in light of several recent examples of mutations introduced into the mouse germline via homologous recombination in embryonic stem (ES) cells (for review and references see Capecchi, 1989). This report documents the successful generation of class II-negative mice and describes their T and B cell compartments.

Generation of ABfo Mice To produce mice lacking MHC class II molecules, one must abrogate expression of both the E and A complexes. The former was accomplished by using the ES cell line D3, which was derived from a 129/Sv mouse (Gossler et al., 1986). This strain is b-haplotype at the H-2 locus and therefore carries a mutation in the E, gene promoter that precludes synthesis of E, protein and, as a consequence, expression of cell surface E complex (Mathis et al., 1983). To accomplish the latter, asoutlined below, adrastic mutation was introduced into the Ar gene on one chromosome of the D3 line, and mice carrying this alteration on both chromosomes were derived. A mutation in one of the D3 AP loci was created via homologous recombination, exploiting the doubleselection technique originally described by Mansour et al. (1988). First, an expressible neomycin (neo) gene was cloned into a unique site in the second exon of a b-haplotype AB genomic clone (Figure 1A). Then, a copy of the herpes simplex virus thymidine kinase gene was added to each end (Figure 1 B). The final construct was linearized and electroporated into 03 cells, and the cells were subjected to either double selection with G418 plus gancyclovir or to single selection with G418 alone. Bona fide AB disruptions were identified by Southern blotting as illus-

Cell 1052

A

mAb

2A2

Y279

40B

Specrficity

Aa

AD

AB ED

Control

AP%

-)I 35S Synthetic Labeling

0 *

Antibody

specificity

Db Aa Ea $

125,

Surface Labeling

EcoRl ES CELLS Figure 1. Production

Bglll ,

ECORI

-

MICE

Dbl AulEaj ;p” 1

.m >

:--

/

of A’$‘”Mice

(A) The bacterial neo gene, driven by a eukaryotic promoter, was introduced into a unique BstEll site in the second exon of a cloned Ai genomic fragment. (B) An EcoRI-Hindlll fragment containing the disrupted gene was excised and introduced between two herpes simplex virus thymidine kinase (HSVTK) genes carried in the Bluescript vector. (C and D) Probe 1, a Bglll-Bgll fragment, detects a 3.6 kb Bglll fragment on Southern blots of DNA from D3 cells (C) and an additional 4.7 kb band on blots of DNA from cells carrying a disrupted A0 gene (D). Probe 2, a Hindlll-EcoRI fragment, detects a 6.4 kb EcoRl fragment on blots of DNA from D3 cells (C) and an additional 4.4 kb band on blots of DNA from cells carrying a disrupted Ae gene (D). (E) Southern blot analysis of a representative ES cell clone containing a disrupted A!$allele. DNA was either digested with EcoRl and hybridized to probe 2 or digested with Bglll and hybridized to probe 1, Molecular weight markers are radiolabeled Hindlll-digested I DNA. (F) Southern blot analysis of representative tail biopsies. Male and female Aj” heterozygotes were mated and several offspring analyzed.

trated in Figures 1C and 1D. Probes from each end of the gene were employed, and multiple integrations were identified by hybridizing digests with a neo probe. Of 193 D3 clones screened, 5 carried an A0 mutation like the one documented in Figure 1 E, and none of these showed additional integrations. Three clones were injected into C578U6 (B6) blastocysts and the embryos reimplanted into foster mothers. All three gave rise to chimeras. On average, 45% of the blastocysts engendered

Figure 2. Cytofluorometric and Class II Molecule Expression

lmmunoprecipitation

Analyses

of

(A) Cytofluorometric analysis. Splenocytes from either an Ai’” mouse or a control littermate were stained with MAbs 2A2, Y279, and 408. (B) lmmunoprecipitation analysis, Splenocytes from either a control A’ E’ or a mutant A;‘” mouse were biosynthetically or ceil surface labeled. Class II complexes were immunoprecipitated with MAbs specific for A, (2A2), E, (14.4.4) or AB and EB (MU1 14). A D%pecific MAb (21.460) served as a positive control. lmmunoprecipitates from equal cell numbers were displayed on SDS-polyacrylamide gels and bands revealed by autoradiography.

progeny and 28% of these were chimeric. Male chimeras were mated with 86 females, and the offspring were scored for germline transmission of the 129/Sv genotype by examining coat color. Chimeras from each of the three D3 clones gave rise to agouti-colored pups. Finally, males and females heterozygous for the AB mutation were crossed to produce A$O homozygotes. As a rule, heterozygous mutaryt mice seem in good health. Homozygotes live to at Iwst 25 weeks of age in a conventional animal facility, although they often show reduced growth as well as impaired breeding performance. Lack of Class II Molecules in Homozygous Mutants To evaluate class II molecule expressron, spleen and lymph node cells from AB’”and c&ntroi mice were labeled with a variety of anti-classff’monoclonal antibodies (MAbs)

Mice Lacking MHC Class II Molecules 1053

and analyzed by cytofluorometry. Figure 2A shows a comparison of splenocytes labeled with three such reagents. There is no detectable staining of cells from the mutant mice by 2A2 (specific for the A, chain), Y-279 (A&, or 40B (AB, Ep). Staining was also not seen with a variety of other reagents: Y3P, 3B9, and 4D5 (specific for A,), Y-21 9 and Y-271 (specific for A& 14.4.4 (E,), and 17.3.3 (Er) (not shown). lmmunoprecipitation of radiolabeled splenocytes provided a more sensitive assay of class II molecule expression. In a first experiment, illustrated in the upper panel of Figure 28, cells were biosynthetically labeled with [35S]methionine for 20 hr. While bands of the expected size are observed in the 2A2 (A,), 14.4.4 (E,), and 408 (AD, EB) lanes for A+E’control animals, no such bands are detected in the equivalent lanes for Al’” mice. In a second experiment, presented in the lower panel of Figure 2B, splenocytes were surface iodinated using lactoperoxidase and glucose oxidase. Again, bands of the expected size are observed for control mice, but none are detected for the mutants. To determine the limits of detection inherent in this and parallel sets of experiments, we quantitated the radioactivity in bands from control lanes versus the equivalent regions from Ag’O lanes by means of a Phosphorlmager. In all cases this ratio was less than 0.01, and in one synthetic labeling experiment it was below 0.001; these results indicate that we should have been able to detect a 1 OO-to 1 OOO-foldreduction in class II molecule expression. Incubation of spleen cell extracts from mutant mice with three polyclonal rabbit anti-human class II sera did precipitate some biosynthetically labeled, but no surface-labeled, protein of the correct size (data not shown). This indicates that A, and/or EB is synthesized and accumulates as isolated chains within mutant splenocytes, but that these molecules are not displayed as mixed dimers at the surface. Finally, immunohistology was performed to verify that rare cell types in A$” mice do not express class II molecules. Figure 3 shows sections of thymus and spleen labeled with MAb M5/114 (anti-Ar, -ED). Control sections show the usual staining of medullary and cortical stroma in the thymus (Figure 3b) and stroma and B cells in the lymph node (Figure 3d). Sections from mutant mice show no staining above background (Figures 3a and 3~). We also saw no evidence of class II molecules in the spleen or intestine (not shown). Considering all of the cytofluorometry, immunoprecipitation, and immunohistology experiments, we made use of four anti-A,, five anti-Ap, one anti-E,, and three anti-Eb reagents, as well as several polyclonal antisera. None of these gave a positive result in A$O mice, indicating a profound lack of MHC class II molecules. T Cells in the Periphery of Ap Mice Visual evaluation of hematoxylin- and eosin-stained sections of lymph nodes and spleen from class II-negative mice revealed no obvious structural defects (not shown). In addition, these organs housed normal numbers of T lymphocytes (not shown). When spleen and lymph node cells from Ag’”mice were double stained with anti-CD4 and anti-CD8 MAbs, a strik-

