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*
Department of Immunology, St. Jude Children’s Research Hospital, Memphis, TN 38105; and
Department of Pathology, University of Tennessee Medical Center, Memphis, TN 38163
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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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6% of CD8+ T cells in the spleens of mice that have
recovered from infection are specific for the immunodominant epitope
(E.J.U. and D.L.W., manuscript in preparation).
The frequency and kinetics of Ag presentation in viral systems have not
been extensively studied. Sendai virus is pneumotropic, infecting only
ciliated and secretory cells in the trachea and bronchi and type II
epithelial cells in the lung 2, 3 . Presentation of viral Ags on
professional APCs occurs when these cells are infected with the virus
or following phagocytosis of infected lung cells or virions. The lung
has a contiguous network of MHC class II-positive dendritic cells (DCs)
that turn over rapidly and may constantly sample the antigenic
environment in the lungs and transport Ag to draining lymph nodes 4 .
These cells increase in number during Sendai virus infection in rats
and remain elevated for 
14 days after infection 5 . Work by
Hamilton-Easton and Eichelberger 6 in the influenza virus system has
shown that T cell hybridomas can be used to detect viral Ag on APCs
after viral infection. These studies showed that cells presenting viral
epitopes were present at 2 days postinfection; however, the numbers of
APCs could not be quantitated accurately. In the current report, we
took advantage of a new technology for generating
lacZ-inducible T cell hybridomas, which are significantly
more sensitive than conventional hybridomas 7, 8 , to quantitate the
numbers of cells presenting viral Ag during the first 10 days after
Sendai virus infection of C57BL/6 mice. The data show that APCs are
readily detectable and persist for several days after the virus has
been cleared from draining lymph nodes, suggesting that viral Ag can
persist in a noninfectious form for 2–3 days.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and housed under specific pathogen-free conditions. Mice were infected at 6–12 wk of age. The Enders strain of Sendai virus and the A/HK-x31 (H3N2) strain of influenza virus were grown, stored, and titrated as described previously 9, 10 . Mice were anesthetized by i.p. injection with Avertin (2,2,2-tribromoethanol) and infected intranasally with 500 50% egg infectious doses of Sendai virus or 240 hemagglutination units of influenza virus.
Abs and cell sorting
All of the Abs used for staining were purchased from PharMingen
(San Diego, CA). MLNs or spleen cells were stained for
three-color cell sorting with combinations of the following Abs:
anti-CD45R phycoerythrin (PE) conjugate (B220; cat no. 01125B),
anti-CD45R Cy5 conjugate (B220; cat no. 01128B), anti-CD11c
FITC conjugate (09704D), CD11c PE conjugate (09705B), anti-CD11b
biotin conjugate (01712D), CD11b PE conjugate (01715B), and
streptavidin Cy-Chrome (13038A). Samples were sorted on a
FACStarPlus flow cytometer (Becton Dickinson, San
José, CA). Four populations of cells were
collected: CD11c+CD45R- (DCs),
CD11b+CD45R- (macrophages),
CD45R+ (B cells), and
CD11c-CD11b-CD45R- cells.
The yields of cells were as follows:
CD11c+CD45R- cells, 1.5–2% in MLNs and
spleen; CD11b+CD45R-, 6–7% from spleen and
1.5–2% from MLNs; CD45R+ cells, 50–60%. In general, the
purity after sorting was 90%.
Cell lines and culture conditions
All cell lines were grown in complete tumor medium containing 10% FCS at 37°C with 10% CO2 11 . L cells transected with the Kb or I-Ab MHC genes have been described previously 12, 13 . The BWZ.36 fusion partner 8, 14 was a gift of Dr. Nilabh Shastri (University of California, Berkeley, CA).
Synthetic peptides
Sendai virus nucleoprotein NP324–332 (FAPGNYPAL) and hemagglutinin-neuraminidase HN421–436 (VYIYTRSSGWHSQLQIG) peptides were synthesized at St. Jude Children’s Research Hospital Center for Biotechnology on an Applied Biosystems model 433A peptide synthesizer (Applied Biosystems, Berkeley, CA). Peptide purity was evaluated using reverse-phase HPLC analysis. Stock solutions of peptides (1 mg/ml) were made in PBS and subsequently diluted in complete tumor medium before use in assays.
