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*
Microbiology and Tumorbiology Center, Karolinska Institutet, Stockholm, Sweden;
Cattedra di Immunologia, Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università di Napoli Federico II, Naples, Italy;
Lymphocyte Biology Section, Division of Rheumatology, Immunology, and Allergy, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115; and
§
Istituto Nazionale per la Ricerca sul Cancro and Dipartimento di Medicina Sperimentale, Università di Genova, Genoa, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because CD94/NKG2A NK cells have been reported to recognize the
nonpolymorphic HLA-E MHC class Ib molecule (or the homologous Qa-1
molecule in mice) (6, 7), these observations led us to ask
whether CD1 molecules can also be recognized by NK cells. CD1 molecules
are nonpolymorphic MHC class I-like glycoproteins that are encoded by a
family of genes outside of the MHC locus. The expression of CD1
proteins does not require TAP-1/2 transporters, although it is
dependent on ß2 microglobulin (8).
The genes and amino acid sequences of CD1 and MHC class I
and class II show a limited but significant level of homology, which
approaches 
30% identity in the 
2 and 3 domains
(8). CD1 genes are evolutionarily old and are present in
rodents and human, as well as in all other mammalian species so far
examined (8). In contrast to HLA-A, -B, -C, and -E Ags,
which are broadly expressed in almost all tissues, CD1 proteins have a
more restricted and cell type-specific expression. All CD1 proteins are
strongly expressed by thymocytes. In extrathymic tissues, CD1a, -b, and
-c are expressed primarily on specialized APC (8).
CD1b and CD1c in humans and CD1d1 in mice have the property to present
lipid Ags (8, 9). Human T cells belonging to either
CD4-CD8- or
CD8+ subsets can recognize
Mycobacterium tuberculosis (M.tb) lipids
presented by CD1b (9, 10, 11, 12). Although not as extensively
studied as for CD1b, it appears that human CD1a and CD1c molecules can
also present mycobacterial lipid Ags to specific T cells (12, 13). In addition, CD1d proteins in both mouse and human have
recently been demonstrated to present synthetic -glycosyl ceramides
to a subset of T cells that bear highly conserved TCRs and coexpress
several NK-associated lectin-type receptor molecules (14, 15)
The purpose of this study was to test the role of CD1 proteins as potential ligands for NK cells and to evaluate the effect of CD1-presented bacterial lipid Ags on NK cytotoxicity. In the current study, we have directly examined the effects of CD1 expression on the lysis of two different NK-sensitive target cells (T2 and HeLa). Our results demonstrated a clear effect of each of the human CD1 proteins on the inhibition of target cell lysis by NK cells. Moreover, the preincubation of CD1b+ target cells with defined bacterial lipid Ags that are known to bind to CD1b led to a significant increase in the inhibitory effect on NK lysis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T-B lymphoblastoid hybrid wild-type cell line T1 and the
TAP-1/2-defective mutant T2 were generated as described
(16). The stably MHC class I-reconstituted T3 cell line
was produced by transfection of human TAP-1/2 cDNA constructs into T2
cells, as described (17). HeLa cells were obtained from
American Type Culture Collection (Manassas, VA). T2 and HeLa
transfected with plasmid constructs of the expression vector pSR-Neo
encoding either CD1a, CD1b, or CD1c were generated and maintained as
described elsewhere (13, 15). Dendritic cell (DC) lines
were generated by treatment of adherent mononuclear cells with GM-CSF
(50 ng/ml) and IL-4 (1000 U/ml) for 7 days as described
(18). M.tb lipid Ags (whole extract, mycolic acids, and
glucose monomycolate) were prepared and reconstituted for cell culture
as described elsewhere (9, 11, 12). Pretreatments of the
T2 and T2-CD1b were performed by adding 10 µg of the M.tb-derived
lipid Ags or commercially obtained mycolic acid (Sigma, St. Louis, MO)
to 106 cells in a final volume of 500 µl of
culture medium and incubating such preparation overnight at 37°C.