ing anomaly was observed. As illustrated in the leftmost panel of Figure 4, there is a massive reduction in the CD4+ T cell population of mutant mice, while the CD8’ subset proportionally increases. Asimilar reduction wasobserved in all mutant animals analyzed, no matter what their age. Some CDCpositive cells do remain, but they represent only a few percent of the usual numbers: 3% to 7% in ten mice analyzed. Essentially all of them are Thy1 + and CD5+, while about 75% and 15% express a8 and y8 T cell receptors(TCRs), respectively (not shown). Several characteristics distinguish the Ag’OCD4-positive cells from those of normal mice. First, their volume profile suggests much larger cells: a mean volume in four experiments of 120.0 versus 88.7 in control littermates (Figure 4, second row of panels). Second, they express CD4 and up TCR at measurably reduced levels: an average 25 channel downshift compared with control mice, representing a roughly P-fold diminution (Figure 4, third row of panels). Third, they are almost all CD44(Pgp-l)hi, while only 15%-250/o of CD4positive cells from control littermates express high levels of this marker (Figure 4, rightmost panels). This difference is not due to segregation of CD44 low and high expressor phenotypes (Lesley et al., 1988; Lynch and Ceredig, 1988) in the mixed 129/86 genetic background, because it was observed with three separate littermate pairs and because it is not seen with the equivalent CD8-positive populations. Finally, the few CD4+ cells in A$” animals show a distinct ’ localization. Normal mice have Tcells primarily in the paracortex of the lymph nodes and periarteriolar lymphoid sheath of the spleen-in discrete “T cell areas.” This can be seen clearly in Figure 3, which shows a lymph node section labeled with anti-IgM in red and anti-CD4 in green (coincident staining should be yellow to orange). The segregation is not perfect, however, because there are always a few CD4+ T cells “misplaced” in the 6 cell follicles, as is evident in the less exposed, higher magnification image of Figure 3h. Ap/Omice, on the other hand, have very few CD4+ T cells in the paracortex or periarteriolar sheath, but do have more or less normal numbers in the B cell follicles (Figures 3e and 39). Given that the few CD4+ T cells in A$” mice appear to constitute a discrete population, we questioned whether they display a distinct repertoire of TCRs. Lymph node cells were triple labeled with antiCD4, antiCD8, and various MAbs specific for particular V,s or VBs. The results of several experiments are presented in Table 1. In general, the repertoires of mutant and control littermates are similar. Worth noting is that for some VBs (e.g., 8 and 1 l), there is great fluctuation in the values for CD4’ cells from the class II-negative mice. This may reflect a certain pauciclonality or, instead, a segregation of positively or negatively selecting coligands in the mixed 129/86 background. Alternatively, this fluctuation may simply reflect the small numbers of mutant CD4’ cells available for staining. T Cells in the Thymus As indicated in the leftmost panels of Figure 5, there is also a sharp reduction in the number of CD4 single-positive cells in the thymus of Ap mice. However, it is not as drastic a diminution as that observed in the periphery: in the sev-

Cdl 1054

Mice Lacking MHC Class II Molecules 1055

CELL VOLUME

CONTROL

abTCR

CD44

5

CD0

Figure 4. Cytofluorometric

-

Analysis of Peripheral

CELL VOLUME

af3TCR

CD44

T Cells

Lymph node cells from control and A$? mice were analyzed by three-color flow cytometry as described in Experimental Procedures. The leftmost panels are dot plots of cells stained for CD4 and CD8. To the right are, sequentially, profiles of volume, a8 TCR staining, and CD44 staining for gated CD4CD8 and CD4-CD8’ populations. Controls were sex-matched littermates.

era1 mice analyzed, there were from 17% to 30% the number of CD4 single-positive cells that were found in control littermates. This residual CD4+CD8- population is peculiar by a number of criteria. First of all, these ceils seem to express very low, though detectable, levels of CD8; such cells do exist in normal mice, but are in low proportion (Figure 5, leftmost panels). In addition, a greater percentage of CD4 single positives are afS TCR (and CD3) negative in mutant mice, and those cells that do express ap TCR do so at reduced levels (approximately 2-fold in multiple experiments) (Figure 5, center panels). Furthermore, almost all the CD4’CD8- thymocytes in AB” mice express the heat-stable antigen recognized by MAbs JllD and M1/69-an unusual feature for purportedly mature cells (Figure 6, right panels). Finally, the CD4 single-positive cells in the mutant mice are cortical cells: they are smaller than normal CD4 single-positive cells (Figure 6, left panels), and thymus sections double labeled with anti-CD4 and ERTR5 (the latter MAb specifically recognizes medullary stromal cells) show that there are almost no CD4+ cells in the medulla of mutant mice (Figures 3i, 3j).

