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*
The Children’s Medical Research Institute,
Gene Therapy Unit, and
Oncology Research Unit, The New Children’s Hospital, Westmead, Australia
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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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CD80 and CD86 are type I integral membrane glycoproteins and members of the Ig supergene family found on the surface of activated T lymphocytes, B lymphocytes, and APC. During the process of lymphocyte activation by costimulation, CD80 and CD86 function as low affinity ligands, or counterreceptors for CD28 and high affinity counterreceptors for CTLA-4. Functionally, CD86 appears to be important in the initiation of a cell-mediated immune response, whereas CD80 is believed to be involved in the maintenance and/or moderation of the immune response (2, 3, 6, 7, 8, 9, 10, 11, 12).
The strategy we are pursuing is aimed at rendering the tumor cells capable of simultaneously providing an Ag-specific signal to an appropriate T lymphocyte, and a second costimulatory signal through the CD28 receptor molecule. The ultimate aim is to activate tumor-specific T lymphocytes without the need for APC, and the results of several in vivo studies with murine tumor models have already validated this hypothesis under certain conditions (5, 13, 14, 15). In developing this strategy, we initially focussed on evaluating retrovirus-mediated gene transfer (transduction) of CD80 and CD86 to human lymphoid (CEM), and nonlymphoid (HeLa) tumor cell lines. After genetically modifying the tumor cells, we observed an unexpected intracellular focal concentration of CD80 and CD86 in transduced CEM cells. Subsequent studies of endogenous CD80 and CD86 gene expression in PBMC revealed a strikingly similar intracellular focal concentration of CD86 protein in monocytes. This intracellular focus was not due to concentration of CD86 in the Golgi, and required intact microtubules to retain structural integrity. This report details the intracellular and membrane localization studies, and describes the temporal regulation of intracellular storage and trafficking of CD86 to the cell surface membrane of monocytes. We discuss the biologic implications of these findings, and hypothesize that this intracellular concentration of CD86 in monocytes represents a functional reservoir of protein. This reservoir may be important in enabling the monocyte to respond to stimulation by rapid and sustained deployment of CD86 to the cell surface membrane.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Psi-2 (16), PA317 (American Type Culture Collection (ATCC), Rockville, MD; CRL 9078), and HeLa (ATCC CCL-2) cells were maintained in DMEM, and CEM (ATCC CCL 119 CCRF-CEM) cells were maintained in RPMI supplemented with 10% heat-inactivated bovine calf serum (Starrate, Bethungra, Australia), penicillin (50 IU/ml), streptomycin (50 µg/ml), and 2 mM glutamine, and cultured at 37°C in a 5% CO2 humidified incubator. Human CD80 (B7-1) cDNA (GenBank accession no. M27533) and CD86 (B7-2) cDNA (GenBank accession no. L25259) were kindly provided by Dr. Gordon Freeman (Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA) in pCDM8 (2, 8). The CD80 and CD86 cDNA inserts were isolated after XhoI digestion and subcloned into the XhoI site of the retrovirus vector plasmid pLXSN to generate plasmids pL80SN and pL86SN, respectively. The integrity and orientation of the cDNA inserts was confirmed by dideoxy nucleotide sequencing.
Psi-2 and PA317 cells were selected in HAT (0.01 mM hypoxanthine, 0.004 mM aminopterin, 0.016 mM thymidine) and HT medium (0.01 mM hypoxanthine, 0.016 mM thymidine) before use for retrovirus packaging. Psi-2 cells were transfected with 3 µg of pLXSN, pL80SN, or pL86SN DNA using 10 µl of LipofectAMINE (Life Technologies, Grand Island, NY) according to the manufacturer’s instructions. After selection in DMEM supplemented with G418 (600 µg/ml active compound; Life Technologies) ecotropic retrovirus corresponding to LXSN, L80SN, or L86SN was harvested, filtered through a 0.45-µm pore size filter, and used to transduce PA317 cells. LXSN-, L80SN-, and L86SN-transduced PA317 cells were then G418 selected (600 µg/ml active compound), and amphotropic retrovirus producer clones obtained by limiting dilution. Amphotropic supernatants were assayed for viral titer and replication-competent recombinant retrovirus as described (17, 18) and used to transduce CEM or HeLa cells, generating L80SN-CEM, L86SN-CEM, L80SN-HeLa, and L86SN-HeLa cells. Each transduced tumor cell population was then G418 selected, and clones derived by limiting dilution. Flow cytometry by FACS and immunofluorescence confocal microscopy (ICM)2 were then used to evaluate expression and cellular localization of CD80 and CD86.
