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Infect Immun, August 1998, p. 3523-3526, Vol. 66, No. 8
Molecular Biology
Institute,1
Department of Pathology and
Laboratory Medicine,2 and
Departments of Medicine and Microbiology and
Immunology,3 UCLA School of Medicine, Los
Angeles, California 90095
Received 10 October 1997/Returned for modification 16 January
1998/Accepted 7 May 1998
Human CD1 is a family of nonpolymorphic major histocompatibility
complex class I-like molecules capable of presenting mycobacterial lipids, including lipoarabinomannan (LAM), to double-negative (DN;
CD4 Human CD1 is a family of
nonpolymorphic major histocompatibility complex (MHC) class I-like
molecules (CD1a to CD1d) (4, 7, 15, 18). Although CD1 is
encoded outside the MHC, its association with Antigens.
Hib strain 10211 (ATCC, Rockville, Md.) was grown
on pyridoxal-supplemented chocolate agar (Becton Dickinson
Microbiological Systems, Cockeysville, Md.) overnight at 37°C in 5%
CO2. Bacteria were scraped from 10 agar plates, pelleted at
1,000 × g, and probe sonicated in 4 ml
phosphate-buffered saline at 50% power continuously for 5 min on ice.
Sonicates were ultracentrifuged at 100,000 × g for
1 h at 4°C, and the supernatants were filter (0.2-µm pore size) sterilized. Protein concentration, as determined by Micro BCA
Protein Assay (Pierce, Rockford, Ill.) was used to standardize the Ag
dose. To prepare nonpeptide Ag, Hib sonicate was treated with
proteinase K (0.7 mg/ml; Boehringer Mannheim, Indianapolis, Ind.) for
30 min at 60°C and then heat inactivated for 10 min at 70°C.
MAbs.
Anti-CD4 and anti-CD8 monoclonal antibodies (MAbs)
used for immunodepletion were purchased from Immunotech (Westbrook,
Maine) and Becton Dickinson (San Jose, Calif.), respectively. Anti-CD1 antibodies were as follows: P3 (murine immunoglobulin G1 [IgG1]) (16), OKT6 (anti-CD1a) (19), BCD1b3.1 (anti-CD1b;
provided by S. A. Porcelli), 10C3 (anti-CD1c) (15), and
L161 (anti-CD1c; Immunotech). F(ab')2 goat anti-mouse
IgG-phycoerythrin (PE) was obtained from Caltag (San Francisco,
Calif.). All other antibodies used in flow cytometry were purchased
from Becton Dickinson.
Preparation of APC.
Peripheral blood mononuclear cells
(PBMC) from a healthy donor were isolated by Ficoll-Paque (Pharmacia,
Uppsala, Sweden) density centrifugation and incubated for 72 h at
106/ml in complete medium (CM; RPMI 1640, 100 mM sodium
pyruvate, 200 mM L-glutamate, 5 U of
streptomycin-penicillin per ml) supplemented with 10% fetal calf serum
(FCS), 200 U of granulocyte-macrophage colony-stimulating factor
(GM-CSF; Genetics Institute, Cambridge, Mass.) per ml, and 100 U of
interleukin-4 (IL-4; Schering Corp., Bloomfield, N.J.) per ml. Adherent
cells were removed with 5 mM EDTA-phosphate-buffered saline (pH 7.3)
for 10 min at 37°C, irradiated with 5,000 rads, and stored in liquid
nitrogen until used. CD1a, CD1b, and CD1c expression was confirmed by
flow cytometry.
Flow cytometry.