The three other major thymocyte populations are readily evident in A$” mice (Figure 5, leftmost panels). The CD4-CDB- cells occur in normal numbers, and there exists both an ab TCR-negative and a TCR-positive subset-just as in littermate controls (center panels). The CD4’CD8+ cells are also found at the usual frequency in mutant mice, but they display noticably elevated levels of CD4 (1.5- to 2-fold, on average) and of ap TCR (2- to 3-fold, on average). The CD4-CD8’ cells are often, but not always, increased in number in the mutant animals, seeming to compensate for the decreased numbers of CD4 single-positive cells. B Cell Maturation To provide an initial view of the B lymphocyte compartment, we stained spleen and lymph node sections with a variety of reagents specific for B lineage cells or for stromal cells implicated in B cell differentiation. Figures 7a and 7b show lymph node sections from an Ap’O mouse and a control littermate double stained with PNA and anti-IgM. In the control (Figure 7a), the paracortex is green due to light staining of T cells by PNA, the B cell follicles are red due to anti-IgM staining, and the germinal centers (GCs)-

Figure 3. Immunohistology:

Absence

of Class II Molecules,

Localization

of Peripheral

CD4’ T Cells, Localization

of CD4’ Thymocytes

(a-c) Sections,of thymus (a, b) and lymph node (c, d) from an Ap mouse (a, c) or a negative littermate (b, d) were stained with an anti-class II MAb. (e-h) Sections of lymph node from an A;” mouse (e, g) or a negative littermate (f, h) were stained with anti-CD4 (green) and anti-IgM (red). Coincident staining should appear yellow to orange. (g) and (h) are higher magnification photographs; the arrows point out CD4+ T cells interspersed with follicular B cells. (i and j) Sections of thymus from an Ag’Oanimal (i) or a control littermate (i) were stained with anti-CD4 (green) and ERTRB, a MAb that stains a subset of medullary epithelial cells (red). In (i) the few green cells in the medulla are larger than typical T cells and may be CD4+ macrophages. M = medulla, C = cortex, F = follicle, PC = paracortex.

Cdl 1056

Table 1. TCR Variable Region Usage in Peripheral

CD4

CD6

T Cells

VP2

VB4

W

‘46

W

Vgll

VP14

v2

Ctl

6.2, 4.1

5.5, 5.0, 4.6 3.9, 1.5, 3.0

16.2, 16.3, 17.8, 19.3, 17.4 19.7, 28.1, 35.0, 29.01, 23.6

4.6, 4.6, 3.2, 3.0 2.3, 12.2, 7.8, 4.9

11.6, 10.1

4.4, 3.8

9.6, 10.0, 8.6. 10.7, 9.2 6.2, 9.4, 6.8, 0.7. 7.1

6.6, 5.3, 9.5

Ai’0

2.3, 3.1, 4.4, 3.9 1.8, 4.0, 8.0

3.0, 3.6, 6.9

3.1, 3.7

Ctl W

5.9, 4.3 6.8, 3.6

2.2, 2.6, 2.0 3.3, 1.7, 3.4

11.1, 14.5 15.0, 19.6

7.4, 13.4, 7.8 9.2, 13.9, 9.8

17.4, 16.0, 16.6 18.4, 21.5

6.2, 4.4 3.0, 6.6

0.4, 0.5 2.3, 2.2

7.8, 6.2 6.2, 6.1

Numbers represent the percentage of T ceils in the CD4’ or CD8’ population displaying a particular VB or V.. Each value comes from triple staining of lymph node cells from an individual mouse. Controls (Ctl) are always negative littermates.

loaded with strongly PNA+IgM+ cells-show up orange. In the mutant (Figure 7b), the T cell areas and 6 cell follicles appear normal, but there are no GCs. Similarly stained spleen sections from mutant animals were also devoid of GCs. Figure 7 demonstrates that the lack of GCs in class II-negative mice is not due to a deficiency in follicular dendritic cells (FDCs), stromal elements that normally express MHC class II molecules and are remarkable for their ability to bind immune complexes (for review see Tew et al., 1990). One observes numerous cells with FDC morphology that are yellow as a result of costaining with an FDC-specific MAb and goat anti-mouse lg. Lastly, Figure 7d shows that, despite their lack of GCs, the mutant animals can produce both IgM+ and IgG’ plasma cells, recognizable by their intense anti-lg staining and nonfollicular localization.

CONTROL

These data indicate that B cell differentiation in class IInegative mice is not grossly perturbed. B cells occur in the spleen and lymph nodes in normal numbers, localize as usual, and are capable of attaining the terminal stage of differentiation. To provide a more refined view of the B lymphocyte compartment, we performed two-color cytofluorometric analysis of spleen and lymph node cells with a variety of reagents known to stain cells of the B lineage. The IgM+ cells from mutant mice are mostly CD45RA(B220)+, Jl 1 d’, CD44(Pgp-l)‘“, CD5(Ly-I)-, and CD1 1 b(Mac-l)-, as are most IgM+ cells from normal animals (data not shown). More discriminating information was provided by analyzing surface expression of the IgD isotype, as illustrated in Figure 8. For 3-week-old mice, like those depicted in Figure 8A, the amount of IgD on IgM+ cells is quite comparable

2 t --. CD8

CD8 Figure 5. Cytofiuorometric

c$TCR

n5TCR

Analysis of Thymocytes

Thymocytes from control and A;@ mice were analyzed by three-color flow cytometry. The leftmost panels are dot plf% of cells stained for CD4 and CD8. The panels on the right are histograms of gated populations stained for a5 TCR.

Mice Lacking MHC Class II Molecules 1057

CELL

Figure 6. Additional from Ag’” Mice

VOLUME

Properties

JIlD

of CD4 Single-Positive

Thymocytes

The left-hand panels show histograms of cell volume (impedancebased measure) for ab TCR-positive and -negative subpopulations. The right-hand panel presents histograms for JilD staining. CD4 single-positive cells were gated after double staining with antiCD4and anti-CD6

in mutants and littermates. However, for older animals differences begin to show up. In the lo-week-old control mouse illustrated in Figure 86, the IgDllgM ratio is elevated, indicative of advancing B cell maturity, but no such increase is apparent in the mutant from the same litter. In the numerous class II-negative mice analyzed, this reduction in IgD-positive cells was found to be quite variable: sometimes there was only a small difference compared with littermate controls; sometimes the difference was as extreme as that observed in Figure 8C. There was no obvious relationship with the animals’ health status, but there was a loose correlation with age. The abnormality in IgD staining could be confirmed in some mice by immunohistology. Figures 7f and 7g show the follicular (IgM’IgD’) and marginal zone (IgM+IgD-) compartments in the spleens of young control and A8’” mice. They appear quite similar. Figure 7h, obtained from a 13-week-old mutant animal, demonstrates a reduced follicle and greatly expanded marginal zone. However, these aberrations were not evident in the lymph nodes of the same mouse (e.g., Figure 7e) and even in the spleen seemed to vary from animal to animal. Antibody Production in A$” Mice The natural antibodies circulating in the serum of several AB@mice and control littermates were quantitated by an enzyme-linked immunosorbant assay (ELISA). As indicated in Figure 9, the most striking difference between the two sets of animals is the concentration of serum IgGl: mutant mice have lo- to lOO-fold lower levels of this isotype. Titers of other immunoglobulin isotypes were not significantly different in mutant and control animals. Circulating antibodies were also tested for autoreactive specificities. There was no measurable reaction to nuclei, double-