Flow cytometry (FACS) and ICM
Flow cytometry was performed on a FACScan cytometer (Becton Dickinson, Mountain View, CA) according to the manufacturer’s protocols. ICM was performed using a Leica (Deerfield, IL) CLSM confocal microscope as described (19). Murine Abs used were anti-BB-1 (CD80) (CAMFolio; Becton Dickinson), CD86 (PharMingen, San Diego, CA), CD4-FITC, CD8-PE, CD3-FITC, CD19-PE, CD28 (Becton Dickinson), HLA-ABC (Silenus, Paris, France), CD14-PE (Dako, Carpinteria, CA), Nucleolus AE-3 (Biodesign, New York, NY), Golgi 58K protein (Sigma, St. Louis, MO), ß-tubulin (SM162; Sternberger Monoclonals, Baltimore, MD), CD4, and CD11b (Dako). Unconjugated Abs were visualized by incubation with FITC-conjugated goat anti-mouse Ig (Becton Dickinson). The mean fluorescence intensity (MFI) was calculated for each FACS profile. All samples examined by ICM were coded until completion of each experiment.
Disruption of the Golgi apparatus and microtubules by brefeldin A (BFA) and nocodazole treatment
As appropriate, L86SN-CEM cells or peripheral blood monocytes were treated with 0.04 µM BFA for 20 min at 37°C to disrupt the Golgi apparatus, or with 50 µM nocodazole (Sigma) for 60 min at 37°C to disrupt microtubules. The cells were then washed with PBS and samples settled onto glass slides; fixed with paraformaldehyde and methanol; and stained with Ab to CD86, anti-Golgi 58K protein, anti-ß-tubulin, or an isotype control, as appropriate, and the secondary FITC-conjugated goat anti-mouse Ab. To reassemble the Golgi apparatus and microtubules, BFA- or nocodazole-treated cells were washed in PBS and incubated for 30 or 60 min, respectively, at 37°C in PBS containing 10% FCS (Life Technologies) to remove the compound, and samples then fixed and stained as described above. All ICM localization patterns were compared with slides of untreated control cells.
PBMC isolation, fractionation, and stimulation in vitro
Heparin-treated blood from normal donors was diluted 1:1 with PBS, and PBMC isolated by density sedimentation for 30 min at 500 x g and 4°C on Ficoll-Hypaque gradients according to the manufacturer’s protocol (Pharmacia, Piscataway, NJ). Five randomly selected healthy blood donors were used in this study to obviate any selection bias. PBMC were collected, washed in PBS, and resuspended in DMEM supplemented with 10% human AB serum, penicillin (50 IU/ml), streptomycin (50 µg/ml), 2 mM glutamine, and 20 U/ml rIL-2 (Boehringer Mannheim, Indianapolis, IN). Monocytes were separated by adherence to plastic, and T lymphocytes were isolated by SRC rosetting as described (20). The monocyte-depleted, nonrosetting population (containing predominantly B lymphocytes) was also collected. An analysis of purity for each cell fraction was determined by FACS. Purified monocytes were stimulated by in vitro culture on plastic in DMEM (nota bene containing bovine calf serum). Cell surface localization of CD4, CD14, HLA-ABC, CD80, CD11b, and CD86 was monitored by FACS and intracellular expression of CD4, CD80, CD86, and CD11b by ICM. Purified T lymphocytes were stimulated by preincubation for 1 h with 5 ng/ml of OKT3 Ab (Janssen-Cilag, Sydney, Australia) and then extensively washed and maintained in rIL-2-supplemented DMEM medium. Cell surface localization of CD4, CD8, CD14, CD19, HLA-ABC, CD80, and CD86 was analyzed on T and B lymphocytes by FACS before and after stimulation, and intracellular expressions of CD80 and CD86 by ICM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Before retrovirus transduction, CEM and HeLa cells were tested by
FACS analysis and ICM and shown to be negative for endogenous CD80 or
CD86 expression (not shown). FACS analysis of stable L80SN-CEM,
L86SN-CEM, L80SN-HeLa, and L86SN-HeLa clones revealed high levels of
CD80 or CD86 cell surface membrane expression, as expected. ICM
analysis of L80SN-CEM and L86SN-CEM cells also revealed a prominent
intracellular focal concentration of CD80 (Fig. 1
A) and CD86 (Fig. 1B), respectively. This intracellular focal
concentration of CD80 or CD86 protein was present in all L80SN-CEM and
L86SN-CEM clones examined. Fluorescence staining was not observed in
the LXSN vector-transduced control CEM cells, or after isotype-control
Ab staining (not shown).