LacZ-inducible T cell hybridomas
MLNs were removed from C57BL/6 mice at 10 days after intranasal
infection with 500 50% egg infectious doses of Sendai virus,
and cell suspensions were cultured with 10 U/ml human rIL-2 for
2 days. Live cells were separated by centrifugation into lymphocyte
separation medium (ICN Biomedicals, Aurora, OH) and cultured overnight
in fresh medium with 10 U/ml human rIL-2. Blast cells were
subsequently harvested and fused with BWZ.36 cells, which contain the
lacZ gene fused to the NF of activated T
cells-activated enhancer from the IL-2 gene. After selection for
peptide specificity, each line was subcloned using the autocloning
facility on a FACStarPlus flow cytometer (Becton
Dickinson). Subclones were screened using L cells transfected with
either Kb or I-Ab molecules plus either peptide
NP324–332 (Kb-restricted hybridomas) or
peptide HN421–436 (I-Ab-restricted hybridomas)
at 10 µg/ml. Those clones that produced 50% or more
lacZ+ cells in response to peptide stimulation
were selected for further study.
APC assays
APCs were added to flat-bottom, 96-well plates (Sarstedt, Newton, NC) plus peptide where appropriate. A total of 105 hybridoma cells were added per well, the cultures were incubated overnight, and a 5-bromo-4-chloro-3-indolyl ß-D-galactoside (X-Gal) assay was performed the following morning. Experiments with L-Kb or L-I-Ab cells used 105 L cells/well. For peptide loading, L cells were incubated with 50 µg/ml peptide at 37°C for 4 h and then washed four times. In some experiments, cells were infected with Sendai virus at 37°C for 1 h at a multiplicity of 10 followed by four washes. For an ex vivo estimation of APC frequency, CLNs, MLNs, and spleens were removed from groups of four to six mice at various times postinfection; next, the organs were pooled, and single-cell suspensions were prepared. Twofold serial dilutions of cells from each organ were made in flat-bottom, 96-well plates starting at 106 cells/well. A total of 105 hybridoma cells were added to each well and cultured overnight; next, an X-Gal assay was performed to identify responding hybridomas. The number of cells in the highest dilution of APCs giving twice the background number of responding hybridomas was taken as the reciprocal frequency. To assess background lacZ expression, hybridomas were cultured with lymph node or spleen cells from mice infected with influenza virus. A lacZ-inducible hybridoma specific for glycoprotein B of murine gammaherpesvirus 68 (4951.5) was used as a further negative control (L. Liu, E. Flano, E.J.U. S. Surman, M. A. Blackman, and D.L.W., unpublished data). This hybridoma was not stimulated in response to APCs presenting Sendai virus epitopes. As a positive control, hybridomas were cultured with spleen or lymph node cells in the presence of 10 µg/ml of the relevant peptide.
X-Gal assay
Cultures in flat-bottom 96-well plates were washed with 200 µl of PBS per well and subsequently fixed by the addition of 100 µl of PBS containing 2% formaldehyde and 0.2% glutaraldehyde for 5 min at 4°C. The plates were washed with PBS and then overlaid with 50 µl of a solution containing 1 mg/ml X-Gal, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, and 2 mM MgCl2 in PBS. Cultures were examined microscopically, and the number of blue cells was counted after 6–8 h of incubation at 37°C or after incubation overnight at 4°C.
Detection of virus in lymphoid tissue
Cell suspensions prepared from lymph nodes and spleen were directly injected into 10-day-old embryonated hen eggs. After incubation at 35°C for 48 h, allantoic fluid from each egg was sampled and assayed for hemagglutinating activity using 0.5% chicken E in a volume of 50 µl. Organs from four mice were pooled at each timepoint, and 107 cells were injected into three eggs each. Samples were scored as positive when at least two of the three eggs contained hemagglutinating activity.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To investigate Ag presentation in Sendai virus-infected mice, we
first generated lacZ-inducible T cell hybridomas specific
for the immunodominant MHC class I- and class II-restricted epitopes.
MLNs were harvested from C57BL/6 mice at 10 days after Sendai virus
infection and cultured in vitro for an additional 3 days in the
presence of IL-2; next, T cell blasts were fused with the BWZ.36 cell
line. A total of 71 hybridomas were generated, two of which recognized
the immunodominant NP324–332/Kb epitope and
five of which recognized the dominant
HN421–436/I-Ab epitope. Subclones were derived
from these hybridomas, and those that gave the strongest response to
peptide (in terms of the percentage of lacZ+cells) were selected. We subsequently tested the sensitivity of
these hybridoma subclones to graded doses of peptide (Fig. 1
). The I-Ab-restricted
hybridomas (5204-E6, 5204-H5, and 5268-B2) responded to peptide
concentrations as low as 1.25 µg/ml to 156 ng/ml, whereas both of the
Kb-restricted hybridomas (5205-D10 and 5205-G9) responded
to concentrations in the 153–76 pg/ml range. The subclones 5204-H5
(HN421–436/I-Ab-restricted) and 5205-G9
(NP324–332/Kb-restricted) were the most
sensitive and consequently were chosen for further study.