To obtain fresh NK cells, PBMC were isolated by centrifugation on Lymphoprep (Nycomed Pharma, Oslo, Norway) gradients from healthy donor buffy coats obtained from the Blood Bank of the Medical School of the Federico II University of Naples. Adherent cells were removed by incubation for 2 h at 37°C in tissue culture flasks, and fresh PBL were stored overnight at room temperature before use as effectors in cytotoxicity assays. IL-2-activated NK cells were generated from PBL cultured in medium supplemented with IL-2 (Cetus, Milan, Italy) (1000 IU/ml) for 48 h before use in cytotoxicity assays. For generation of NK cell lines, freshly isolated nonadherent lymphocytes were washed and resuspended in IMDM medium (BioWhittaker, Berlin, Germany) at a final concentration of 2.5 x 105/ml and cultured together with irradiated T2 cells (0.5 x 105/ml) for 5–7 days of culture. All effectors were depleted of CD3-positive cells by immunomagnetic separation using magnetic beads (Dynal, Oslo, Norway) coated with anti-CD3 mAb. The depletion procedure was repeated twice, and 98% of the remaining cells were CD56+CD3- as assessed by FACS analysis. NK clones were generated as previously reported (4). The stable NK effector line NKL was a gift of Dr. M. J. Robertson (Bone Marrow Transplantation Program, Indiana University, Indianapolis, IN). Generation of NK clones and measurement of NK cytotoxicity by 51Cr release assay were performed according to previously published methods (4).
mAbs, F(ab')2 production, and flow cytometry
Anti-CD1 mAbs used in this study included OKT6 (IgG1, anti-CD1a; obtained from American Type Culture Collection), BCD1b3.1 (IgG1, anti-CD1b; Ref. 13), F10/21A3 (IgG1, anti-CD1c; S. Porcelli, unpublished observations), and 7C6 (IgM, anti-CD1c; Ref. 9). All IgG mAbs were purified by protein G column affinity chromatography from ascites fluid (OKT6) or culture supernatants (BCD1b3.1 and F10/21A3). IgM mAbs (7C6) were used as ascites fluid dialyzed extensively against RPMI 1640. F(ab')2 were prepared from IgG1 anti-CD1 mAbs by enzymatic digestion of purified Ig using immobilized ficin columns (Pierce, Rockford, IL) and purified by passage over a protein A Sepharose column followed by dialysis against PBS. T2 CD1 transfectant cell lines were incubated with 4 µg/ml of specific anti-CD1 F(ab')2 for 30 min at room temperature, washed twice, and used in the cytotoxicity assay. Phenotypic characterization of monocyte-derived DCs was performed using FITC- and PE-labeled mAbs against CD40, CD80, CD86 (PharMingen, San Diego, CA), CD83 (Immunotech, Marseille, France), HLA class I (Sigma), CD3, CD14, HLA-DR, and isotype-matched control mAbs (Becton Dickinson, Mountain View, CA). Immunofluorescence staining, flow cytometry, and data analysis were performed as described (4).