Cytokine-activated PBMC containing 20%
monocytes (105/sample) were resuspended in Hanks balanced
salt solution-1% bovine serum albumin (FACS [fluorescence-activated
cell sorting] buffer); 100-µl cell suspensions were stained with 0.3 µg of unlabeled monoclonal antibodies to CD1a, CD1b, and CD1c per ml
for 45 min on ice. P3 was used as an IgG1 isotype control. Cells were
washed with FACS buffer and then stained with 2 µl of PE-conjugated
F(ab')2 goat anti-mouse IgG. 7-Amino actinomycin D
(Calbiochem) was used as a dead cell discriminator; 5,000 to 10,000 live events per sample were analyzed with a FACStar Plus flow cytometer
and Lysis II software (Becton Dickinson Immunocytometry Systems,
Mountain View, Calif.). T cells were analyzed similarly except that
they were incubated with fluorescein isothiocyanate- or PE-conjugated
antibodies for 30 min.
Isolation of T-cell populations.
To prepare autologous
CD1+ APC, PBMC were resuspended to 2 × 106/ml in CM supplemented with 20% FCS, GM-CSF (400 U/ml),
and 200 IL-4 (U/ml). Cell suspensions were plated at 100 µl/well in a 96-well round-bottom plate for 24 h and then irradiated with 5,000 rads. Hib Ag was then added at 1 µg/ml (final protein concentration), and double-negative (DN; CD4 Proliferation assays.
T cells were plated in triplicate at a
concentration of 104/well in the presence of
105 heterologous, irradiated (5,000 rads) PBMC containing
20% CD1+ monocytes per well. Assays were performed in
96-well flat-bottom microtiter plates in 200 µl of CM-2% pooled
human serum-8% FCS. Hib Ag was added to a final concentration of 10 µg/ml. In blocking experiments, cells were cultured in the presence
of anti-CD1 MAbs at a final concentration of 20 µg/ml or 1:400
ascites fluid. Wells were pulsed with 0.5 µCi of
[3H]thymidine during the last 4 h of a 72-h
incubation period. Cells were harvested onto glass fiber filters, and
tritiated thymidine incorporation was measured by scintillation
counting (Beckman Instruments, Fullerton, Calif.).
Autologous APC were prepared by treating blood monocytes with
GM-CSF and IL-4 to induce expression of CD1. Substantial surface expression of all three CD1 gene products was detected in these induced
monocytes. By flow cytometry, the frequencies of CD1+
monocytes were 8.5, 6.3, and 10.6% (anti-CD1a, anti-CD1b, and anti-CD1c, respectively; 0.6% for isotype control staining). The mean
fluorescence intensity units were 272, 95, and 83, respectively (11 for
isotype control staining). Thus, comparable expression levels of the
different CD1 isoforms is achieved by this induction method.
Healthy donor lymphocytes were depleted of CD4+ and
CD8+ T cells in order to prepare a population enriched for
CD1-restricted T cells. These DN T cells were cocultured with Hib
Ag-pulsed CD1+ APC and then screened for both Hib Ag
reactivity and CD1 restriction by a standard proliferation assay in the
presence of blocking antibodies specific for CD1a, CD1b, or CD1c.
Forty-six T-cell cultures were obtained from four different donors and
analyzed. Most of these were unstable in passage and therefore
incompletely characterized. Fifteen T-cell lines had demonstrable
anti-Hib nonpeptide activity, distributed among all four donors. This
observation supports the interpretation that T cells reactive with Hib
nonpeptides are a common trait in independent human donors.
Two T-cell populations, Hib16 and Hib19, proliferated well to Hib Ag
and were selected for more detailed study (Fig.
1). Ag-specific proliferation of Hib16
was blocked 98% by anti-CD1c MAb (Fig. 1A), whereas that of Hib19 was
blocked 88% by anti-CD1b MAb (Fig. 2B). In addition, four Hib19
subclones proliferated to Hib Ag in a CD1b-restricted manner (Fig. 1C).
CD1+ APC derived from several unrelated donors also
supported Ag-specific and CD1b-restricted proliferation of Hib19,
indicating further that Ag recognition was not MHC restricted (Fig.
