MOLECULAR AND CELLULAR BIOLOGY, Aug. 2008, p. 5014–5026
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 28, No. 16
MPYS, a Novel Membrane Tetraspanner, Is Associated with Major
Histocompatibility Complex Class II and Mediates Transduction
of Apoptotic Signals?†
Lei Jin,1Paul M. Waterman,1Karen R. Jonscher,2Cindy M. Short,1
Nichole A. Reisdorph,1and John C. Cambier1*
Integrated Department of Immunology, University of Colorado School of Medicine and National Jewish Medical and Research Center,
Denver, Colorado 80206,1and Department of Anesthesiology, University of Colorado Health Sciences Center,
Denver, Colorado 802622
Received 19 April 2008/Accepted 30 May 2008
Although the best-defined function of type II major histocompatibility complex (MHC-II) is presentation of
antigenic peptides to T lymphocytes, these molecules can also transduce signals leading alternatively to cell
activation or apoptotic death. MHC-II is a heterodimer of two transmembrane proteins, each containing a
short cytoplasmic tail that is dispensable for transduction of death signals. This suggests the function of an
undefined MHC-II-associated transducer in signaling the death response. Here we describe a novel plasma
membrane tetraspanner (MPYS) that is associated with MHC-II and mediates its transduction of death
signals. MPYS is unusual among tetraspanners in containing an extended C-terminal cytoplasmic tail (?140
amino acids) with multiple embedded signaling motifs. MPYS is tyrosine phosphorylated upon MHC-II
aggregation and associates with inositol lipid and tyrosine phosphatases. Finally, MHC class II-mediated cell
death signaling requires MPYS-dependent activation of the extracellular signal-regulated kinase signaling
The ability of major histocompatibility complex class II
(MHC-II) to transmit signals was first recognized more than 20
years ago (6, 10). Depending upon the species and cell type,
anti-MHC-II monoclonal antibody (MAb) stimulation induces
tyrosine phosphorylation, calcium mobilization, cAMP produc-
tion, and mitogen-activated protein kinase, AKT, and protein
kinase C activation (for a review, see reference 1). In activated
murine B cells, MHC class II signals trigger tyrosine phosphor-
ylation and calcium mobilization via associated CD79a and -b
(18). These responses are also induced by T-cell receptor
(TCR) binding to the MHC-II/antigenic peptide complexes on
B cells (18).
Several recent studies have shed light on the biological im-
portance of MHC-II signaling, particularly MHC-II-mediated
death signaling. It was found that cross-linking of MHC-II by
MAb and via cognate MHC-II–TCR interaction can lead to
Fas-independent antigen-presenting cell (APC) death (21, 25).
Dendritic cells (DCs) undergo accelerated clearance from the
lymphoid organs after interacting with antigen-specific T cells
(15). Prolonged DC survival can lead to autoimmunity (9).
Thus, it has been proposed that MHC-II-mediated death sig-
naling functions to limit potentially dangerous uncontrolled
immune responses by elimination of APCs after they have
served their antigen-presenting purpose (25). Given the similar
response of certain B cells to MHC-II aggregation, it seems
likely that under certain circumstances they too are eliminated
by this mechanism.
The ability to mediate cell death signaling makes MHC-II a
candidate therapeutic target for treatment of certain malignan-
cies (4). Anti-MHC-II MAbs (1D09C3 and Hu1D10) are being
tested in clinical trials involving patients with refractory and
relapsed non-Hodgkin’s lymphoma or relapsed low-grade or
follicular lymphoma (5, 7). These anti-MHC-II MAbs exhibit
rapid and potent in vitro tumoricidal activity for lymphoma/
leukemia cells with no long-lasting hematological toxicity in
The molecular basis of MHC-II-mediated signaling of cell
death is unknown. This response is independent of comple-
ment and Fc receptors (22). Effective humanized anti-MHC-II
MAbs are all immunoglobulin G4 (IgG4) isotype antibodies
(Abs) that are devoid of complement-dependent cytotoxicity
and do not mediate Ab-dependent cell-mediated cytotoxicity
(21). All of these MAbs induce death efficiently in lymphoma/
leukemia tumor cells (21). Thus, death induced by these Abs,
and by extension T-cell antigen receptors, is thought to be
mediated by MHC-II signaling (20). Consistent with this pos-
sibility, protein kinase C activation is reportedly required for
MHC-II-mediated death in Raji human B-cell lymphoma, ma-
ture DCs, and activated THP-1 monocytes but not in primary
human plasmacytoid DCs (2, 12, 14, 27). MHC-II-mediated
signaling of death also appears to occur independently of Src
family kinase (14, 27) and caspase activation (8, 13) in Raji and
Ramos cells. Reactive oxygen species production and Jun N-
terminal protein kinase (JNK) activation have been implicated
in MHC-II-mediated death in lymphomas line JVM-2 and
GRANTA-519 (7). Thus, data are fragmentary, and there is no
* Corresponding author. Mailing address: University of Colorado
School of Medicine and National Jewish Medical and Research Cen-
ter, Integrated Department of Immunology, Room 1004 Goodman
Building, 1400 Jackson St., Denver, CO 80206. Phone: (303) 398-1352.
Fax: (303) 270-2325. E-mail: email@example.com.
† Supplemental material for this article may be found at http://mcb
?Published ahead of print on 16 June 2008.
consensus regarding how MHC class II molecules transduce
Here we describe studies aimed at dissection of the molec-
ular signaling pathways by which MHC class II molecules
transduce signals leading to apoptotic death of B lymphoma
cells. We report identification of a novel MHC-II-associated
membrane protein, termed MPYS, and demonstrate that it
mediates death signaling via activation of the extracellular sig-
nal-regulated kinase (ERK) pathway.
MATERIALS AND METHODS
Induction and assay of cell death. K46 cells were suspended in 5% complete
IMDM (catalog no. 10-016-cv; Mediatech Inc.) at a concentration of 106cells/ml
and then transferred to round-bottomed 96-well plates (100 ?l/well) and cultured
at 37°C for 1 h. Biotinylated MHC-II MAbs were added to the wells and
incubated for 12 min. Avidin was then added to the wells, and cells were cultured
for the indicated time. Propidium iodide (PI) (2 ?g/ml) and annexin V-Alexa
Fluor 488 (1 ?l/100 ?l cells) (A-13201; Molecular Probes) or DiOC6 (3) (50 nM)
and PI (2 ?g/ml) were used to measure apoptotic or dead cells by flow cytometry.
The ERK inhibitor PD98059 (513000; Calbiochem), AKT inhibitor LY294002
(440202; Calbiochem), and p38 inhibitor SB203580 (559389; Calbiochem) were
used in this study. The inhibitor was added to cells at 5 ? 106cells/ml and
cultured for 4 h. Samples were then split into 1 ? 106cells/ml, and cell death was
induced and measured as describe above. To inhibit src kinase, PP2 (529573;
Calbiochem) was added to cells and cultured for 30 min before cell death was
induced as described above.