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, C and D,
respectively). A series of ICM images of immunolabeled cells were
acquired in the plane (x-y-axis) of the
slide throughout the entire depth (z-axis) to
demonstrate the discrete localization of the intracellular
concentration of CD86 in L86SN-CEM cells (Fig. 1E)
and the absence of any focal concentration of CD86 protein in
L86SN-HeLa cells (Fig. 1F). Similar results to those
shown in Figure 1, E and F, were obtained for
CD80 expression in L80SN-CEM, and L80SN-HeLa cells, respectively (not
shown). Characterization of the intracellular concentration of CD86 protein in L86SN-CEM cell clones
To determine whether the intracellular concentration of CD86 in
L86SN-CEM cells was associated with the nucleolus, cells were
immunostained with the nucleolus-specific Ab AE-3 (Fig. 2A, left),
anti-CD86 Ab (Fig. 2A, center), or
both Abs simultaneously (Fig. 2A, right)
and analyzed by ICM. Double immunostaining (Fig. 2A,
right) clearly demonstrated that the intracellular
reservoir of CD86 was not associated with the nucleolus. ICM analysis
of L86SN-CEM cells stained with anti-Golgi 58K protein Ab (Fig. 2B, left) showed a single focal
concentration of fluorescence that was very similar in appearance and
localization to that observed for intracellular CD86 staining (Fig. 2B, center), and double staining with both
Abs (Fig. 2B, right) suggested possible
Golgi localization of the intracellular CD86 protein. To examine this
possibility further, L86SN-CEM cells were treated with BFA to
disorganize the Golgi apparatus, and then immunostained and analyzed by
ICM. The normal staining pattern observed on untreated L86SN-CEM cells
with anti-Golgi 58K protein Ab (Fig. 2C,
left) was no longer evident after BFA treatment (Fig. 2C, center), and reappeared as the Golgi
apparatus reassembled when BFA was washed from the culture (Fig. 2C, right). No observable change in the
pattern of CD86 localization in L86SN-CEM cells (Fig. 2D,
left) was observed after BFA treatment (Fig. 2D, center), or after BFA was washed from
the cells to reassemble the Golgi apparatus (Fig. 2D,
right).
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E, left)
was no longer evident after nocodazole treatment (Fig. 2E,
center), and reappeared as the microtubules
reassembled when nocodazole was washed from the culture (Fig. 2E, right). The intracellular focal
concentration of CD86 in L86SN-CEM cells (Fig. 2F,
left) was also disorganized after nocodazole
treatment (Fig. 2F, center), although cell
surface CD86 localization was still apparent. When nocodazole was
washed from the culture, the intracellular focal concentration of CD86
reappeared (Fig. 2F, right). These results
were reproduced in six independent experiments with three different
L86SN-CEM cell clones. Cell surface localization of CD80 and CD86 in unstimulated and OKT3-stimulated peripheral blood T lymphocytes does not involve an intracellular concentration of either protein
Localization of CD80 and CD86 in peripheral blood T lymphocytes
was performed by FACS and ICM analysis on unstimulated and
OKT3-stimulated cells. Unstimulated T lymphocytes did not express
detectable cell surface CD80 or CD86 by FACS analysis (not shown), and
there was no evidence of an intracellular focal concentration of either
protein by ICM (Fig. 3, A and
D, respectively). After OKT3 stimulation, the T lymphocytes
were examined daily, and at day 8, cell surface CD80 and CD86 was first
detected by FACS (not shown) and ICM, peaked in intensity by day 10
(Fig. 3, B and E, respectively), and began to
decline again by day 14 (Fig. 3, C and F,
respectively). At no time during this 14-day period was an
intracellular focal concentration of either protein observed in a total
of seven independent experiments. Isotype-control Ab staining was
negative at each time point by FACS analysis and ICM (not shown).