stranded DNA, thyroglobulin, or rheumatoid factor (J.-L. Pasquali, unpublished data). The ability of mutant mice to respond to type II T-independent antigens was evaluated by injecting three different carbohydrate antigens (Figure 1 OA). Both control and mutant mice make specific antibody to dextran, phosphorylcholine, and levan. There is large scatter in the antibody titers but no clear-cut differences between the two sets of animals. In addition, the profiles of lg isotypes produced in the control and mutant mice are very similar (data not shown). Finally, the capacity to mount T-dependent antibody responses was evaluated after repeated immunization with keyhole limpet hemocyanin (KLH), ovalbumin (Ova), or the branched copolymer poly(Tyr, Glu)-poly(Ala)-poly(Lys) (TGAL) (Figure 1 OB). The first two antigens can provoke strong antibody production in essentially all mouse strains and can elicit both A- and E-restricted T helper cells, while TGAL is a classical A-restricted antigen. Mutant mice make no response to any of them, even after repeated boosts. Discussion We produced mice lacking MHC class II molecules in order to facilitate study of their diverse influences on the immune response. To avoid expression of E complexes, we began with an ES cell line derived from the 129/Sv mouse, a strain that carries a deletion in the promoter region of the E, gene. To eliminate expression of A complexes, we introduced a drastic mutation in the AB gene via homologous recombination, generated mice that transmit the mutation through thegermline, and mated them to produce homozygotes. The resulting Ag’”mice were found to be devoid of class II A and E molecules by extensive cytofluorometric, biochemical, and immunohistological analyses. Certain of our assays permit a confidence level of 0.1%; i.e., we should have been able to detect class II expression even if it was lOOO-fold lower than that observed normally. These analyses also argue strongly against the expression of isotype-mismatched class II molecules-in particular, A,:EB, whose assembly remains theoretically possible in the Ap’Omice. A battery of reagents was employed, including MAbs that specifically recognize individual A!, Al, E,, or E$ chains, as well as several pan-class II antibodies that detect most mouse and even some human class II complexes. The complete inability of these reagents to label any cell type would seem to rule out significant expression of A,:EB molecules. Evidence against them is also provided by the absence of any antibody response to complex, multiepitope antigens like KLH and Ova. Indeed, A,:EB complexes have not previously been demonstrated, even though the reciprocal E.:AB complex has been documented repeatedly (Germain and Quill, 1988; Malissen et al., 1988; Sant et al., 1987; Sant and Germain, 1989; Spencer and Kubo, 1989; Kimoto et al., 1989; Anderson and David, 1989; Anderson et al., 1989; Matsunagaet al., 1990). Thus, the ApI0 mice appear to provide a useful system for studying the influence of MHC class II molecules on

Cell 1058

Mice Lacking MHC Class II Molecules 1059

A

Figure 6. Cytofluorometric

B (IO weeks)

(3 weeks)

Analysis of IgD on Splenic B Cells

Ai’” mice and negative littermates

were double stained with anti-IgM and anti-IgD. (A) Animals were aged 3 weeks; (B) 10 weeks; (C) 13 weeks.

the differentiation and operation of the immune system. In addition, they may allow us to detect unusual subsets of CD4+ T cells that are normally masked by the dominant class II-restricted set. T Cells in the Periphery The most striking effect of abrogating class II molecule expression is the near-complete elimination of CD4’ T NATURAL

ANTIBODIES

I-

7 e

cc,f-----TIgGl

IkIM

Figure 9. Concentrations

Of

IgGZa

Circulating

id ~~I IgGZo lgG3

Antibodies

Blood was taken from several Ai’” (open circles) and control (closed circles) animals, and the concentrations of various lg isotypes were quantitated by ELISA and normalized as explained in Experimental Procedures.

Figure 7. lmmunohistology

C

cells from the peripheral lymphoid organs. This result was foretold by experiments with mice injected from birth with anti-class II mAbs (Kruisbeek et al., 1983, 1985) and by experiments with animals carrying rearranged TCR transgenes derived from class II-restricted T cells (Berg et al., 1989a; Kaye et al., 1989). It nevertheless provides clear-cut confirmation of the assertion that CD4’ T cells need to undergo a positive selection event mediated by cells displaying MHC class II molecules. Our result brings into question past observationson individuals presenting with combined immunodeficiency owing to a congenital defect in MHC class II gene expression (for review see Griscelli et al., 1989). Such individuals have been reported to harbor normal numbers of CD3’ lymphocytes in the periphery and often only slightly reduced numbers of CD4-positive ceils. This observation has led some to doubt that a class II-mediated positive selection event is necessary for CD4’ cells to emerge from the thymus. But are the thymuses of these individuals really devoid of class II complexes? (See, for example, Schuurman et al., [ 19851.) Surprisingly, some CD4’ T cells-a few percent of the usual number-are found in the periphery of A$” mice. However, these appear to be a quite specific population. They are relatively large cells, express a8 TCR at a reduced level, and display high levels of CD44, all consistent with their being in an activated state. Intriguingly, they reside preferentially in B cell follicles rather than in the usual T cell areas of the spleen and lymph nodes.

of B Lineage Cells

(a and b) Lymph node sections (x 60) from control (a) or A$ (b) mice were stained with PNA (green) and anti-IgM (red). (c)A spleen Section from a mutant mouse was stained with an anti-FDC MAb (green) and goat anti-mouse lg (red). FDCs are large cells with irregular extensions. (d) A spleen SeCtiOn (x 160) from a mutant mouse was stained with anti-IgM (red) and anti-lgG (green). (e) A lymph node section (x 160) from a 1 O-week-old AI’” mouse was stained with anti-IgM (red) and anti-IgD (green). (f-h) Spleen sections from a 3-week-old control mouse ( x 320) (9, an Ai” littermate ( x 320) (g), and a 1 O-week-old A;” mouse ( x 160) (h) were stained with anti-IgM (red) and anti-lgD (green). D = germinal center; F = fOlliCle; M = marginal zone.

Cell 1060

TINDEPENDENTRESPONSES

Figure 10. Antibody Responses to T-lndependent and T-Dependent Antigens Ai’” (open circles) and control (closed circles) mice were challenged with various type II T-independent (A) or T-dependent(B) antigens. At the indicated times after injection, blood was drawn and specific antibody titers were quantitated by ELISA. Open arrows indicate the days on which antigen boosts were given.