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, both hybridomas responded to smaller
numbers of peptide-loaded APCs than virus-infected APCs. This
difference probably reflects a higher density of peptide-MHC complexes
after peptide loading. The
NP324–332/Kb-restricted 5205-G9 hybridoma
required 30 peptide-loaded cells or 2000 virus-infected cells to
give a response above twice the background (untreated APCs). The
HN421–436/I-Ab-restricted 5204-H5 hybridoma
required 250 peptide-loaded cells or 8700-virus infected cells.
Experiments using whole spleen cells as APCs yielded similar results
(data not shown). These data indicate that Sendai virus-specific
lacZ-inducible hybridomas can detect very low numbers of
APCs presenting viral epitopes and could potentially be used to detect
APCs from virus-infected mice.
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Little is known about the frequency of cells presenting particular viral epitopes during viral infection, so we designed an experiment to determine 1) whether APCs were present at a sufficient level to be detectable and 2) how long Ag presentation lasted after the clearance of virus. We infected C57BL/6 mice with Sendai virus, and then removed CLNs, MLNs, and spleens at days 3–10 postinfection. Graded numbers of cells from these organs were incubated with the lacZ-inducible 5205-G9 and 5204-H5 hybridomas. As a negative control, C57BL/6 mice were infected with influenza x31 virus; at each timepoint, the lymph nodes and spleens of these control mice were titrated in the same way as for Sendai virus-infected mice. This gave an estimate of the background level of activation of the hybridomas in response to APCs from mice during an immune response to an unrelated virus.
Due to the small size of the MLN in the first 2 days after infection,
our study began at day 3 postinfection. At this time, we could detect
relatively high frequencies of cells in the MLN presenting viral Ags
(1:9 x 103 cells presenting
HN421–436/I-Ab and 1:9.7 x
104 cells presenting NP324–332/Kb)
(Fig. 3). APCs presenting the
NP324–332/Kb epitope in the MLN remained at a
frequency of 1:105 cells until day 10, when the frequency
fell to <1 in 106. HN421–436/I-Ab
presentation in the MLN began at a frequency of 1:104
cells at days 3–5 and then steadily declined to undetectable levels by
day 10. In the CLN, the pattern was similar, except APC frequencies
were 10- to 20-fold lower. Detection of APCs in the spleen was
variable, and the frequency was at the limit of detection of these
assays (1:106) when unfractionated cells were used.
These figures provided a minimal estimate of the frequency of APCs, as
none of the hybrids appeared to recognize <30 cells/well, and APCs
expressing a low density of Ag were probably not detectable. It should
be noted that the data in Fig. 3 indicate that there is a two-log
variation in the titers of APCs presenting
NP324–332/Kb or
HN421–436/Ab epitopes. Given the fact that
viral titers can vary by several logs in the lungs of mice infected
with the same dose of virus, these data probably reflect the inherent
variation between individual infections. Experiments under controlled
conditions using exogenously added peptides indicate that the hybridoma
assay of APC frequencies is very consistent and usually only shows
variation within one to two doubling dilutions.
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). When presented in this way, the data
reveal a large increase in APC numbers between days 3 and 5,
corresponding to the dramatic increase in size of the MLN. This was not
the case for the HN421–436/I-Ab
response in the CLN, where the numbers of APCs remained approximately
the same between days 3 and 6. At the peak, the number of
HN421–436/I-Ab-presenting cells was
approximately threefold higher than
HN324–332/Kb-presenting cells in the MLN (note
different scales on Fig. 4), whereas these numbers were more similar in
the CLN.
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). Virus was not detected at any
timepoint studied in the CLN or spleen but was detectable in the MLN at
days 3 and 6 postinfection. This finding is consistent with previous
reports 15 . Because cells presenting both MHC class I and MHC class
II epitopes were present in the absence of detectable virus (CLN and
MLN after day 6), the data indicate that viral Ag is persisting in some
form in these tissues. Clearly, more experiments are needed to
determine the mechanism and form of Ag persistence.