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Peripheral blood B lymphocytes were isolated from the purified PBMC fraction by depletion of T lymphocytes and monocytes, and the B lymphocytes identified and characterized by FACS analysis. As expected, CD80 and CD86 were not detectable on the surface of unstimulated B lymphocytes by FACS analysis. After 24 h of stimulation by in vitro culture, both CD80 and CD86 were detectable by FACS analysis and ICM, and the cells remained positive for a least 48 h in vitro (not shown). At no time during the 48 h in vitro culture of peripheral blood B lymphocytes was an intracellular focal concentration of CD80 or CD86 protein observed by ICM (not shown).
Induction of CD80 expression in peripheral blood monocytes involves cell surface localization, but no intracellular concentration of the protein
Freshly isolated monocytes had no detectable cell surface CD80 by
FACS analysis (Fig. 4A, red
line, MFI 8), and there was no evidence of an intracellular
concentration of CD80 (Fig. 4B) detected by ICM.
After 24 h of stimulation by in vitro culture, approximately 70%
of the monocyte population demonstrated cell surface CD80 localization
by FACS (Fig. 4A, green line, MFI 103), but no intracellular
concentration of protein was observed by ICM (Fig. 4C). After 48 h of stimulation by in vitro
culture, more than 90% of the monocytes demonstrated surface
localization of CD80 (Fig. 4A, blue line, MFI 156) by FACS
analysis, but no intracellular concentration of the protein was
observed by ICM (Fig. 4D). Isotype-control Ab
staining was negative by FACS and ICM (not shown). The results were
reproduced in three independent experiments with monocytes isolated
from different PBMC donors.
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Although there was some variation among the PBMC donors used in
the study, between 60 and 80% of the purified peripheral blood
monocytes consistently and constitutively expressed CD86 immediately
after isolation, and cell surface fluorescence staining was clearly
evident by FACS analysis (Fig. 4E, red line, MFI 67) and ICM
(Fig. 4F). ICM also revealed an intracellular focal
concentration of CD86 in the monocytes (Fig. 4F) that
was similar in appearance to that observed in L86SN-CEM cells (Fig. 1B). After 24 h of stimulation by in vitro
culture, the surface CD86 fluorescence intensity had increased (Fig. 4E, green line, MFI 177) and the intracellular CD86 focal
concentration had disappeared from approximately 50% of the monocyte
population (Fig. 4G). After 48 h of stimulation
by in vitro culture, more than 90% of the purified monocytes were
positive for cell surface CD86 by FACS analysis (Fig. 4E,
blue line, MFI 268), and the intracellular concentration of CD86 was
only detectable in less than 5% of the population (Fig. 4H). This relationship between increasing cell
surface localization of CD86 and decreasing intracellular concentration
of the protein as a function of time in vitro was reproduced in three
independent experiments with monocytes from different PBMC donors.
Percentages were derived by counting more than 150 cells in each ICM
field.
The intracellular reservoir of CD86 protein in peripheral blood monocytes is not due to concentration within the Golgi complex, and requires a network of intact microtubules to retain structural integrity
A series of BFA and nocodazole studies was performed on peripheral
blood monocytes from normal donors to characterize the intracellular
reservoir of CD86 protein. To examine any relationship between the
reservoir and the Golgi, freshly isolated monocytes were treated with
BFA to disorganize the Golgi apparatus, and then immunostained and
analyzed by ICM. The ICM staining pattern observed when untreated
monocytes were incubated with an anti-Golgi 58K protein Ab (Fig. 5A, left)
was no longer evident after BFA treatment (Fig. 5A,
center), and reappeared as the Golgi apparatus
reassembled when BFA was washed from the culture (Fig. 5A,
right). In contrast, there was no observable change
in the intracellular concentration of CD86 in monocytes incubated with
anti-CD86 Ab (Fig. 5B, left) after BFA
treatment (Fig. 5B, center), or when BFA
was washed from the culture to reassemble the Golgi apparatus (Fig. 5B, right).