DAYS AFTER IMMUNIZATION

DAYS AFTER IMMUNIZATION

DAYS AFTER ,MMUNlZATlON

TDEPENDENTRESPONSES OVA

TGAL

DAYS AFTER IMMUNIZATION

DAYS AFTER iMMUNlLATlON

DAYS AFTER lMM”NlZATlON

We do not yet know the developmental history of these cells, but there seem to be several possibilities. One possibility is that they were positively selected on nonconventional, class II-like molecules. Some years ago, it was demonstrated that the murine MHC contains two genes, A82 (recently renamed OB) and Ep2, that are about 50% homologous to conventional class II 8 chain genes (for review and references, see Robinson and Kindt, 1989). Both of these loci are transcribed, although at reduced levels and in a distinct set of cells when compared with the orthodox A and E loci (Larhammer et al., 1985; Wake and Flavell, 1985; Braunstein and Germain, 1986). OB appears also to be translated (Karlsson et al., 1991). More recently, three other class II-like genes have been discovered in the murine MHC: two S-like genes and one a-like gene, which are less than 50% homologous to their orthodox counterparts (J. Monaco, personal communication). A second possibility is that the few CD6positive cells in Ag’Omice were positively selected on MHC class I molecules. In general, the CD4lclass II, CDB/class I dichotomy is very stringent, but there have been some recent reports of class l-restricted CD4’T cells (Macphail and Stutman, 1987, 1988; Matsubayashi et al., 1989). A third possibility is that the A$” CDCpositive cells were not positively selected on MHC molecules. Precedent for such a scenario exists: CD4-CD8- cells expressing high levels of up TCR, and which are presumably mature, have been described in both transgenic (Kisielow et al., 1988; Berg et al., 1989b) and nontransgenic (Crispe et al., 1987a; Budd et al., 1987; Fowlkes et al., 1987; Ceredig et al., 1987; Miescher et al., 1987; Hashimoto et al., 1987) systems. In those cases studied so far, generation of such cells seems not to reflect the usual positive selection on MHC molecules (Scott et al., 1989; Benoist and Mathis, 1989).

Whatever its origin, the small population of CD4’ T cells in class II-negative mice is intriguing because of its preferential localization. In a normal mouse, the great majority of T lymphocytes residing in the peripheral lymphoid organs are found in the paracortex of the lymph nodes and the periarteriolar lymphoid sheath of the spleen; however, a few percent of the CDCpositive cells can be found in the B cell follicles, especially in germinal centers when they arise (Gutman and Weissman, 1972; Rouse et al., 1982). That these “misplaced” T cells serve an important function is suggested by the fact that they also occur in humans (Velardi et al., 1986a, 1986b), rats (Heinen et al., 1988) and even toads (M. Cooper, personal communication). The human cells seem not to exhibit conventional helper activity, being very poor producers of IL-2 and BCGF (Velardi et al., 1986a, 1986b); interestingly, however, they often coexpand in patients with B cell chronic lymphocytic leukemia or multiple myeloma (Velardi et al., 1985). One is tempted to speculate that the CDCpositive cells observed in the follicles of A$Omice represent the same population as those in the follicles and eventually the germinal centers of normal animals. How they became activated and achieved their localization are unanswered questions; meanwhile, one is tempted to suggest an influence by molecules on B cells (e.g., Igs, superantigens). T Cells in the Thymus Given the striking results on peripheral T cells and considering current models of positive selection (von Boehmer, 1990; Robey et al., 1991; Borgulya et al., 1991), we expected to find-very few CD4+CD8 cells in the thymuses of A$” mice. But this was not the case. There were significant numbers of CD4 single-positive &f&as many as 30% the usual number in some indi&duals. However, more refined analyses revealed that these are probably nonma-

Mice Lacking MHC Class II Molecules 1061

ture cells and that there are few (if any) truly mature CD4 single-positive thymocytes in class II-negative mice. About 40% of this residual CD4’CD8- population is TCR, probably representing asubset recently discovered in normal animals (Matsumoto et al., 1989; Hugo et al., 1990, 1991; Egerton et al., 1990). This subset is almost certainly a counterpart to the immature CDCCD8’TCR population described some years ago (Paterson and Williams, 1987; Bluestone et al., 1987; Fowlkes et al., 1987; Crispe et al., 1987b). The other 80% of the CD4+CD8- population in mutant mice express a8 TCRs, but differ from mature thymocytes in several respects: these cells almost always display low levels of CD8, express heat-stable antigen, are very small (like cortical cells), and, in fact, appear to be confined to the cortex. These cells may or may not be equivalent to a recently discovered nonmature subset in normal mice (Vernachio et al., 1989; Fowlkes and Pardoll, 1989; Nikolic-Zugic and Bevan, 1990). The existence in class II-negative mice of a CD4+CD8-““subset expressing high levels of TCRs appears, at first glance, to be problematic for the instructional model of positive selection (Robey et al., 1991; Borgulya et al., 1991). According to this model, commitment to the CD4 versus CD8 lineage results from class II versus class I molecule engagement by the TCR on double-positive thymocytes, resulting in a specific signal that calls for downregulation of CD8 or CD4. However, in our mice, CD4 single-positive thymocytes with high levels of TCR exist in the apparent absence of class II molecule engagement. Yet, for this result to be considered a true challenge to the instructional model, it is necessary to rule out other possibilities: first, that the residual CD4+CD8-““TCR” subset in A$” mice may not have a counterpart in normal animals, arising because of some perturbation in the normal differentiation pathway caused by adearth of class II molecules; second, that such cells may exist in normal animals but represent a dead-end subset that never progresses further; and third, that these cells may be equivalent to a previously unrecognized population in normal mice that develops independently of positive selection, i.e., is not MHC restricted. Labeling and transfer experiments should allow us to explore these possibilities. The lack of class II molecules in A$” mice also has an interesting effect on double-positive thymocytes: both TCR and CD4 levels are up-regulated. This observation is highly reminiscent of findings by Singer and colleagues. They treated animals or thymus cultures with anti-CD4 antibodies and saw a very similar up-regulation of TCR levels (McCarthy et al., 1988; Zuniga-Pflucker et al., 1989; Nakayama et al., 1990; Bonifacino et al., 1990). Surprisingly, their parallel experiments with anti-class II reagents showed no such effect (McCarthy et al., 1988; ZunigaPflucker et al., 1989; Nakayama et al., 1990). Taken together, our data and those of Singer and colleagues indicate that stromal cell class II complexes engage CD4 molecules on double-positive thymocytes and transmit a negative signal that maintains low TCR levels. Whether and why such dampening is required for subsequent thymocyte differentiation remain open questions. Lastly, Ap mice are capable of generating the enigmatic