Cytotoxicity assay
Cytotoxicity was measured using a conventional 4-h 51Cr release assay. Target cells were labeled with Na251CrO4 (100 mCi/2 x 106 cells), and the percent of specific lysis was calculated as 100 x [(cpm in experimental wells) - (cpm in wells with target cells alone)]/(total cpm incorporated into target cells). The spontaneous 51Cr release from target cells cultured alone was consistently <20% of the total cpm. In mAb blocking experiments, 5 µg/ml of the indicated anti-KIR Abs were added at the beginning of the cytotoxicity test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because previous studies have shown that CD1 proteins are not
dependent on TAP-1/2 for their assembly and can be expressed
efficiently in T2 cells, we used this cell line as a recipient in which
to test the effects of expression of human CD1 proteins on NK cell
lysis. CD1 transfectants were then tested in cytotoxicity experiments
for sensitivity to lysis by various NK cell effector populations. These
included freshly isolated CD3-depleted PBL (Fig. 1
, A–C), the NKL cell line
(Fig. 1D), a freshly established polyclonal NK cell line
(Fig. 1E), and short-term IL-2-activated NK cells (Fig. 1, F and G). A clear, albeit not complete, reduction
of NK susceptibility was found for transfected T2 cell targets
expressing each of the different CD1 proteins. This was observed with
all of the different NK cell effector populations tested. Note that in
some donors no protection or even a slight increase in NK cytotoxicity
was observed with some of the CD1 molecule isoforms. However, NK cells
from all donors were inhibited by at least one of the CD1
molecules.
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H shows a
representative experiment with polyclonal NK cells generated from a
donor recognizing all three CD1 isoforms. Pretreatment of
CD1-transfected HeLa cells with the specific anti CD1
F(ab')2 reduced the protection substantially
(Table I). These data confirmed that the
protection from NK lysis observed for cells transfected with CD1 was
specifically due to the expression of CD1 glycoproteins.
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G), consistent with previously reported results
(19).
This may be due to the expression of other dominant inhibitory ligands
by C1R or possibly to the lower CD1 surface expression on C1R
transfectants (see Discussion). Therefore, the expression of
MHC class I and CD1 molecules were measured on the T2, HeLa, and C1R
cell lines and their related CD1 transfectants (Fig. 2). The MHC class I expression was not
affected by CD1 transfection in all transfectants cell systems studied;
on C1R cells, an appreciable level of MHC class I molecules was
detected (Fig. 2A). In contrast, the highest CD1 levels were
detected on T2 and HeLa, while C1R CD1 expression was approximately
half of that observed for T2 and HeLa transfectants (Fig. 2B). A bright staining for CD1 group 1 molecules was
observed as well on DC (Fig. 2C).
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The NK cell killing against each of the CD1 transfectants was
significantly augmented by pretreatment of the target cells with
F(ab')2 specific for the form of CD1 expressed by
the target (Fig. 3). This indicated that
the expression of the intact, native form of each of the different CD1
proteins on the surface of the target cells was responsible for the
observed inhibition of NK cell-mediated lysis of the T2 transfectants.
Similar results were seen with anti-CD1c IgM (mAb 7C6) pretreatment
of the CD1c transfectants (data not shown).
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A),
whereas F(ab')2 against CD1c alone (Fig. 4, A–C) or CD1b or CD1a alone (data not shown) had no effect.
The pretreatment with anti-MHC class I Ab also increased the DC
recognition by polyclonal NK cells. The combination of anti-MHC
class I and single anti-CD1 Abs induced a strong synergistic effect
on NK recognition (Fig. 4, B and C). This
synergistic effect between MHC class I and CD1 blockade was donor
dependent (Fig. 4, B and C). This result was
consistent with the notion that each individual form of CD1 as well as
MHC class I has some capacity to independently inhibit the NK cell
effector population.
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Because differences between donors with respect to CD1 recognition
were noticed (Figs. 1 and 4), we assessed the variation in CD1
recognition in a panel of nine established NK clones from four
different donors. This revealed a complex and heterogeneous recognition
pattern (Fig. 5). For the majority (seven
of nine) of clones, target cell lysis appeared to be inhibited by at
least one CD1 protein. The exceptions were NK clones 28 and 48, which
were not inhibited by expression of any of the CD1 proteins studied. NK
clones 1 and 3 appeared to be inhibited by all three CD1 Ags, although
with varying degrees of efficiency. Notably, the pattern varied between
clones, although the clones derived from an individual donor appeared
to show a trend toward inhibition by the same CD1 protein (e.g., clones
from donor A were inhibited most strongly by CD1b, whereas three of
five clones from donor D were inhibited by CD1a).