2). Although the human CD1 molecule is
strictly nonpolymorphic, we found that CD1+ APC differed in
the ability to support both Ag-specific and background proliferation of
Hib19. This ability was unrelated to their level of expression of CD1b
(Fig. 2), which suggested either that the CD1-T-cell receptor (TCR)
interaction was modulated by a polymorphic molecule or that the levels
of putative costimulatory molecules differed among the APC tested
(5).
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
CD1 Presents Antigens from a Gram-Negative
Bacterium, Haemophilus influenzae Type b
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ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
CD8
) as well as CD8+ T
cells. Structural similarities between LAM and the capsular polysaccharides of gram-negative bacteria led us to consider the latter
as candidate CD1 ligands. We derived two CD1-restricted DN T-cell
populations which proliferated to Haemophilus influenzae type b (Hib) antigen. One T-cell population also proliferated to
proteinase K-treated Hib antigen, suggesting that it recognized a
nonpeptide. Our work thus expands the universe of T cell antigens to
include nonpeptides distinct from mycobacterial lipids and suggests a
potential role for CD1-restricted T cells in immunity to Hib.
![]()
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
2-microglobulin
relates it structurally to MHC class I. CD1 molecules are expressed on
immature thymocytes (19) and antigen-presenting cells (APC)
including cytokine-activated macrophages (13), B cells
(22, 23), and dermal dendritic cells (9). Recent
studies have revealed that CD1 possesses the unique function of
presenting nonpeptide antigen (Ag) to T cells (3, 17, 21,
24). A prototypic Ag presented in the context of CD1 is
lipoarabinomannan (LAM), a mannose polymer substituted at one end with
arabinose and at the other with a phosphatidic acid containing
tubulostearic and palmitic acids. De-O-acylation of LAM totally
abrogated T-cell responsiveness, suggesting that the lipid moiety was
required for Ag recognition (21). Since gram-negative
bacteria contain lipoglycans structurally analogous to LAM (2, 11,
14, 20), we sought to isolate CD1-restricted T cells which
recognize antigens from Haemophilus influenzae type b (Hib),
a representative gram-negative bacterium.
![]()
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
CD8
) T cells
(see below) were added at 104 to 105 per well
to give a final culture volume of 200 µl. After 72 h of culture,
autologous APC and Ag were replenished. Heterologous APC and fresh Ag
were again added after 72 h and every 2 weeks thereafter. Between
Ag stimulations, IL-2 (10 U/ml; Schiaparelli, Columbia, Md.) was added,
and cells were split every 3 to 4 days to maintain near confluency.
CD4+ T-cell populations derived by coculturing nondepleted
peripheral blood leukocytes with irradiated autologous PBMC in the
presence of Hib Ag. T-cell subclones were isolated at limiting
dilution, using 105 heterologous CD1+ APC per
well.
![]()
RESULTS AND DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References

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FIG. 1.
Hib Ag-specific, CD1-restricted proliferation of Hib16
(A) and Hib19 (B) T-cell populations and of Hib19 T-cell clones (C). T
cells were cocultured with irradiated CD1+ APC and Hib Ag
in the presence of blocking antibodies specific for CD1a, CD1b, or
CD1c. During the last 4 h of a 72-h incubation, cultures were
pulsed with tritiated thymidine, the cellular incorporation of which
was determined by scintillation counting and expressed as counts per
minute.

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FIG. 2.
Hib Ag-specific, CD1b-restricted proliferation of Hib19
T cells. Hib19 T cells were cocultured with irradiated CD1+
APC (isolated from seven unrelated donors [D1 to D4 and D7 to D9])
and Hib Ag in the presence of CD1b blocking antibodies. Proliferation
was measured as for Fig. 1. The fold stimulation and mean fluorescence
intensity of CD1 expression are indicated for each donor.