Immunoprecipitation and immunoblotting. Cells were lysed in 0.33% CHAPS
buffer (150 mM NaCl, 10 mM Tris [pH 7.5], 10 mM sodium pyrophosphate, 2
mM Na3VO4, 10 mM NaF, 0.4 mM EDTA, 1 mM phenylmethylsulfonylfluoride,
and 1 ?g/ml each of aprotinin, ?1-antitrypsin, and leupeptin) (40 ? 106/ml) at
4°C for 1.5 h or overnight. Cell lysates were centrifuged at 12,000 ? g at 4°C for
20 min. Supernatants were analyzed on a 10% sodium dodecyl sulfate-poly-
acrylamide gel electrophoresis (SDS-PAGE) gel. The gel was transferred to a
polyvinylidene difluoride membrane, and proteins were probed with Abs and
visualized using an Odyssey infrared imaging system (Li-Cor). The Abs used
were 4G10 phosphotyrosine Ab, antihemagglutinin (anti-HA) MAb 16B12
(MMS-101P; Covance Research), antiactin Ab (I-19) (sc1616; Santa Cruz), anti-
SHP-1 Ab (12660), anti-SHIP Ab (26), anti-CD22 Ab (12045), anti-JNK 1 Ab
(c-17) (sc474; Santa Cruz), anti-pan-ERK (610123; Transduction Lab), and Abs
from Cell Signaling Technology: anti-p38 (no. 9212), anti-p-P38 (no. 9211),
anti-pERK (no. 9101), anti-AKT (no. 9272), anti-pJNK (no. 9251), and anti-
pSer473-AKT (no. 9271).
Analysis of intracellular free calcium concentration. Cells were loaded with
Indo-1AM (Molecular Probes, Eugene, OR) for 30 min at 37°C as recommended
by the manufacturer. Anti-MHC-II MAb (10 ?g/ml) was then added to the
suspension for another 12 min at 37°C before cells were washed and suspended
in IMDM supplemented with 2% fetal calf serum. The cells (106cells/ml) were
analyzed before and after stimulation via cross-linking with avidin (20 ?g/ml).
Data were analyzed using the Flow-Jo software program (Tree Star, Inc., San
Nano-LC-MS/MS. K46 cells (?5 ? 108) were lysed in 0.33% CHAPS buffer,
and immunoprecipitation was performed as previously described (18). Proteins
were eluted from Ab-bound beads with 0.1 M citric acid (pH 2.0). Eluates in
citric acid were neutralized with 2 M Tris (pH 10.0). The combined eluates were
buffer exchanged into 25 mM ammonium bicarbonate (40867; Fluka) using Zeba
desalting spin columns (89890; Pierce). The protein ammonium bicarbonate
solution was dried, reconstituted in 8.0 M urea (U-4883; Sigma), reduced in
dithiothreitol (50 mM; D5545; Sigma), alkylated with iodoacetamide (100 mM;
I1149; Sigma) and trypsin (1 ?g; V511A; Promega), and digested overnight at
37°C. The digests were analyzed by reverse-phase nanospray liquid chromatog-
raphy-tandem mass spectrometry (nano-LC-MS/MS) (Agilent 1100 high-perfor-
mance liquid chromatography system and Agilent Ultra ion trap). Proteins were
identified using the Spectrum Mill (Agilent) database search algorithm.
Molecular cloning of the murine MPYS gene. Primers (MPY5 and MPY3)
were designed to amplify the full-length mpys sequence from a K46 cDNA library
(see Table S2 in the supplemental material). PCR was carried out using Ex Tag
DNA polymerase (RR001; TaKaRa) and sequenced.
Making MPYS-EGFP, MPYS-HA, and MPYS-Flag constructs. Primers
(MPYS-EGFP-For and -Rev) were designed to include restriction enzyme sites
in the end of the mpys coding sequence (see Table S2 in the supplemental
material). The PCR product was cloned into the EcoRI-NotI sites of a pMXI-
EGFP fusion construct. A reverse primer (HA-MPY-Rev) was designed to
include an XhoI site and an HA tag sequence (see Table S2 in the supplemental
material). HA-MPYS was amplified using the primers MPYS-EGFP-For and
HA-MPY-Rev. The PCR product was cloned into the EcoRI-XhoI sites of a
pMXI-IRES-EGFP vector. For the LEL-Flag construct, a Flag sequence was
inserted between V73and Q74of MPYS. For the SEL-Flag construct, the Flag
sequence was inserted between E149and K150of MPYS. The insertion was
performed as described previously (30). The construct was confirmed by se-
Generation of polyclonal anti-MPYS Abs. Primers (GST-MPYS-For and
-Rev) were designed to amplify cDNA sequence encoding the C-terminal 101
amino acids (aa) of MPYS (see Table S2 in the supplemental material). The
PCR product was cloned into the BamHI-NotI sites of pGEX-5X-1 (27-4585-01;
Amersham Biosciences) to make the peptide, which was used to immunize a
rabbit. The polyclonal Ab was affinity purified using peptide-coupled Sepharose
shRNA knockdown of MPYS expression. Two short-hairpin RNAs (shRNAs)
were designed to target sequences in exons 5 and 7 of mpys (sh5, 5?GGATCC
GAATGTTCAATCA-3 ?; sh7, 5?GCACATTCGTCAGGAAGAA-3 ?, respec-
tively), using the pSicoOligomaker 1.5 software program (28). The forward and
reverse primers (1 ?l each of 1 ?g/?l) (see Table S2 in the supplemental
material) were annealed (28), and the annealed oligonucleotides were ligated
into pLL3.7 through HpaI and XhoI sites. An shRNA that targets the luciferase
gene was used as a control (31). Lentiviruses were generated as previously
Cell surface biotinylation. Cells (25 ? 106cells/ml) were suspended in phos-
phate-buffered saline. Sulfo-NHS-LC-Biotin (21335; Pierce) (0.5 mg/ml) was
added, and cells were incubated with rotation for 30 min at room temperature.
Glycine (50 mM) was added to the cell suspension for an additional 5 min to
quench excess succinimidyl ester. The cells were then centrifuged and lysed in
0.33% CHAPS buffer.
Live-cell imaging. K46 cells expressing green fluorescent protein (GFP)-
tagged MPYS (MPYS-GFP) (5 ? 104) were loaded into Lab-Tek chamber
(177445; Nunc, Denmark) slides and allowed to adhere for 12 h. Cells were
loaded with 2 ?g/ml Hoechst 34580 (H21486; Invitrogen) and 1 ?M dihydro-
rhodamine 6G (D633; Invitrogen) for 30 min. Images were collected using a 63?
objective in an inverted Zeiss 200 M microscope.