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C, left) was no longer evident
after nocodazole treatment (Fig. 5C,
center), but did reappear as the microtubules
reassembled when nocodazole was washed from the culture (Fig. 5C, right). Similarly, the intracellular
reservoir of CD86 in freshly isolated monocytes incubated with
anti-CD86 Ab (Fig. 5D, left) was also
disorganized after nocodazole treatment (Fig. 5D,
center), and reappeared rapidly when nocodazole was
washed from the culture (Fig. 5D, right).
These results were reproduced in three independent experiments with
monocytes isolated from different PBMC donors. The type I integral membrane proteins CD11b and CD4 do not colocalize with intracellular CD86 in peripheral blood monocytes
The ICM pattern of intracellular CD86 concentration in peripheral
blood monocytes incubated with anti-CD86 Ab (Fig. 6A) was distinctly
different from that obtained when monocytes were incubated with
anti-CD11b Ab and examined by ICM (Fig. 6B). The
CD11b protein was found to be predominantly associated with the cell
surface membrane of freshly isolated monocytes, with some punctate
intracellular fluorescence activity also evident (Fig. 6B). This punctate pattern of CD11b localization,
however, was entirely different from that observed for intracellular
CD86 (Fig. 6A). When incubated with anti-CD4 Ab,
freshly isolated monocytes were only weakly positive for cell surface
CD4 by FACS analysis, and no intracellular concentration of CD4 protein
was detected by ICM (not shown). As a control for CD4 immunostaining,
L86SN-CEM cells were incubated with anti-CD4 Ab, and examined by
ICM. Again, a punctate intracellular staining pattern was observed
(Fig. 6C) that differed significantly from the
typical intracellular CD86 localization pattern (Fig. 6A).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A series of experiments was performed on L86SN-CEM cells to determine the nature of the intracellular concentration of CD86 protein. We eliminated the possibility of an interaction with the CEM cell nucleolus by colocalization studies with anti-CD86 and a nucleolus-specific Ab. We then addressed the possibility that the intracellular concentration of CD86 was due to storage within the Golgi complex. ICM localization studies were performed on L86SN-CEM cells before and after treatment with BFA to disrupt the Golgi apparatus, and revealed that the intracellular concentration of CD86 was not due to storage within the Golgi. The relationship between the intracellular concentration of CD86 and the integrity of the microtubules was then examined by treating L86SN-CEM cells with nocodazole, a compound that disrupts microtubule organization. The intracellular focal concentration of CD86 in L86SN-CEM cells was disrupted by nocodazole treatment, and rapidly restored after the compound was washed from the cultures. Evidently, intact microtubules are required to retain the structural integrity of the intracellular focal concentration of CD86 in transduced CEM cells.
Although the results of these initial studies were interesting, they were based on a genetically modified T lymphoblastoid tumor cell line. To ensure that the observations were not peculiar to CEM cells, we extended our studies to an analysis of endogenous CD80 and CD86 expression in PBL and peripheral blood monocytes. Consistent with previous reports, FACS analyses revealed that 1) only stimulated T lymphocytes, B lymphocytes, and monocytes expressed CD80; 2) only stimulated T and B lymphocytes expressed CD86; and 3) both unstimulated and stimulated monocytes expressed CD86 (2, 6, 7, 9, 10, 11, 12). We then examined purified PBL and peripheral blood monocytes by ICM for any intracellular concentration of CD80 or CD86 protein. The ICM experiments revealed that only monocytes, which are known to constitutively express CD86, had an intracellular focal concentration of CD86 protein. Moreover, the intracellular localization of CD86 in peripheral blood monocytes was very similar in appearance to that observed in the L86SN-CEM cells. The observation of an intracellular focal concentration of CD86 in monocytes, and the absence of a similar concentration of CD80 in these cells, suggested that constitutive gene expression was important in the process. This hypothesis would explain why no intracellular concentration of CD80 or CD86 was ever observed in T or B lymphocytes, and was consistent with the CEM cell experiments in which both genes were constitutively expressed from the retrovirus promoter. The absence of an intracellular concentration of CD80 or CD86 in the transduced HeLa cells, however, may indicate that the process is restricted to certain cell types. Further studies will be required to address this question.