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As we could readily detect APCs ex vivo, we subsequently asked
what are the major cell types presenting viral epitopes in the primary
response? We used FACS to fractionate MLNs or spleen cells from
infected mice into enriched populations of B cells
(CD45R+), DCs (CD45R-CD11c+), or
macrophages (CD45R-CD11b+). Day 6 was chosen
for these studies, as this was a time when a high frequency of APCs was
detectable in unfractionated lymph nodes. As shown in Table II, we observed a large enrichment in the
frequency of HN421–436/I-Ab- and
NP324–332/Kb-presenting cells in the
macrophage and DC fractions. In contrast, the B cell fraction was
depleted of I-Ab- and Kb-presenting cells. This
observation that very few B cells were presenting viral epitopes during
the infection was surprising in view of the vigorous B cell response to
the virus 16 . The major APCs at 6 days postinfection were DCs and
macrophages.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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LacZ-inducible T cell hybridomas have been used successfully to identify new minor histocompatibility Ags by expression cloning 17 and also to study the cells presenting Ag after DNA vaccination 18 . The hybridomas were found to have responses that were identical with those of conventional hybridomas, and the peptide sensitivity and signal to noise ratio was comparable 8 . Because of the ability to detect activation in single cells, the lacZ-inducible hybridomas were 50- to 100-fold more sensitive than conventional hybridomas at detecting small numbers of APCs. Our T cell hybridoma specific for NP324–332/Kb responded to 30 peptide-loaded APCs or 2000 virus-infected cells/well, and the HN421–436/I-Ab hybridoma recognized 250 peptide-loaded or 8700 virus-infected APCs. These figures were established using nonprofessional APCs (L cells), and it is possible that the detection limit may be lower using the professional APCs located in lymphoid organs. Therefore, the APC frequencies obtained in this study should be taken as a minimum estimate. Despite this limitation, APCs were easily detectable ex vivo and could be detected several days after the clearance of infectious virus from lymphoid tissue. An alternative approach to quantifying Ag/MHC complexes has been described by Dadaglio et al. and Porgador et al. using Abs against specific peptide/MHC complexes 19, 20 . The Ab approach has an advantage in that it allows direct quantification of peptide/MHC complexes on the cell surface and can be used in situ. However, it has a disadvantage in that it is difficult to generate and confirm the specificity of Ab reagents, particularly if reagents for several epitopes are required. It is not clear how the hybridoma and Ab techniques compare with each other in terms of sensitivity, although both approaches are not likely to detect low levels of peptide/APC complexes.
The number of APCs reached a peak in lymph nodes at 5 days postinfection, before the peak CTL response, which is consistent with the fact that naive CTLs require activation by APCs in the draining lymph node before they can migrate to the lung as differentiated effector CTLs. At this time after infection, viable virus can be detected in the MLN using a sensitive egg infection assay. However, no detectable virus is present after 6 days postinfection in the draining lymph node, but NP324–332/Kb- and HN421–436/I-Ab-presenting cells are still detectable up to day 9 postinfection. It is unlikely that the lacZ-inducible hybridoma assay is more sensitive than the egg infection assay for detecting infectious virus. This implies that viral Ags persist in a noninfectious form for several days. There are several possible sources for this persistent Ag. Viral proteins produced within infected APCs may be degraded slowly, so that peptide epitopes are generated over several days until the supply is exhausted. Alternatively, there may be an extracellular depot of Ag that is constantly sampled by DCs which are able to present nonreplicating virus particles on MHC class I molecules 21 in addition to MHC class II molecules. Yet another possibility is that there may be limited transcription from persisting viral genomes.
It has been assumed that in respiratory infections Ag presentation occurs exclusively in the draining lymph nodes and that specific T cells only migrate to the spleen following activation. In fact, we observed APCs in the spleen, albeit at very low levels. This observation would imply that antiviral T cells can be activated directly at this site, rather than the spleen being merely a repository for T cells that were activated elsewhere. This would explain recent data in the influenza virus system showing a low but detectable level of specific CTLs in the spleen at early times after infection (0.1–0.5% of CD8+ T cells at 7 days postinfection) 22 .