Table S1 in the supplemental material lists proteins identified by nano-LC/
MS/MS in the MHC-II complex with at least two unique peptides; Table S2 lists
all the primers used in the study. Figure S1 in the supplemental material shows
the mass spectra of peptides from MHC-II ?/? chains, CD20, CD37, and MPYS
identified in the MHC-II complex (panel A) and MHC-II ?/? chains and MPYS
in the MPYS complex (panel B).
Nucleotide sequence accession number. The mouse mpys cDNA sequence
(accession no. DQ910493) has been deposited in GenBank.
MHC-II MAb induces time and dosage-dependent death of
K46 B lymphoma cells. We studied MHC-II-induced cell death
in a murine B lymphoma line, K46 (17). Cross-linking of
MHC-II by MAbs in K46 cells has been shown to induce
tyrosine phosphorylation, calcium flux, and phosphatidylinosi-
tol 3-kinase activation (3). As shown in Fig. 1A and B, MHC-II
cross-linking by biotinylated I-Ab,d,q/Ed,k-reactive MAb M5/
114 (rat IgG2b) and avidin induced time and dosage-depen-
dent K46 B lymphoma exposure of phosphatidylserine as in-
dicated by annexin V staining. The anti-Fc receptor MAb
2.4G2 (rat IgG2b) was used as an isotype control. This MHC-II
MAb response, which suggests a commitment to cell death,
was observed as early as 1 h and peaked at 5 h (Fig. 1A). The
cell death response was also measured by dual staining with PI
and DiOC6(3)/PI (Fig. 1C and D). PI staining reflects cell
permeability, and loss of DiOC6(3) staining reflects mitochon-
drial membrane depolarization. The control 2.4G2 MAb did
not induce cell death. Our findings demonstrate that MHC-II
molecules transduce apoptotic cell death signals in K46 cells
VOL. 28, 2008MPYS: A TRANSDUCER OF MHC-II DEATH SIGNALS5015
and this response involves depolarization of mitochondrial
PP2 inhibits MHC-II MAb-induced tyrosine phosphoryla-
tion and calcium mobilization but not the death response.
Cross-linking of MHC-II by MAbs in K46 cells is known to
induce protein tyrosine phosphorylation and calcium flux (3).
To explore whether these signaling events are involved in the
death response, we assessed the effect of the Src kinase inhib-
itor PP2 on induction of cell death. As expected, MHC-II
MAb-induced tyrosine phosphorylation and calcium responses
were blocked by PP2 treatment (Fig. 2a and b). However,
MHC-II MAb-induced cell death remained largely intact after
treatment with inhibitor (Fig. 2c). This finding is consistent
with results in previous studies, which showed that tyrosine
phosphorylation and calcium flux are not required for the
MHC-II-mediated death response of human B cells (14, 16).
MHC-II MAb-induced ERK activation but not AKT and p38
activation is required for the death response. MHC-II aggre-
gation can lead to activation of AKT and mitogen-activated
protein kinase kinases ERK, p38, and JNK in monocytes and
human B cells (1). We investigated whether these signaling
events are activated upon MHC-II MAb stimulation of K46
cells. Indeed, cross-linking of MHC-II by biotinylated M5/114
and avidin induced strong and sustained ERK and p38 activa-
tion in K46 cells (Fig. 3a and b). AKT was weakly activated by
MHC-II cross-linking, as measured using phospho-AKT Ab
blotting (Fig. 3A). There was weak basal JNK activation mea-
sured by phosphor-JNK Ab. However, MHC-II activation did
not increase JNK phosphorylation (Fig. 3C). Thus, MHC-II
cross-linking activates AKT, ERK, and p38 but not JNK in K46
We then studied requirements for these signaling events in
the cell death induced by MHC-II MAb. Treatment of K46
cells with LY294002 inhibited MHC-II-mediated AKT activa-
tion but had no effect on the death response (Fig. 3D). MHC-
II-mediated p38 activation was completely inhibited by
SB203580, but the inhibitor also had no effect on the death
response (Fig. 3E). Interestingly, at 50 ?M, the ERK-specific
FIG. 1. MHC class II MAb induces time and dosage-dependent apoptosis of K46 B lymphoma cells. (A) K46 cells were treated with 15 ?g/ml
biotinylated anti-MHC class II (M5/114; rat IgG2b) MAb or its isotype control, biotinylated anti-mouse Fc?RIIB MAb ((2.4G2; rat IgG2b),
followed by 20 ?g/ml avidin for the indicated time. Cell death was measured by annexin V/PI dual staining (n ? 3). (B) Cells were treated as for
panel A at the indicated Ab doses for 5 h. Cell death was measured as for panel A (n ? 3). (C and D) Cells were treated with 15 ?g/ml biotinylated
M5/114 or 2.4G2 plus 20 ?g/ml avidin for 5 h. Cell death was measured by annexin V/PI (C) or DiOC6/PI dual staining (D) (n ? 3). Numerical
annotation reflects the percentage of cells in each quadrant. Error bars represent standard deviations of triplicates.
5016JIN ET AL.MOL. CELL. BIOL.
inhibitor PD98059 inhibited MHC-II-mediated cell death by
?50% (Fig. 3F). However, this dose did not completely inhibit
MHC-II-induced ERK activation, explaining the residual
cell survival and implicating ERK in the cell death response
In conclusion, although cross-linking MHC-II with MAb
leads to activation of AKT, ERK, and p38, only ERK activa-
tion is required for the death response of these cells.
Identification of a novel MHC-II-associated membrane pro-
tein using nano-LC-MS/MS. The signaling circuitry by which
MHC-II aggregation activates ERK is unknown. MHC-II ?
and ? chains contain cytoplasmic tails of only 12 and 18 amino
acids, respectively, and deletion of these tails does not affect
the death response (16). Thus, we postulated that MHC-II
must transmit death signals through an associated cell surface
protein(s). The B-cell-specific proteins CD19, CD20, and
CD79a/b have been shown to be physically and functionally
associated with MHC-II and therefore were candidates (18,
19). However, unlike the MHC-II death response, CD19,
CD20, and CD79a/b expression is B cell specific, suggesting
that these molecules are not likely to be involved in this re-
sponse. MHC-II is also known to associate with tetraspanins,
including CD9 and CD37 (19). However, these molecules have
very short cytoplasmic tails (8 to ?14 aa) that lack defined
signaling motifs. Therefore, we hypothesized that a novel
MHC-II-associated protein(s) might function in transduction
of signals that mediate the death response. To explore this
possibility, we undertook proteomic analysis of MHC-II-asso-
ciated proteins. We prepared lysates of K46 cells using a mild
detergent (CHAPS) that preserves weak protein-protein inter-
actions and then immunoprecipitated MHC-II and associated
proteins using anti-MHC-II MAb beads. We eluted proteins
from the beads and identified them using nano-LC-MS/MS.