The monocyte expression studies also revealed an interesting temporal relationship between the intracellular concentration of CD86 and the surface membrane localization of the protein. As the peripheral blood monocytes were stimulated by in vitro culture, both the absolute number of cells positive for surface membrane CD86 and the mean fluorescent intensity of the population increased as a function of time. Simultaneously, the intracellular concentration of CD86 protein decreased. The most likely explanation for this observation was that CD86 was being transported from an intracellular reservoir to the monocyte surface membrane in response to in vitro stimulation. In an attempt to further explore this antegrade CD86 transport hypothesis, a series of experiments was performed on peripheral blood monocytes to determine the nature of the intracellular reservoir. Similar to experiments described for the L86SN-CEM cells, BFA and nocodazole were used to treat freshly isolated peripheral blood monocytes to investigate any relationships between the intracellular CD86 reservoir, the Golgi, and the microtubules. Consistent with the earlier findings, BFA treatment revealed that the intracellular reservoir of CD86 in peripheral blood monocytes was not due to storage within the Golgi. In addition, nocodazole treatment revealed that intact microtubules were necessary for the intracellular reservoir of CD86 to retain structural integrity in peripheral blood monocytes. Both of these results further support a model in which the intracellular concentration of CD86 represents a functional reservoir of translated protein available for rapid and sustained deployment to the monocyte cell surface membrane in response to stimulation.
As mentioned earlier, CD86 is a type I integral membrane glycoprotein of the Ig supergene family. A question raised by our observations was whether or not all constitutively expressed integral membrane proteins of this family would be concentrated within the intracellular reservoir in monocytes. To address this question, we performed FACS analyses and ICM localization studies on peripheral blood monocytes with Ab to CD4 and CD11b. CD4 is a type I integral membrane protein of the Ig supergene family constitutively expressed by monocytes, and CD11b is a type I integral membrane protein of the integrin family constitutively expressed by approximately 80% of monocytes. ICM analysis revealed no intracellular concentration of CD4 in peripheral blood monocytes, and a punctate pattern of intracellular CD11b that was entirely different from the typical focal concentration observed with CD86. Clearly, not all constitutively expressed type I integral membrane proteins concentrate within the reservoir.
Collectively, the biologic significance of our observations may be related to the temporal regulation of lymphocyte costimulation. The interaction between CD86 on the surface of an APC, and CD28 (which is expressed on 95% of resting CD4+ T lymphocytes) is thought to be crucial for the initiation of a significant subset of T lymphocyte-mediated immune responses (6, 7, 8, 9, 10, 12). Since CD86 is constitutively expressed by monocytes, this costimulation and activation of T lymphocytes can occur immediately if an appropriate Ag is present. In contrast, the interaction between CD80 on an APC and CTLA-4 on a T lymphocyte, which is believed to be more important in the maintenance and/or moderation of the immune response, can only occur some hours after both cells have been stimulated to express these counterreceptors (21, 22, 23). Therefore, the presence of an intracellular reservoir of CD86 that could allow rapid and sustained transport of the protein to the monocyte surface membrane would seem to be biologically advantageous and entirely consistent with the current tenets of lymphocyte activation by costimulation.
Structurally, the intracellular reservoir described in this report probably represents a highly organized concentration of transport vesicles capable of rapid deployment of CD86 to the monocyte surface membrane. Although similarities exist between our results and a recent report describing intracellular storage and rapid release of Fas ligand from activated monocytes (24), the fluorescence localization patterns of CD86 and Fas ligand appear to be completely different. More striking parallels appear to occur between the CD86 reservoir in monocytes and the recently identified neuronal cell storage organelle, the botrysome. The botrysome is a discrete organelle located between the Golgi and the endoplasmic reticulum, and has been shown to be involved in intracellular protein trafficking in response to cellular activation (25). Similarities between the subcellular location and size of the neuronal cell botrysome (1–4 µm), and the intracellular reservoir for CD86 in monocytes are compelling, and future studies will attempt to examine any relationship that might exist. These studies, in addition to precise kinetic analyses of the trafficking and concentration of CD86 in monocytes, will be necessary to fully understand this novel reservoir, and may provide further insights into the temporal regulation of APC function in the cell-mediated immune response.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: ICM, immunofluorescence confocal microscopy; MFI, mean fluorescence intensity; BFA, brefeldin A.
Received for publication December 30, 1997. Accepted for publication February 5, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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