CDCCD8-TCR+ subset of thymocytes. This finding negates the view that these cells result from simultaneous engagement of CD4 and CD8 molecules on doublepositive cells (von Boehmer, 1988; Wu et al., 1990; Egerton and Scollay, 1990), a view that was already challenged by observations that these cells seem not to be subject to the usual positive selection on MHC molecules (Benoist and Mathis, 1989). B Ceil Maturation and Function Despite their lack of MHC class II molecules, the AB’”mice are able to produce B cells in the usual numbers. Furthermore, the fact that they contain plasma cells, have normal levels of serum lg, and are capable of responding to T-independent antigens indicates that B cell differentiation can proceed to its terminal state. We did notice a reduction in the percentage of cells that express both IgM and IgD on the surface, particularly in older mice. An enrichment in IgM’IgD- cells might have been due to an accumulation of CD5+ B cells-a population thought by some to be of distinct lineage, known to make a major contribution to both natural antibodies and T-independent responses, and observed to express only low levels of surface IgD (for reviews see Herzenberg and Stall, 1989; Hardy, 1990). However, we did not detect any increase in the percentage of CD5+ (or Mac-l+) B cells in the spleens of mutant mice. At this time, we do not know whether the enrichment in IgM’IgD- cells represents an accumulation of immature cells that have not yet expressed IgD, of mature but activated cells that have downregulated this isotype, or of ceils that will never express IgD because they are off the normal pathway. Our results are superficially similar to those obtained on mice treated from birth with an anti-class II MAb (Fultz et al., 1982). The B cells of these animals were also enriched in IgM’IgD- cells. However, unlike our mutant mice, they showed a drastic reduction (m90%) in total numbers of B lymphocytes. It would seem that the MAb treatment actually killed off the majority of B cells. An interesting comparison can also be made with humans with class II-negative immunodeficiency. Like class II-negative mice, these individuals have normal B lymphocyte numbers, and some (but not all) of them show reduced expression of IgD on IgM’ cells (Griscelli et al., 1989). The Ag’” mice are able to mount effective antibody responses against T-independent antigens such as dextran, phosphorylcholine, and levan. At first glance, this is not surprising; evidence has been offered, however, that T-independent responses do actually require an MHCrestricted interaction between B cells and monocytes (Chused et al., 1976; Boswell et al., 1980a, 1980b; Morrissey et al., 1981). And, of late, the division between T-dependent and T-independent responses has been obscured because the latter may be much more efficient in the presence of T cells (see discussion in Mond and Brunswick, 1987). Our results indicate that no MHCmediated interaction is required for effective responses to type II T-independent antigens, and would suggest that if such responses are augmented by T helper-derived cytokines, these cytokines can also be produced by mono-

Cell 1062

cytes, Bcells, CD8’Tcells,orthefewCD4+Tcellsresiding in B cell follicles. Both in the unstimulated state and in response to T-independent antigens, the Afi’Omice are able to generate antibodies of both the IgG and IgM isotypes. This result was expected, given ample evidence of IgG antibodies in nude mice (for references see Holub, 1989). Interestingly, the mutant mice do not have normal levels of IgGl in their blood. This isotype has been thought to be the most T helper dependent, but in vitro and in vivo experiments have not produced consistent results on the cytokine control of switching to IgGl (for review see Cebraet al., 1984; Esserand Radbruck, 1990; Finkelman and Holmes, 1990). Viewed in its ensemble, the B cell compartment of class II-negative mice is almost surprisingly normal. There are certain defects, but they appear not to be debilitating. Conclusions The A$Omice have allowed us to confirm some old notions about the role of MHC class II molecules in T and B cell differentiation, but they have provided some surprises. These mutants should be useful for addressing some major outstanding questions: Under what conditions do CD4’ T cells regulate the response of CD8’ T cells? Do class II molecules significantly shape the y6 T cell repertoire? What role do class II molecules play in the rejection of various organ grafts? They may also prove valuable as models for human immunodeficiency diseases-in particular, those provoked by HIV infection or by a congenital defect in MHC class II gene expression. Experimental

Procedures

Construction of A0 Recombinants The recombinant used to disrupt the AB gene was designed for implementing the double-selection technique described by Mansour et al. (1988). The starting material was a 6.3 kb EcoRl fragment from an AB genomic clone carried in the plasmid pcEXV-n (Miller and Germain, 1988). The fragment’s coding potential was interrupted by insertion of a 1 .l kb segment of the pMC1 Neo-PolyA plasmid (Stratagene, Inc., La Jolla, CA) into a unique BstEll site located in the second exon. The above construct was then flanked on both the 5’and 3’ends by the herpes simplex virus thymidine kinase gene (McKnight, 1980). This was accomplished in two steps. First, Sall fragments containing individual copies of the gene were self-ligated, doublets were isolated, and the purified doublets were blunt-end ligated into the Bluescript plasmid (Stratagene, Inc.). Second, the resulting plasmid was linearized with Sal1 and blunt-end ligated with a purified EcoRI-Hindlll fragment from the f$-neo plasmid. The resulting construct was linearized with Notl, purified by phenol-chloroform extraction, and precipitated with ethanol before transfection. Transfection and Selection of ES Cells The D3 embryonic stem cell line was derived from a mouse of the 1291 Sv strain (Gossler et al., 1986) and was provided by Dr. Rolf Kemmler (Freiburg, Germany). Experiments were initiated with cells from the 14th passage. Cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 15% fetal calf serum, 1 m M sodium pyruvate, 0.1 m M P-mercaptoethanol, 1000 U/ml leukemia inhibitory factor (Smith et al., 1988) and 400 uglml gentamycin sulfate. They were routinely carried on gelatinized plates in a 37OC incubator gassed with 10% CO,. and passaged by trypsinization with Hanks balanced salt solution supplemented with 0.25% trypsin and 0.04% EDTA. For electropoiation, l-2 x 10’ D3 cells were suspended in 500 @I of growth medium in the presence of 5 to 10 ug of linearized vector DNA, and were shocked with a Bio-Rad Gene Pulser equipped with