The phenotypes of Hib16 and Hib19 T-cell populations and subclones were
determined by two-color flow cytometry. Both T-cell populations lacked
expression of CD4. Although both T-cell populations expressed variable
levels of CD8dim, they contained a large fraction which
were DN. CD56 was expressed on 90 and 31% of Hib16 and Hib19 T cells,
respectively. The expression of CD56, a natural killer (NK) cell
marker, on both T-cell populations parallels the expression of NK1.1 on
a significant fraction of mouse CD1-restricted T cells (6).
Although the role of CD56 in CD1-restricted T-cell responses has not
been addressed, the CD4
CD8
/dim
CD56+ phenotype of Hib16 and Hib19 nevertheless indicated
they were not conventional T-helper cells. Finally, Hib16 expressed
predominantly (94%) TCR
. Whereas only 68% Hib19 expressed
TCR
(the remainder expressed TCR
), four Hib19 subclones
expressed exclusively TCR
. This finding argues that the Ag
reactivity and CD1 restriction of Hib19 (Fig. 1B and C) required
expression of the TCR
heterodimer. Thus, the phenotypes of both
Hib16 and Hib19 mirrored those of other mycobacterium-specific,
CD1-restricted T-cell populations (3, 17, 21, 24).
To determine whether the Ag to which Hib16 and Hib19 reacted was a nonpeptide, proteinase K-treated Hib Ag was tested in a proliferation assay. Proteinase K-treated Hib Ag was shown to be devoid of protein Ag by two criteria. First, Hib Ag proteins resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized with Coomassie blue stain disappeared after treatment with proteinase K (data not shown). Second, to ensure that small immunogenic peptides did not persist, proteinase K-treated Hib Ag was tested for the ability to stimulate several CD4+, MHC class II-restricted, Hib Ag-specific T-cell populations. All of nine CD4+ T-cell populations which proliferated specifically to Hib Ag failed to proliferate to proteinase K-treated Hib Ag (data for four representative populations are shown in Fig. 3). In contrast, proteinase K treatment did not affect the proliferative response of Hib19 to Hib Ag (Fig. 3), indicating that the Ag was a nonpeptide.
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Since Hib expresses neither LAM nor mycolic acids, we have speculated that either of two lipoglycan Ags, lipo-oligosaccharide (12) and the capsular polysaccharide (capPS) (1, 8) may be presented by CD1. LAM is structurally related to the capPS of several gram-negative bacteria, including Hib (2, 14), groups A to C of Neisseria meningitidis (2), and several K serotypes of Escherichia coli (11, 20). What is not generally appreciated about capPS is that they are naturally lipidated, a modification responsible for anchoring these highly charged molecules into bacterial outer membranes (2). Like LAM, capPS are substituted with a phosphatidic acid containing palmitic acid. This structural similarity between LAM and capPS led us to propose that palmitic acid anchors into the hydrophobic groove of CD1 diverse covalently attached polysaccharides for direct recognition by T cells (10). Accordingly, we hypothesized that CD1-restricted T cells play an important role in host immunity to encapsulated bacteria by virtue of their ability to recognize capPS. Although only mycobacterium-reactive CD1-restricted T cells have been identified to date, the demonstration here that CD1-restricted T cells can recognize Hib Ag suggests a role for CD1 Ag presentation in the immune response to gram-negative bacteria. Future studies designed to identify the Hib Ag and the immunological roles of these CD1-restricted T cells should provide novel insights into antibacterial immunity.
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ACKNOWLEDGMENTS |
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This work was supported by NIH grants AI38545 and CA 12800, the UCLA Medical Scientist Training Program, the Jonsson Comprehensive Cancer Center, and The Gustavus and Louise Pfeiffer Research Foundation.
We thank S. Stenger for critical reading of the manuscript.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Pathology and Laboratory Medicine, UCLA School of Medicine, CHS 13-222, 10833 Le Conte Ave., Los Angeles, CA 90095-1732. Phone: (310) 825-0650. Fax: (310) 206-0657. E-mail: jbraun{at}pathology.medsch.ucla.edu.
Editor: J. R. McGhee
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