Forty-one proteins were recovered from the MHC-II immu-
FIG. 2. MHC-II signaling of cell death does not require activation of Src family tyrosine kinases. (A) K46 cells were treated with 25 ?M PP2
or dimethylsulfoxide (DMSO) for 30 min at 37°C and then stimulated with biotin–MHC-II (M5.114; 20 ?g/ml) and avidin (20 ?g/ml) for the
indicated times. SDS-PAGE-fractionated whole-cell lysates were probed with the indicated Abs (n ? 3). (B) The intracellular free calcium
concentration ([Ca2?]i) was measured before and during K46 stimulation with biotin–MHC-II (M5.114; 10 ?g/ml) and avidin (10 ?g/ml). Cells
were pretreated with DMSO or PP2 as described for panel (A) (n ? 3). (C) K46 cells treated as for panel A were stimulated with biotin–anti-
MHC-II (M5.114; 10 ?g/ml; shaded bars) or biotin-anti-Fc?RIIB (2.4G2; 10 ?g/ml; open bars) and avidin (10 ?g/ml) for 5 h, and death was
measured by annexin V-PI staining as described in Materials and Methods (n ? 3). Error bars represent standard deviations of triplicates.
VOL. 28, 2008 MPYS: A TRANSDUCER OF MHC-II DEATH SIGNALS5017
FIG. 3. ERK function is required for MHC-II-mediated cell death. (A to C) K46 cells were stimulated with biotin–MHC-II (M5/114; 20 ?g/ml)
plus avidin (20 ?g/ml) for the indicated times and detergent lysates prepared. Transfers of SDS-PAGE-fractionated whole-cell lysates were probed
with the indicated Abs (n ? 4). (D to F) K46 cells were treated with the indicated doses of LY294002, SB203580, or PD98059 as described in
Materials and Methods and then stimulated, lysed, and probed as for panels A to C. Cell death was stimulated and measured as for panel C of
Fig. 2. Shaded bars denote biotin–anti-MHC-stimulated samples. Open bars denote biotin–anti-FcR-stimulated samples. Error bars represent
standard deviations of triplicates (n ? 3).
5018JIN ET AL.MOL. CELL. BIOL.
noprecipitates based on the detection of at least two unique
peptides from each (see Table S1 in the supplemental mate-
rial). MHC-II ? and ? chains were identified by 6 and 14
peptides, respectively, confirming the effectiveness of the af-
finity purification (see Fig. S1a in the supplemental material).
We were particularly interested in MHC-II-associated proteins
containing a transmembrane domain(s) and cytoplasmic signaling
motifs. A hypothetical protein, RIKEN cDNA 2610307O08, was
implicated by three unique peptides predicted by its DNA se-
quence (see Fig S1a in the supplemental material). This hypo-
thetical protein is predicted to contain four transmembrane
domains by the SOSHI (http://bp.nuap.nagoya-u.ac.jp/sosui/),
and TMpred (http://www.ch.embnet.org/software/TMPRED_form
.html) software programs, along with multiple signaling motifs pre-
dicted by the ELM (http://elm.eu.org/) and Scansite (http://scansite
based inhibitory motif (ITIM), SVY244EIL) (Fig. 4A). We
designated it MPYS based on its N-terminal methionine-proline-
tyrosine-serine amino acid sequence.
We cloned the mpys gene from a cDNA library produced
from the murine K46 B lymphoma cell line (17). The gene
encoded a 378-aa protein with a predicted mass of 42 kDa (Fig.
4A). The sequence does not show significant homology to
known or predicted proteins, suggesting MPYS belongs to a
novel, single-member class of proteins. Human MPYS is
?80% homologous with mouse MPYS, and no invertebrate
homologues of mpys were found in the database (Fig. 4A).
To begin to explore protein expression and function, we
raised a polyclonal Ab against the C-terminal 101 aa of
murine MPYS and used it to immunoprecipitate endoge-
nous MPYS from K46 cell lysates. We then analyzed eluates
by nano-LC-MS/MS. Ab reactivity with MPYS was con-
firmed by detection in eluates of 15 unique peptides cover-
ing more than 50% of the total amino acid sequence (see
Fig. S1b in the supplemental material), including peptides
from the N and C termini of the protein (see Fig. S1b in the
supplemental material). Consistent with MPYS association,
two and four peptides derived from MHC-II ? and ? chains,
respectively, were found in the immunoprecipitate (see Fig.
S1b in the supplemental material). This association was
confirmed by immunoblotting (Fig. 4B).
To evaluate the cell surface expression of MPYS, we ex-
pressed mpys-HA in K46 cells. These cells are designated
KHA. Anti-HA immunoprecipitation (IP) of lysates of surface-
biotinylated K46 cells, followed by SDS-PAGE, transfer, and
avidin blotting, revealed that MPYS is biotinylated (Fig. 4c)
and therefore is on the cell surface.
To confirm the predicted topology of the MPYS protein,
we made two Flag-tagged mpys constructs. One has a Flag
tag inserted in the predicted large extracellular loop (LEL-
Flag). The other has a Flag tag in the predicted small ex-
tracellular loop (SEL-Flag). We expressed these two con-
structs in K46 cells and stained the cells with anti-Flag Ab.
As shown in Fig. 4D, the Flag-tagged MPYS proteins were
detected on the cell surface. On the contrary, our own poly-
clonal Ab, which is against the predicted cytoplasmic tail of
MPYS, did not stain intact cells (data not shown). These
data suggest that the predicted four-transmembrane topol-
ogy is likely correct (Fig. 4E).
To investigate MPYS distribution in cells, we made a
MPYS-GFP fusion construct and expressed it in K46 cells.
Confocal microscopy showed that while some MPYS was
found on the cell surface, a large proportion was actually lo-
calized to mitochondria (Fig. 4F).
The transmembrane region of MPYS contains four charged
residues and two cysteines. Such residues often mediate inter-
or intraprotein interactions. To determine if MPYS can form
protein complexes, we used the membrane-permeative chem-
ical cross-linker dithiobis(succinimidyl)propionate to form co-
valent bonds between neighboring proteins and performed
SDS-PAGE analysis on K46 whole-cell lysates on a nonreduc-
ing SDS-PAGE gel. In addition to the monomer, MPYS im-
munoblotting revealed an ?80-kDa band, a band double the
size of the MPYS monomer (Fig. 4G). This suggested that
most MPYS exists as a dimer within cells. The blot was
stripped and reblotted with MPYS Abs in the presence of a
blocking peptide (the 101 aa used to generate the MPYS Ab).