a capacitance extender set at 1000 V/cm and 125 uF. After 5 min at room temperature, the cells were diluted and plated at 2 x lo6 cells per 10 cm plate. After 2 hr, the medium was changed and supplemented with 150 uglml genicitin (G418) and 2 pM gancyclovir. The medium was changed every 2 days throughout the IO day selection period. Resistant colonies were harvested by scraping and aspirating into a sterile drawn microcapillary pipette and were transferred onto fibroblast feeder layers in a 48-well plate. Clones were monitored daily and were passed every 2 or 3 days on gelatinized petri plates. An aliquot of each clone was frozen, and DNA was prepared from the remainder. Ten microgram DNA samples from the ES cell clones were digested with EcoRl and analyzed by Southern blotting for homologous recombination using probe 2 illustrated in Figures IC and 1D. A homologous recombination event should result in replacement of the normal 6.4 kb band by one of 4.4 kb. Cell lines considered positive by this assay were analyzed from the opposite end of the gene by a second Southern blot: a Bgl II digest probed with probe 1 shown in Figures IC and 1 D. Finally, the absence of a second, nonhomologous recombination event was ruled out by reprobing these blots with the neo insert. Production of Ap” Mice All embryo manipulations were performed essentially as described by Robertson (1987). Blastocysts were obtained from superovulated B6 female mice at day 3.5 of pregnancy. Mature blastocysts were collected and immediately microinjected. Morula were collected and cultured for about 4 hr to allow blastocyst formation. Between 8 and 12 ES cells were injected into the blastocoel cavity. Successfully injected blastocysts were cultured for 3 hr at 37% to allow repair of the membrane prior to reimplantation. Blastocysts were surgically reimplanted into the uteri of 96 x SJL mice. Chimeric mice were scored by looking at coat color; chimeras should have streaks of 129-derived agouti hair on the normally black hair of B6 mice. Chimeric males were crossed with 66 females, and the offspring were scored for transmission of the 1291S.v genotype through the germline by examining coat color. Transmission of the mutated Ar allele in agouti offspring was confirmed by blotting EcoRI-digested DNA from tail biopsies and hybridizing the blots to probe 2. Finally, heterozygous offspring were mated to produce homozygotes. Since the A:‘O homozygotes do not breed very well (if at all), the mutation is carried in the heterozygous state by backcrossing onto B6 animals. The homozygotes used for the studies in this paper result from mating males and females from the first, second, or third backcrosses against B6. Cytofluorometric Analyses The following mouse MAbs were used in MHC class II molecule characterization: Y-219, Y-271, and Y-279, all specific for As (Landais et al., 1986); 2A2, 4D5, 369, and Y3P, specific for A, (Landais et al., 1986; Becket al., 1986); 14.4.4, for E. (Ozato et al., 1980; Germain and Quill, 1986); 17.3.3, for EB (Ozato et al., 1980; Quill et al., 1986); and 408, which sees A and E of diverse haplotypes (Pierres et al., 1981)-more specifically, A0 (C. B., unpublished data) and, by analogy, En. The following rat MAbs were used in the analysis of T cells: KT3. specific for CD3 (Tomonari, 1988); H129-19, specific for CD4 (Pierres et al., 1984); H57-597, for a8 TCR (Kubo et al., 1989); GL3, for y6 TCR (Goodman and Lefrancois, 19891); ?OH12, for Thy1 determinants (Ledbetter and Herzenberg, 1979); Jll D and Ml 169, for heat-stable antigen (Bruce et al., 1981; Springer et al., 1978); IM7 and l42/5, for CD44 (Trowbridge et al., 1982; gift of J. Gutierrez-Ramos, Basel, Switzerland); 820-6.5, for VB2 (Malissen et al., unpublished data); KT4.10, for Vr4 (Tomonari et al., 1990); MR 9.4, for $,5 (Bill et al., 1990); 44.22.1, for V,6 (Acha-Orbea et al., 1985); KJl8, for Vr8 (Haskins et al.. 1984); RR3.15, for V&11 (Bill et al., 1989); 14.2, for Vr14 (Liao et al., 1989); and B-20.1. for V,2 (Malissen et al., unpublished data). Anti-B cell reagents were as follows: 44-D7, for IgD (gift of K. Rajewsky, Cologne, Germany); 53.7.3, for CD5 (Ledbetter and Herzenberg, 1979); RA3682. for CD45RA (6220) (Coffman and Weissman, 1981); and M1170. for CD1 1 b (Mac-l) (Springer et al., 1978). Fiuorescein isothiocyanate (FITC)-conjugated anti-CD8 and phycoery&rin-conjuplated anti-CD4 reagents were from Becton-Dicktnson.Attffborescentconjugated antirat and anti-mouse antibodies. were from Jackson lmmunoresearch

Mice Lacking MHC Class II Molecules 1063

Laboratories, Inc. (West Grove, PA). Fluorescent avidin conjugates were purchased from Biomeda (Foster City, CA). Thymus, lymph node, and spleen cell suspensions were prepared in Dulbecco’s modified Eagle’s medium. The spleen erythrocytes were lysed by hypotonic shock. Cell pellets were resuspended in ice cold phosphate-buffered saline (PBS) supplemented with 30 m M HEPES (pH 7.4). 5% heat-inactivated horse serum, and 0.1% sodium azide. Staining was performed in this same buffer. Test antibodies were added to 2 x lo5 cells in a total volume of 50 ul in go-well microtiter dishes. Incubations were for 30 min on ice with two washes between steps and three washes after the last step. Cells were then fixed in a 1% paraformaldehyde in PBS and stored at 4OC until analyzed. Flow cytometry was performed on an ODAM ATC 3000 equipped with logarithmic amplifiers. lmmunohistology Lymphoid organs ware frozen in liquid nitrogen and mounted for cryostat sectioning. Sections4 pm thick were dried and fixed with acetone. All antibody incubations were for 1 hr at room temperature in a humidified box; all dilutions and washes were in PBS. First-stage reagents were M5/114 (anti-A, -E [Bhattacharya et al., 1981]-specific for AB and, by analogy, EB [Braunstein and Germain, 19871) GK1.5 (antiCD4; Dialynas et al., 1983) ER-TR5 (anti-medullary epithelium; van Vleit et al., 1984), PNA-FITC (Vector Labs, Burlington, CA), 4Cll (anti-FDC; Gray et al., 1990) goat anti-mouse Ig-Texas red (Southern Biotechnology Assoc., Birmingham, AL), goat anti-mouse IgG-FITC (Southern Biotechnology Assoc.), and 11-26~ (anti-mouse IgD; gift of J. Kearney, Birmingham, AL). Mouse MAbs were detected with a polyclonal rat anti-mouse IgG-FITC conjugate (Jackson lmmunoresearch Labs, Hamburg, Germany). Counterstaining for IgM was with goat anti-mouse IgM-Texas red (Southern Biotechnology Assoc.). After staining, sections were washed thoroughly and mounted in Mowiol (Hoechst, Frankfurt-am-Main, Germany) plus 25 mglml 1 ,Cdiazobicyclo(2,2,2)octane to prevent fading. Slideswereviewed under aZeiss Axiophot fluorescence microscope using 580-585 nm and 450-495 nm filters for Texas red and FITC, respectively. lmmunopreclpitation Experiments Techniques used for labeling cells and precipitating class II molecules were essentially as described recently (Kaufman et al., 1990a, 1990b). Briefly, equal numbers of mouse spleen cells (approximately IO’ per sample) were biosynthetically labeled with [YS]methionine for 3 hr or 20 hr or were cell surface iodinated using lactoperoxidase and glucose oxidase. Equal aliquots of Nonidet P-40 lysates prepared from these labeled cells were precleared with normal mouse serum and incubated with various MAbs followed by protein G-Sepharose for immunoprecipitation. The immunoprecipitates were analyzed by electrophoresis in SDS using 12% polyacrylamide gels followed by autoradiography and, if necessary, fluorography. The amount of radioactivity in each band was quantitated using a Phosphorlmager (Molecular Dynamics). The MAbs were as follows: 21.460 (anti-Db; gift of B. Stockinger, Basel, Switzerland), 2A2 (anti-A,; Beck et al., 1986) 14.4.4 (anti-E,; Ozato et al., 1960; Germain and Quill, 1986) 408 (anti-A+, -Er; Pierres et al., 1981; C. B., unpublished data), 17.3.3 (anti-E@; Ozato et al., 1980; Quill et al., 1986); M5/114(anti-A,, -EB; Bhattachatyaet al., 1981; Braunstein and Germain, 1987). Serum lmmunoglobulins Concentrations of various lg isotypes in the serum were evaluated in an ELISA. Plates were coated overnight with a broadly reactive rabbit anti-mouse lg antibody; after blocking with PBS-BSA, the wells were incubated with serial dilutions of sera from A$” mice and normal littermates. Individual isotypes were revealed by a battery of phosphatase-conjugated anti-iSOtype reagenk. ISOtype concentrations were calculated from the linear portions of the dilution curves and were normalized by reference to average values from the several normal fittermates included in each experiment. T-Independent Responses All of the reagents used in assaying type II T-independent responses were kindly provided by J. Kearney. Mice were injected with 50 ug per mouse of al ,6 dextran, 20 pg per mouse of Aerobacter levanicum (levan), or 2 x IO8 formalin-fixed StreptOCOCCus pneumoniae rough