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CD1b was shown in previous studies to present M.tb-derived lipid
and glycolipid Ags to T cells (9, 10, 11, 12). Upon this
recognition, T cells were triggered to kill infected presenting cells
and to produce IFN-
. Here, we investigated how NK cells react to the
CD1b presentation of a M.tb whole lipid extract or two of its purified
constituents: mycolic acid and glucose monomycolate. T2 CD1b and T2
mock transfectants were incubated overnight at 37°C with M.tb-derived
lipid Ags at a final concentration of 10 µg/ml. Fig. 6, A and B shows
one representative experiment on the NK recognition of T2 CD1b and
T2-mock cell lines after preincubation with M.tb lipid Ags. The
preincubation of T2-CD1b cells with whole extract and the purified
glucose monomycolate led to a moderately increased inhibitory effect,
while purified mycolic acid caused a marked increase in the inhibition
of NK killing. No significant effects were found when the T2-mock cells
were exposed to the same lipid preparations (Fig. 6B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B). Partial
inhibitory effects mediated by these molecules may mask the effects of
CD1 proteins. Another explanation may relate to the possible HLA-E
expression by C1R. HLA-E expression is up-regulated by HLA-Cw04 leader
sequence-derived peptides (6) and could thus mediate an
inhibition that masks the effect of coexpression of CD1 proteins by
these cells. In contrast to the negative results with C1R cell transfectants, inhibitory effects of CD1a, -b, or -c expression on NK cell-mediated lysis were readily observed in CD1-transfected T2 and HeLa cells. T2 have a deletion of the entire class II region including the TAP-1 and -2 peptide transporter genes. Paradoxically, presentation of signal peptides by HLA-E is TAP dependent, and therefore T2 is not expected to express this CD94/NKG2A ligand (20). HeLa cells were also protected by CD1 transfection, and these express low levels of MHC class I comparable with T2 and remarkably high levels of CD1 on their cell surface.
The specificity of the inhibition of NK cells by CD1 proteins was confirmed using F(ab')2 of mAbs specific for each CD1 protein to reverse the observed inhibition. Because the anti-CD1 mAbs used are known to be reactive with the mature cell-surface form of the CD1 proteins that they recognize, these results indicate that the intact CD1/ß2-microglobulin complex must be responsible for the observed inhibition of NK cell-mediated killing of T2 and HeLa cell transfectants and of DCs. Thus, it is unlikely that the inhibitory effect of CD1 expression can be accounted for by indirect mechanisms, such as the presentation of CD1 leader peptides by HLA-E and subsequent interaction with CD94/NKG2A. Instead, we propose that one or more specific receptor molecules on NK cells must interact directly with cell-surface CD1 proteins to generate the putative inhibitory signals.
Using a variety of different NK effector populations, we found that the
expression of a single form of CD1 on transfected T2 cells provided
significant but incomplete protection from NK cell-mediated killing. In
the various experiments performed, inhibition of NK cell lysis of T2
cells by CD1 expression ranged between a 30% to 70% reduction of the
level achieved with control target cells. In one set of experiments
(see Fig. 1D), the levels of protection achieved on T2 cells
transfected with TAP-1/2 to reconstitute normal MHC class I levels was
compared with that achieved for cells transfected with the different
forms of CD1. Notably, the restoration of MHC class I expression on the
target cell membrane almost completely abolished NK killing at all E:T
ratios used, whereas the expression of any of the CD1 proteins was able
to inhibit the NK killing only partially. The lower potency of CD1
expression compared with MHC class I reconstitution was especially
apparent at high E:T cell ratios, at which the protective effects of
CD1 but not MHC class I expression were largely overcome (Fig. 1D). However, at low E:T cell ratios, the same level of
inhibition was found for either MHC class I-reconstituted or
CD1-expressing T2 cells. The lower expression of CD1 on T2
transfectants compared with the levels of MHC class I on the
TAP-1/2-reconstituted cells (data not shown), or the fact that TAP-1/2
reconstitution generates multiple inhibitory MHC class I targets on the
cell surface, may account for the apparent difference in the potency of
inhibition we observed.