As show in Fig. 4G, no protein bands were recognized, indi-
cating that MPYS Ab blotting is specific. Sequential blotting
for CD19 provided an additional control for equivalent loading
To assess tissue distribution of MPYS, we probed SDS-
PAGE fractionated and transferred lysates of various B and T
cells (Fig. 4H). Anti-MPYS reacted with a predominant 40-
kDa species in spleen and thymus tissue (Fig. 4H). Splenocytes
have higher MPYS expression than thymocytes, which is con-
sistent with higher level-expression in B cells (Fig. 4H). MPYS
was also present in dendritic cells (Fig. 4I).
To assess potential changes in MPYS expression during B-
cell development and differentiation, whole-cell lysates from
B-lineage tumors were probed with MPYS Ab. MPYS Ab
recognized a 40-kDa band that was enhanced in K46 cells
expressing the HA-tagged mpys gene (Fig. 4J). MPYS was
highly expressed in cells representing mature stages of B cells
(Bal17 line) but weakly expressed in pre-B cells (70Z/3 line),
immature B cells (WEHI 231 line), and memory B-cell stages
(A20 line). It was not detected in plasma cells (J558L line)
(Fig. 4J). Thus, it appears to be expressed throughout the B
lineage prior to the plasma cell stage but occurs at highest
levels in mature B cells.
MPYS possesses inhibitory signaling function. The cyto-
plasmic tail of MPYS contains ITIMs, motifs known to recruit
the inhibitory signaling effectors SHP-1 and SHIP (11). To
assess the ability of MPYS to serve an inhibitory function, K46
cells were stimulated with anti-MHC-II MAb for 2 min before
by SDS-PAGE, and subjected to immunoblotting analysis. An-
tiphosphotyrosine blotting revealed that MPYS is tyrosine phos-
phorylated upon MHC-II cross-linking (Fig. 5A). Reprobing the
blot with anti-SHP-1 and SHIP Abs showed that phosphorylated
MPYS bound SHP-1 and SHIP (Fig. 5A). Notably, a 32-kDa
unknown tyrosine-phosphorylated protein was also associated
with MPYS. Thus, MPYS engages negative signaling effectors
when tyrosine is phosphorylated, consistent with its ITIM.
Studies of various inhibitory receptors suggest that SHP-1
and SHIP recruitment results in inhibition of calcium mobili-
zation (11). To assess whether MPYS mediates this function,
we overexpressed MPYS in K46 cells (Fig. 4J). MPYS overex-
pression led to dramatically reduced MHC-II-mediated cal-
VOL. 28, 2008 MPYS: A TRANSDUCER OF MHC-II DEATH SIGNALS5019
cium mobilization (Fig. 5B). These data suggest that MPYS act
in feedback regulation of some MHC-II signals, i.e., those that
lead to calcium mobilization.
We also found that cells overexpressing MPYS tend to be lost
from populations during culture, suggesting MPYS may have a
negative effect on cell growth. To further explore this possibility,
we expressed an MPYS-GFP fusion construct in A20 cells. Sorted
MPYS-GFP-positive A20 B lymphoma cells lost MPYS-GFP ex-
pression within 10 days (Fig. 5C). In contrast, expression of GFP
alone in A20 was maintained indefinitely (data not shown). These
data indicate that MPYS functions as a negative regulator of cell
5020 JIN ET AL.MOL. CELL. BIOL.
Knockdown of MPYS expression in K46 cells inhibits MHC
class II aggregation-induced cell death and ERK activation.
To further study the role of MPYS in MHC-II signaling, an
shRNA targeting exon 5 (sh5) or exon 7 (sh7) of the mpys gene
was prepared and used to knock down MPYS expression in
K46 cells. Cells expressing sh5 RNA displayed a ?90% reduc-
tion in MPYS expression, while in cells expressing sh7-RNA
MPYS, expression decreased by ?80% (Fig. 6A). Surface ex-
pression of IgM, MHC-II, CD19, CD45, CD80, and CD86 was
not altered by shRNA expression (data not shown). Contrary
to the case with MPYS overexpression (Fig. 5C), MPYS
knockdown increased the growth rate of K46 cells (Fig. 6B).
We next examined the role of MPYS in the death response
by assessing anti-MHC-II induction of death in K46 cells ex-
pressing MPYS knockdown constructs. K46 cells expressing a
control luciferase shRNA (luc) or MPYS sh5 knockdown
shRNA (sh5) were stimulated with biotinylated MAb M5/114
and avidin, and cell death was measured by annexin V/PI dual
staining (Fig. 6C and D). Biotinylated MAb 2.4G2 was used as
an isotype control. MHC-II MAb-induced cell death was re-
duced significantly in K46 cells expressing the sh5 MPYS con-
struct (where MPYS expression is diminished by ?90%) (Fig.
6C and D). An effect of MPYS knockdown was noted at all
doses of anti-MHC-II MAb used (Fig. 6C). Similar results
were observed in K46 cells expressing the sh7 MPYS knock-
down construct (data not shown). The fact that two shRNAs,
targeting different regions of mpys, caused similar outcomes
argues strongly that this is not an off-target effect. Similar
results were observed when other anti-MHC-II Abs were used
for stimulation (data not shown). We conclude that MPYS
expression is essential for anti-MHC-II MAb induction of B-
To extend earlier findings that MHC-II-mediated activation
of ERK is required for the death response, we investigated the
FIG. 4. Identification of a novel MHC-II-associated membrane protein. MHC-II-associated proteins were isolated by coimmunoprecipitation
from K46 cell CHAPS detergent lysates and analyzed using nano-LC-MS/MS. One candidate, designated MPYS, was identified based on detection
of three peptides predicted by the DNA sequence. (A) Alignment of mouse and human MPYS orthologs with annotation of their predicted
signaling motifs. (B) K46 cells were lysed in CHAPS buffer. MHC-II IP was accomplished using M5/114-coupled Sepharose beads; CD22 was
immunoprecipitated using Cy34-coupled Sepharose beads. MPYS, MHC-II, and CD22 were detected with the indicated Abs (n ? 3). (C) HA IP
was performed using 1% NP-40 lysates of surface-biotinylated K46 cells (5 ? 106) expressing HA-MPYS (KHA) or vector using HA MAb 16B12
(vec). Electrophoretic transfers were probed with indicated Abs (n ? 3). (D) K46 cells expressing LEL-Flag, SEL-Flag, or vector were stained using
biotin-Flag (M2; Sigma) and streptavidin-PE (n ? 2). (E) A cartoon illustrates plasma membrane disposition of MPYS and its four transmembrane
domains. Key residues conserved between human and mouse MPYS are annotated. (F) K46 cells expressing MPYS-GFP were stained with
dihydrorodamine 6G (red) and Hoescht (blue). Arrows indicate MPYS-GFP localization on the cell surface (n ? 3). (G) K46 cells were treated
with dithiobis(succinimidyl)propionate (DSP) (?) or DMSO (?) and lysed in radioimmunoprecipitation assay buffer as described previously (16).