strain R36A (phosphorylcholine) (Lamers et al., 1989). All injections were done in normal saline i.p. At the indicated times, animals were bled and the serum assayed for specific antibody production by ELISA essentially as described in Lemeur et al. (1985). For the levan assay, plates were coated overnight at 5°C with 5 ugl ml levan. For al,6 dextran, plates were coated with a 5 uglml solution of dextran-BSA, and the MAb 42-2A7-2 (IgM, “b” allotype) was titrated (starting at 500 ng per well) as a positive control. For the phosphorylcholine assay, plates were coated overnight with a 1 uglml solution of phosphorylcholine-BSA, and a serial dilution of MAb PCA (IgM, “b” allotype, anti-phosphorylcholine) was used as a positive control. T-Dependent Responses KLH was purchased from Calbiochem (San Diego, CA) and TGAL from INC (Lisle, IL). Mice 6-8 weeks of age were injected with 50 ug of KLH (Lp.), 50 pg of TGAL (s.c.), or Ova (s.c.) in complete Freund’s adjuvant. Serum was collected after 12 days. The mice were further boosted at 21 days with an additional 25 ug of these antigens i.p. in saline. Serum was collected 7 days later. In some experiments, additional boosts were given. Specific antibodies to these antigens were measured by sandwich ELISA as described previously (Lemeur et al., 1985). Acknowledgments We thank H. von Boehmer and A. Singer for critically reading the manuscript; J. Kearney and Ft. Ceredig for generously providing reagents and advice; R. Kemler for the gift of D3; P. Dellabona, S. Chan, and J.-L. Pasquali for their help; K. Rajewsky, G. Hammerling, J. Gutierrez-Ramos, F. Ronchese, B. Stockinger, and R. Germain for donating antibodies and plasmids; T. Mak for leukemia inhibitory factor; C. Waltzinger for performing the cytofluorometric analyses; P. Gerber, J. Hergueux, C. Ebel, M. Digelman, and L. Heydler for technical assistance; and P. Michel, N. Zinck, and S. Metz for careful husbandry. This work was supported by institute funds from the INSERM and the CNRS and by grants to D. M. and C. B. from the Association pour la Recherche sur le Cancer. The Base1 Institute of Immunology was founded and is supported by F. Hoffman-La Roche Ltd., Basel, Switzerland. D. C. received fellowships from the North Atlantic Treaty Organization and the Fondation pour la Recherche Medicale Francaise. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received June 27. 1991 References Acha-Orbea, H., Zinkernagel, R. M., and Hengartner, H. (1985). Cytotoxic T cell clone-specific monoclonal antibodies used to select clonotypic antigen-specific cytotoxic T cells. Eur. J. Immunol. 75, 31-36. Anderson, G. D., and David, C. S. (1989). In vivo expression and function of hybrid la dimers (EaA6) in recombinant and transgenic mice. J. Exp. Med. 770, 1003-1011. Anderson, G. D., Banerjee, S., and David, C. S. (1969). MHC class II Aa and Ea molecules determine the clonal deletion of V86+ T cells. J. Immunol. 143, 3757-3765. Ashida, E. R., and Scofield, V. L. (1987). Lymphocyte major histocompatibilitycomplex-encoded class II structures may act assperm receptors. Proc. Natl. Acad. Sci. USA 84, 3395-3399. Beck, 8. N., Buerstedde, J.-M., Krco, C. J., Nilson, A. E., Chase, C. G., and McKean, D. J. (1986). Characterization of cell lines expressing mutant I-Aband I-Ak molecules allows the definition of distinct serologic epitopes on A. and Ar polypeptides. J. Immunol. 136, 2953-2961. Benoist, C., and Mathis, D. (1989). Positive selection of the T cell repertoire: where and when does it occur? Cell 58, 1027-1033. Berg, L. J., Pullen, A. M., Fazekasde St. Groth, B., Mathis, D., Benoist, C., and Davis, M. M. (1989a). AntigenlMHC-specific T cells are preferentially exported from the thymus in the presence of their MHC ligand. Cell 58, 1035-l 046.

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Berg, L. J., Fazekas de St. Groth, B., Pullen, A. M., and Davis, M. M. (1989b). Phenotypic differences between up versus p T-ceil receptor transgenic mice undergoing negative selection. Nature 340,559-562. Bhattacharya, A., Dorf, M. E., and Springer, T. A. (1981). A shared alloantigenic determinant on la antigens encoded by the I-A and I-E subregions: evidence for I region gene duplication. J. Immunol. II, 2488-2495. Bill, J., Kanagawa, O., Woodland, D. L., and Palmer, E. (1989). The MHC molecule I-E is necessary but not sufficient for the clonal deletion of VBII-bearing T cells. J. Exp. Med. 769, 1405-1419. Bill, J., Kanagawa, O., Linter, J., Utsunomiya, Y., and Palmer, E. (1990). Class I and class II MHC gene products differentially affect the fate of VP5 bearing thymocytes. J. Mol. Cell. Immunol. 4, 269-280. Bluestone, J. A., Pardoll, D., Sharrow, S. O., and Fowlkes, B. J. (1987). Characterization of murine thymocytes with CD3-associated T-cell receptor structures. Nature 326, 82-84. Bonifacino, J. S., McCarthy, S. A., Maguire, J. E., Nakayama, T., Singer, D. S., Klausner, Ft. D., and Singer, A. (1990). Novel posttranslational regulation of TCR expression in CD4+CD8+ thymocytes influenced by CD4. Nature 344, 247-251. Borgulya, P., Kishi, H., Mtiller, U., Kirberg, J., and von Boehmer, H. (1991). Development of the CD4 and CD8 lineage of T cells: instruction versus selection. EMBO J. 10, 913-918. Boswell, H. S., accessory cells accessory cells subpopulation.

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