DC appear to represent a major site of expression of most CD1 proteins in humans, and therefore it is intriguing to note that treatment of monocyte-derived DCs with a mixture of anti-CD1a, -b, and -c mAb increased the sensitivity of these cells to NK lysis in vitro. Abs against a single form of CD1 did not reverse the protection, except when used in combination with anti-MHC class I mAbs, indicating that to overcome the inhibitory effect it is necessary to block NK recognition of most or all expressed CD1 proteins simultaneously or block at the same time MHC class I and CD1.
The observed synergistic effect of the anti-MHC class I and single anti-CD1 F(ab')2 on reversal of protection from NK cells can be interpreted in at least two different ways: 1) one NK receptor may interact with both MHC nonclassical class I and CD1, or 2) two distinct inhibitory receptors recognizing MHC class I and CD1 molecules are involved in NK-DC interactions. This issue is under active investigation in our laboratory. The full DC protection by NK recognition is achieved only when a full inhibitory signal is delivered from the NK receptor(s) for MHC class I and CD1 molecules. Thus the protective effect of CD1 proteins on DCs is apparent despite the presumably normal expression of classical MHC class I and HLA-E molecules on these cells. Although the reason for the failure of these latter proteins to completely protect DCs from NK lysis is not entirely clear, we speculate that this may relate to the extremely prominent expression of costimulatory molecules present on these cells (21). CD1 could also have an important role in virus-infected DC in which the normal TAP-dependent MHC class I pathway is perturbed and fails to protect DC until they can prime T cells. Our results also indicate that the presentation of bacterial lipid Ags by CD1b may be able to significantly augment the protection against NK cell lysis conferred by this CD1 protein. The mechanism for the effect of lipid Ags is currently under investigation. We speculate that this enhanced protection from NK lysis may be important in the setting of bacterial infection. In such circumstances, the NK cytolytic response may be damped by lipid Ag presentation in the context of CD1, while specific double negative and CD8+ T cells may be efficiently activated by such presentation.
While the present study was being conducted, it was reported (22) that cytolysis by mouse lymphokine-activated killer cells is inhibited by target cell expression of the murine CD1 molecule, mCD1d1. The closest human homologue of mCD1d1 is the human CD1d protein, which was not directly assessed for NK cell inhibitory activity in our study. Nevertheless, the inhibitory effect of mCDd1 on mouse lymphokine-activated killer cells adds further support to our hypothesis that CD1 molecules are ligands of specific inhibitory receptors expressed by NK cells.
Based on the initial results reported here, we envision an important role for negative signaling by CD1 recognition in infections, particularly in the setting of global MHC class I down-regulation by microorganisms.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Ennio Carbone at his current address: Microbiology and Tumorbiology Center (MTC)/Karolinska Institute, Box 280, S-171 77 Stockholm, Sweden.
3 Abbreviations used in this paper: KIR, killer cell-inhibitory receptor; M.tb, Mycobacterium tuberculosis; DC, dendritic cell.
Received for publication October 5, 1999. Accepted for publication March 28, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß T cell pool. J. Immunol. 162:366.. J. Exp. Med. 179:1109.This article has been cited by other articles:
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), T2-CD1a (•),
T2-CD1b (
), T2-CD1c (
), and T3 (
); (G) C1R-mock
transfectant (
), or pretreated with specific
anti-CD1 F(ab') 2 reacting with CD1 forms expressed by
transfectants (•). CD1a (B), CD1b (C),
and CD1c (D) targets are indicated.
); with a mixture of
anti-MHC class I and anti-CD1c fragments (
), and T2-CD1c (
) targets
as indicated. The E:T ratio was 2:1.