Whole-cell lysates were run on 5% nonreducing gels, transferred, and probed with MPYS Ab and then stripped and reprobed with MPYS Ab plus
the blocking peptide. Afterward, the blot was probed again with anti-CD19 Ab. (H and I) Transfers of whole-cell detergent lysates from
AutoMACS (Miltenyi Biotec)-selected CD4?, CD8?, B220?cells, and cultured BMDC were probed with anti-MPYS Ab (n ? 3). (J) Transfers
of whole-cell lysates from various B-lineage lymphomas were probed with anti-MPYS Ab (n ? 3).
VOL. 28, 2008 MPYS: A TRANSDUCER OF MHC-II DEATH SIGNALS5021
effect of MPYS knockdown on ERK activation. We probed
fractionated and transferred lysates of luc or sh5 shRNA-ex-
pressing K46 cells using phospho-ERK Ab. MPYS knockdown
inhibited MHC-II MAb-induced ERK activation (Fig. 6E).
These findings indicate that MHC class II signaling of cell
death is dependent on MPYS-linked ERK activation.
MHC-II-mediated, MPYS-dependent activation of ERK is
Src family kinase independent. If, as shown in Fig. 2, anti-
MHC class II-induced death is not dependent on Src family
kinase-mediated tyrosine phosphorylation but is ERK depen-
dent, one would predict that ERK activation by class II signals
should not be inhibited by PP2. As shown in Fig. 6F, this is the
case. Taken together, these data indicate that the death re-
sponse is mediated by Src family kinase-independent activation
of MPYS and downstream ERK. Further, PP2-sensitive anti-
class II-activated signaling events, including calcium mobiliza-
tion and MPYS tyrosine phosphorylation and association with
SHIP-1 and SHP-1, are not required for the death response
(Fig. 2; also data not shown).
Finally, we addressed whether the MPYS dependence of
ERK activation reflects a requirement that MPYS interact
directly with ERK. Cells were activated with MHC-II MAb
before being lysed and subjected to MPYS immunoprecipita-
tion. Immunoprecipitates were analyzed by SDS-PAGE and
ERK immunoblotting. Under conditions in which we observed
SHP-1 recruitment to MPYS, we did not detect recruitment of
ERK (Fig. 6G).
MHC-II-independent aggregation of MPYS leads to cell
death signaling. To test the ability of MPYS to transduce
death signals when aggregated independently, we stimulated
K46 cells expressing Flag-tagged MPYS (LEL-MPYS) with
anti-Flag and measured death by PI staining. As shown in Fig.
7, anti-Flag treatment led to a 10-fold increase in PI staining of
cells within 15 h of stimulation. This response, while clearly
significant and Ab dose dependent, was somewhat less than
that induced by anti-MHC-II. We conclude that MPYS may
function in isolation as a transducer of death signals and thus
could mediate death signaling in MHC-II-negative cells, such
as T cells.
The heterodimeric MHC class II protein complex mediates
binding of antigenic peptides and presentation of these pep-
tides to CD4?T cells for their consequent activation, prolif-
eration, and differentiation. It is becoming increasingly clear
that under some circumstances, it is important that cells pre-
senting antigen be eliminated once they have served their func-
tion. Available evidence indicates that this may occur by TCR-
induced MHC class II-mediated induction of apoptotic death.
Importantly, such a mechanism would ensure elimination of
only APC that had presented specific, potentially offensive
antigens and would function to terminate responses to those
antigens. Although this is not surprising for dendritic cells, it
FIG. 5. MPYS negatively regulates MHC-II signaling. (A) K46 cells were stimulated using biotinylated anti-MHC-II (M5/114, 20 ?g/ml) and
avidin (40 ?g/ml) for 2 min. Cells were lysed in CHAPS buffer. MPYS was immunoprecipitated using MPYS polyclonal Ab and protein G. Normal
rabbit serum IgGs and protein G were used as a control IP in the lane designated C. The blot was probed with the indicated Abs (n ? 3). (B) K46
cells expressing vector alone or HA-MPYS were stimulated and calcium mobilization measured as for Fig. 2B (n ? 3). (C) Sorted GFP-positive
MPYS-GFP-expressing A20 cells were cultured and GFP expression levels monitored on the indicated days (n ? 5).
5022JIN ET AL.MOL. CELL. BIOL.
FIG. 6. Knockdown of MPYS expression in K46 cells inhibits MHC class II-mediated cell death and ERK activation. (A) K46 cells expressing
the control shRNA targeting the luciferase gene (luc) or exon 5 of the mpys gene (sh5) or exon 7 of the mpys gene (sh7) were lysed, SDS-PAGE
fractionated, transferred, and blotted with indicated Abs (n ? 4). (B) K46 cells expressing different shRNA constructs were cultured, and the cell
growth rate was measured using trypan blue (15250061; Invitrogen) (n ? 3). (C) K46 cells expressing luc shRNA (luc) or mpys-sh5 shRNA (sh5)
were stimulated with biotin-m5/114 and avidin for 5 h in cells and death measured by annexin V staining (n ? 3). (D) Time course of cell death
induced in shRNA-expressing populations by biotin-m5/114 (10 ?g/ml) and avidin (20 ?g/ml) (n ? 3). Biotinylated anti-FcR MAb 2.4G2 (10
?g/ml) and avidin (20 ?g/ml) were used as the isotype control (n ? 3). (E) K46 cells expressing luc shRNA or sh5 shRNA were activated as for
Fig. 3A. The blot was probed with the indicated Abs (n ? 3). (F) Cells were treated with PP2 (10 ?M) for 2 min as for Fig. 2 and were then
stimulated and analyzed as for Fig. 3F. (G) MPYS IP was done as for Fig. 5A. The blot was probed with indicated Abs (n ? 2). Error bars in all
panels represent standard deviations of triplicate assays. Data shown are representative of at least three experiments.
VOL. 28, 2008MPYS: A TRANSDUCER OF MHC-II DEATH SIGNALS 5023
seems somewhat counterintuitive that such a mechanism
would eliminate B-cell APC, some of whose daughters would
be expected to become Ab-secreting cells. Nonetheless, there
is compelling evidence that some ex vivo B cells die an apop-
totic death following aggregation of their MHC class II (23).