Fractionation of the APC population revealed a large enrichment for cells presenting viral epitopes in the macrophage and DC populations, which is consistent with data obtained in influenza infection using conventional hybridoma cells 6 . In contrast, APC frequency was markedly depleted in the B cell fraction, suggesting that B lymphocytes are not a major APC population in the primary Sendai virus infection. In the influenza virus study, B lymphocytes taken from the BAL were shown to present Ag. However, this may be due to the fact that B cells in the lung are exposed to a very large amount of Ag, so that even inefficient Ag presentation would be sufficient to stimulate T cell hybridomas. Ag presentation locally in the draining lymph node is likely to be of more importance physiologically, as it is here, rather than in the lung, where naive T cells become activated. It would be interesting to test whether B cells are more important APCs in a secondary response, because there would be an elevated number of Sendai virus-specific B cells expressing high affinity Ab, which is known to enhance the APC function of B cells 23 .
In conclusion, new and sensitive techniques now allow us to detect relatively low frequencies of APCs ex vivo. In this study, we used an infection where the virus is completely cleared in a short period of time; however, the system is broadly applicable, and it would be interesting to apply this technique to a persistent infection. Study of the kinetics of Ag presentation is important, because efficient Ag presentation is a general requirement for T cell-mediated immune responses. Understanding this vital step may lead to a better understanding of the processes that interrupt the generation of an efficient immune response and may provide insight for the development of more rational vaccines.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. David L. Woodland, Department of Immunology, St. Jude Children’s Research Hospital, Memphis, TN 38105. E-mail address:
3 Abbreviations used in this paper: MLN, mediastinal lymph node; CLN, cervical lymph node; BAL, bronchoalveolar lavage; DC, dendritic cell; PE, phycoerythrin; X-Gal, 5-bromo-4-chloro-3-indolyl ß-D-galactoside; HN, hemagglutinin-neuraminidase; NP, nucleoprotein.
Received for publication October 1, 1998. Accepted for publication December 17, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cell involvement in the pneumonia caused by Sendai virus. Cell. Immunol. 143:183.[Medline]
interferon but not the immunoglobulin response in mice that vary in susceptibility to Sendai virus pneumonia. J. Virol. 69:5592.[Abstract]
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C. M. Jones, S. C. Cose, R. M. Coles, A. C. Winterhalter, A. G. Brooks, W. R. Heath, and F. R. Carbone Herpes Simplex Virus Type 1-Specific Cytotoxic T-Lymphocyte Arming Occurs within Lymph Nodes Draining the Site of Cutaneous Infection J. Virol., March 1, 2000; 74(5): 2414 - 2419. [Abstract] [Full Text]
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V. Loyer, P. Fontaine, S. Pion, F. Hetu, D.-C. Roy, and C. Perreault The In Vivo Fate of APCs Displaying Minor H Antigen and/or MHC Differences Is Regulated by CTLs Specific for Immunodominant Class I-Associated Epitopes J. Immunol., December 15, 1999; 163(12): 6462 - 6467. [Abstract] [Full Text] [PDF]
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L. Liu, E. J. Usherwood, M. A. Blackman, and D. L. Woodland T-Cell Vaccination Alters the Course of Murine Herpesvirus 68 Infection and the Establishment of Viral Latency in Mice J. Virol., December 1, 1999; 73(12): 9849 - 9857. [Abstract] [Full Text]
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E. J. Usherwood, R. J. Hogan, G. Crowther, S. L. Surman, T. L. Hogg, J. D. Altman, and D. L. Woodland Functionally Heterogeneous CD8+ T-Cell Memory Is Induced by Sendai Virus Infection of Mice J. Virol., September 1, 1999; 73(9): 7278 - 7286. [Abstract] [Full Text]
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M. A. Coppola, E. Flano, P. Nguyen, C. L. Hardy, R. D. Cardin, N. Shastri, D. L. Woodland, and M. A. Blackman Apparent MHC-Independent Stimulation of CD8+ T Cells In Vivo During Latent Murine Gammaherpesvirus Infection J. Immunol., August 1, 1999; 163(3): 1481 - 1489. [Abstract] [Full Text] [PDF]
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) or loaded with
NP324–332 or HN421–436 peptide, respectively
(
), and then used as APCs. Background was assessed by culturing the
hybridomas with the appropriate untreated L cells. Hybridoma 5205-G9
(NP324–332/Kb-specific) had very low
background numbers of lacZ+ cells (usually <5 cells/well),
whereas the background with hybridoma 5204-H5
(HN421–436/I-Ab-specific) was more variable
and generally higher (typically 20–30 cells/well).