This fate may be reserved for a certain B-cell subpopulation
whose continued antigen presentation and/or Ab production
might be disadvantageous for the animal. For example, this
mechanism may be important in elimination of autoreactive B
cells. Indeed, one report has described T-cell-contact-induced
death of anergic B cells (24).
This study focused on understanding how MHC class II
molecules transduce apoptotic signals. This response is known
not to be dependent on the short, 12- and 18-aa cytoplasmic
tails of MHC-II (16), suggesting the function of an MHC-II-
associated transmembrane protein in transduction of these
signals. Although multiple candidate-associated molecules
have been advanced, for reasons discussed earlier none of
these are likely possibilities. Therefore, we embarked on a
search for associated proteins that mediate MHC class II trans-
duction of death signals. Nano-LC-MS/MS analysis of MHC
class II-associated proteins revealed a previously unknown
FIG. 7. Aggregation of surface MPYS induces K46 cell death. (A) K46 cells expressing the Flag-tagged-MPYS (LEL-MPYS) were stimulated
for 15 h with different doses of 2.4G2 anti-FcR MAb (FcR), anti-Flag MAb (MPYS), or D3.137 anti-MHC-II MAb (MHC II). Cell death was
measured by PI staining and forward light scatter properties. Shown are dead cells as a percentage of the total (n ? 2). (B) Cytograms displaying
PI staining as a function of forward light scatter of cells described for panel A (n ? 2).
5024 JIN ET AL.MOL. CELL. BIOL.
membrane tetraspanner that is associated with class II and
expressed in both mature B cells and dendritic cells (Fig. 4):
cells that undergo apoptotic death in response class II aggre-
gation. Consistent with a transmembrane signaling function,
the protein, termed MPYS, is found on the cell surface and
contains multiple sites of predicted protein-protein interaction
in its 140-aa cytoplasmic tail (Fig. 4).
Based on these features, we explored the role of MPYS in
signaling. MPYS was tyrosine phosphorylated following MHC
class II aggregation (Fig. 5). Consistent with its content of
ITIMs, this phosphorylation was associated with its binding of
SHP-1 and SHIP-1 (Fig. 5). Consistent with the previously
demonstrated role of Src family kinases in tyrosine phosphor-
ylation of ITIMs (29), the Src inhibitor PP2 blocked MPYS
tyrosine phosphorylation and recruitment of SHP-1 and
SHIP-1 (data not shown). Suggesting that inhibitory signaling
by these molecules is operative during class II signaling, MPYS
overexpression inhibited class II calcium signaling and placed
cells at a competitive disadvantage in terms of growth (Fig. 5).
Conversely, MPYS knockdown promoted growth (Fig. 6).
Taken together, these data indicate that at least two signaling
pathways emanate from MHC class II on these cells. One of
them acts through Ig-?/? and SRC family kinase activation to
mediate calcium mobilization (18) (Fig. 2 and 8). A second,
acting through MPYS tyrosine phosphorylation and recruit-
ment of SHP-1 and/or SHIP-1, mediates inhibition of calcium
signaling and cell growth.
In parallel experiments, MPYS expression was found to be
required for MHC class II transduction of signals leading to
ERK activation and apoptotic death. These responses were
found to be linked, since the ERK inhibitor PD98059 blocked
both ERK phosphorylation and the cell death response (Fig.
3). Parenthetically, the class II death response was not affected
by SB203580 or Ly294002, blockers of the Akt and p38 re-
sponses that also accompany class II signaling (Fig. 3). Sur-
prisingly, however, neither MHC class II-mediated activation
of ERK nor apoptosis was inhibited by the Src family tyrosine
kinase inhibitor PP2 (Fig. 6 and 2). These findings indicate that
a third signaling pathway emanates from MHC class II (Fig. 8).
This pathway involves tyrosine phosphorylation-independent,
but MPYS-dependent, ERK activation and leads to cell death.
It can be activated by “direct” Ab-mediated aggregation of
MPYS (7), suggesting that this signaling pathway is autono-
The role of MPYS in death responses involving mitochon-
drial membrane depolarization is particularly interesting in
view of the demonstrated localization of MPYS both on the
cell membranes and in mitochondria (Fig. 4). It is tempting to
speculate that MPYS function somehow involves shuttling be-
tween these subcellular compartments. More studies will be
required to address this question.
We thank Ryan Young and Yosef Refaeli for providing the shRNA
knockdown protocol and the pLL3.7 vector, Aimee Pugh-Bernard for
the K46 cDNA library, and Rick Willis for bone marrow-derived den-
This work is supported by a grant from the National Institute of
Allergy and Infectious Diseases, 5R01AI020519-22, to J.C.C. J.C.C. is
an Ida and Cecil Green Professor of Immunology. The CNRU Mass
Spectrometry Core is supported by NIH/NIDDK grant P30 DK048520.
We have no conflicting interests to declare.
1. Al-Daccak, R., N. Mooney, and D. Charron. 2004. MHC class II signaling in
antigen-presenting cells. Curr. Opin. Immunol. 16:108–113.
2. Bertho, N., V. M. Blancheteau, N. Setterblad, B. Laupeze, J. M. Lord, B.
Drenou, L. Amiot, D. J. Charron, R. Fauchet, and N. Mooney. 2002. MHC
class II-mediated apoptosis of mature dendritic cells proceeds by activation
of the protein kinase C-delta isoenzyme. Int. Immunol. 14:935–942.
3. Bobbitt, K. R., and L. B. Justement. 2000. Regulation of MHC class II signal
transduction by the B cell coreceptors CD19 and CD22. J. Immunol. 165:
4. Bridges, S. H., A. M. Kruisbeek, and D. L. Longo. 1987. Selective in vivo
antitumor effects of monoclonal anti-I-A antibody on B cell lymphoma.
J. Immunol. 139:4242–4249.
5. Brown, K. S., D. J. Levitt, M. Shannon, and B. K. Link. 2001. Phase II trial
of Remitogen (humanized 1D10) monoclonal antibody targeting class II in
patients with relapsed low-grade or follicular lymphoma. Clin. Lymphoma
6. Cambier, J. C., M. K. Newell, L. B. Justement, J. C. McGuire, K. L. Leach,
and Z. Z. Chen. 1987. Ia binding ligands and cAMP stimulate nuclear
translocation of PKC in B lymphocytes. Nature 327:629–632.
7. Carlo-Stella, C., M. Di Nicola, M. C. Turco, L. Cleris, C. Lavazza, P.
Longoni, M. Milanesi, M. Magni, M. Ammirante, A. Leone, Z. Nagy, W. R.
Gioffre, F. Formelli, and A. M. Gianni. 2006. The anti-human leukocyte
antigen-DR monoclonal antibody 1D09C3 activates the mitochondrial cell
death pathway and exerts a potent antitumor activity in lymphoma-bearing
nonobese diabetic/severe combined immunodeficient mice. Cancer Res. 66:
8. Carmagnat, M., B. Drenou, H. Chahal, J. M. Lord, D. Charron, J. Estaquier,
and N. A. Mooney. 2006. Dissociation of caspase-mediated events and pro-
grammed cell death induced via HLA-DR in follicular lymphoma. Oncogene
9. Chen, M., Y. H. Wang, Y. Wang, L. Huang, H. Sandoval, Y. J. Liu, and J.
Wang. 2006. Dendritic cell apoptosis in the maintenance of immune toler-
ance. Science 311:1160–1164.
10. Chen, Z. Z., K. M. Coggeshall, and J. C. Cambier. 1986. Translocation of
protein kinase C during membrane immunoglobulin-mediated transmem-
brane signaling in B lymphocytes. J. Immunol. 136:2300–2304.
11. Dal Porto, J. M., S. B. Gauld, K. T. Merrell, D. Mills, A. E. Pugh-Bernard,
and J. Cambier. 2004. B cell antigen receptor signaling 101. Mol. Immunol.
12. Drenou, B., L. Amiot, N. Setterblad, S. Taque, V. Guilloux, D. Charron, R.
Fauchet, and N. Mooney. 2005. MHC class II signaling function is regulated
during maturation of plasmacytoid dendritic cells. J. Leukoc. Biol. 77:560–
13. Drenou, B., V. Blancheteau, D. H. Burgess, R. Fauchet, D. J. Charron, and
N. A. Mooney. 1999. A caspase-independent pathway of MHC class II anti-
gen-mediated apoptosis of human B lymphocytes. J. Immunol. 163:4115–
14. Guo, W., J. G. Castaigne, N. Mooney, D. Charron, and R. Al-Daccak. 2003.
Signaling through HLA-DR induces PKC beta-dependent B cell death out-
side rafts. Eur. J. Immunol. 33:928–938.
15. Ingulli, E., A. Mondino, A. Khoruts, and M. K. Jenkins. 1997. In vivo
detection of dendritic cell antigen presentation to CD4(?) T cells. J. Exp.
16. Jin, L., J. C. Stolpa, R. M. Young, A. E. Pugh-Bernard, Y. Refaeli, and J. C.
Cambier. 2008. MHC class II structural requirements for the association
with Ig?/?, and signaling of calcium mobilization and cell death. Immunol.
17. Kim, K. J., F. I. Weinbaum, and R. Asofsky. 1978. Functional characteristics
of BALB/c T cell lines: suppression in the mixed lymphocyte response and
cell-mediated lysis. J. Immunol. 121:2299–2304.
FIG. 8. Model: MPYS and ERK mediate MHC-II signaling-in-
duced cell death.
VOL. 28, 2008MPYS: A TRANSDUCER OF MHC-II DEATH SIGNALS5025
18. Lang, P., J. C. Stolpa, B. A. Freiberg, F. Crawford, J. Kappler, A. Kupfer, Download full-text
and J. C. Cambier. 2001. TCR-induced transmembrane signaling by peptide/
MHC class II via associated Ig-alpha/beta dimers. Science 291:1537–1540.
19. Leveille, C., R. Al-Daccak, and W. Mourad. 1999. CD20 is physically and
functionally coupled to MHC class II and CD40 on human B cell lines. Eur.
J. Immunol. 29:65–74.
20. Longo, D. L. 2002. DR’s orders: human antibody kills tumors by direct
signaling. Nat. Med. 8:781–783.
21. Nagy, Z. A., B. Hubner, C. Lohning, R. Rauchenberger, S. Reiffert, E.
Thomassen-Wolf, S. Zahn, S. Leyer, E. M. Schier, A. Zahradnik, C. Brunner,
K. Lobenwein, B. Rattel, M. Stanglmaier, M. Hallek, M. Wing, S. Anderson,
M. Dunn, T. Kretzschmar, and M. Tesar. 2002. Fully human, HLA-DR-
specific monoclonal antibodies efficiently induce programmed death of ma-
lignant lymphoid cells. Nat. Med. 8:801–807.
22. Nagy, Z. A., and N. A. Mooney. 2003. A novel, alternative pathway of
apoptosis triggered through class II major histocompatibility complex mol-
ecules. J. Mol. Med. 81:757–765.
23. Newell, M. K., J. VanderWall, K. S. Beard, and J. H. Freed. 1993. Ligation
of major histocompatibility complex class II molecules mediates apoptotic
cell death in resting B lymphocytes. Proc. Natl. Acad. Sci. USA 90:10459–
24. Rathmell, J. C., S. E. Townsend, J. C. Xu, R. A. Flavell, and C. C. Goodnow.
1996. Expansion or elimination of B cells in vivo: dual roles for CD40- and
Fas (CD95)-ligands modulated by the B cell antigen receptor. Cell 87:319–
25. Setterblad, N., V. Blancheteau, A. Delaguillaumie, F. Michel, S. Becart, G.
Lombardi, O. Acuto, D. Charron, and N. Mooney. 2004. Cognate MHC-TCR
interaction leads to apoptosis of antigen-presenting cells. J. Leukoc. Biol.
26. Tamir, I., J. C. Stolpa, C. D. Helgason, K. Nakamura, P. Bruhns, M. Daeron,
and J. C. Cambier. 2000. The RasGAP-binding protein p62dok is a mediator
of inhibitory Fc?RIIB signals in B cells. Immunity 12:347–358.
27. Truman, J. P., M. L. Ericson, C. J. Choqueux-Seebold, D. J. Charron, and
N. A. Mooney. 1994. Lymphocyte programmed cell death is mediated via
HLA class II DR. Int. Immunol. 6:887–896.
28. Ventura, A., A. Meissner, C. P. Dillon, M. McManus, P. A. Sharp, L. Van
Parijs, R. Jaenisch, and T. Jacks. 2004. Cre-lox-regulated conditional RNA
interference from transgenes. Proc. Natl. Acad. Sci. USA 101:10380–10385.
29. Vivier, E., and M. Daeron. 1997. Immunoreceptor tyrosine-based inhibition
motifs. Immunol. Today 18:286–291.
30. Wang, W., and B. A. Malcolm. 1999. Two-stage PCR protocol allowing
introduction of multiple mutations, deletions and insertions using
QuikChange site-directed mutagenesis. BioTechniques 26:680–682.
31. Wille, L., M. L. Kemp, P. Sandy, C. L. Lewis, and D. A. Lauffenburger. 2007.
Epi-allelic Erk1 and Erk2 knockdown series for quantitative analysis of T cell
Erk regulation and IL-2 production. Mol. Immunol. 44:3085–3091.
5026 JIN ET AL.MOL. CELL. BIOL.