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*
Epimmune, San Diego, CA 92121; and
Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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secretion in vitro in response to the vaccine was reduced compared with
healthy volunteers. A similar pattern of cytokine secretion was
observed following mitogen stimulation, suggesting a general altered
balance of Th1/Th2 responses. Further analysis indicated that HTL
recall responses to whole tetanus toxoid protein were reduced in
chronically infected subjects, and reduced responsiveness correlated
with the outcome of Theradigm-HBV immunization. Finally, experiments in
HBV transgenic mice indicated that the nonnatural Pan DR HTL epitope,
PADRE, is capable of inducing high levels of IFN- secretion
and that its inclusion in a lipopeptide incorporating an immunodominant
Ld-restricted CTL epitope resulted in breaking tolerance at
the CTL level. Overall, our results demonstrate an alteration in the
quality of HTL responses induced in chronically infected HBV patients
and suggest that use of a potent HTL epitope may be important to
overcome CTL tolerance against specific HBV Ags. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The crucial role of CTL in the resolution of HBV infection has been further underlined by studies in HBV transgenic mice. Adoptive transfer of HBV-specific CTL into mice transgenic for the HBV genome resulted in suppression of virus replication. This effect was primarily mediated by a nonlytic, lymphokine-based mechanism 4, 5, 6, 7 .
As is the case for class I-restricted responses, class II-restricted T cell responses are usually detected in patients with acute hepatitis but are absent or weak in patients with chronic infection 8 . However, in contrast with the clear association of CTL responses with the resolution of HBV infection, the association of an HBV-specific helper T lymphocyte (HTL) response with clearance of HBV infection has been less clearly documented. Because hepatocytes do not express MHC class II molecules, HTL responses are likely to recognize specific viral Ags phagocytosed and presented by professional APC 9 . In this context, although HTL responses may directly contribute to the clearance of HBV infection through the secretion of cytokines that suppress viral replication 10 , their primary role in disease resolution is probably mediated by supporting the induction and expansion of virus-specific CTL and B cells.
These data suggest that a vaccine capable of inducing HBV-specific CTL responses similar in quality and magnitude to those observed in acute hepatitis could represent a safe and effective treatment for chronic HBV infection. The modular immunotherapeutic, Theradigm-HBV, was designed based on this line of reasoning 11 .
Theradigm-HBV is composed of an HLA-A2-restricted, HBV-derived CTL epitope covalently linked to an universal HTL epitope and palmitoylated at the N-terminus. The palmitic acid moiety enhances the immunogenicity of the construct and obviates the need for an oil-based adjuvant 11 . The HLA-A2-restricted hepatitis B core Ag (HBcAg) 18–27 CTL epitope was selected for inclusion in Theradigm-HBV. This epitope is recognized by specific CTL derived from HLA-A2.1 patients with acute, resolving HBV infection 1, 2 . The HBcAg 18–27 epitope is also highly conserved among different HBV isolates, binds all common HLA-A2 subtypes analyzed to date, and is immunogenic in HLA-A2Kb transgenic mice and primary human CTL cultures 12, 13, 14 .
The tetanus toxoid (TT)-derived peptide 830–843 was selected as the universal HTL epitope. This epitope has been shown to be highly degenerate in its HLA-DR binding and is capable of functioning as an HTL epitope in individuals expressing many different DR types 15 . Furthermore, an HTL epitope unrelated to HBV was intentionally selected to avoid potential problems related to Ag-specific tolerance in the class II HTL compartment resulting from chronic HBV infection.
Previous studies in normal, uninfected volunteers have shown that Theradigm-HBV is highly immunogenic in humans 11, 16 . The magnitude of the CTL responses induced by Theradigm-HBV immunization was in fact found to be comparable to CTL responses associated with clearance of acute viral infection 16 . A strong correlation between the induction of CTL and HTL responses was also apparent, demonstrating that HTL responses are crucial for the development or maintenance of CTL responses 11, 16 .
Subsequent clinical studies demonstrated that while individuals chronically infected with HBV were overall hyporesponsive to immunization, nonetheless Theradigm-HBV was capable of inducing HBV-specific CTL responses in a dose-dependent fashion (J. Heathcote et al., manuscript in preparation). These data indicate that although some level of Ag-specific T cell tolerance at the CTL level is associated with chronic HBV infection, HBV-specific CTL precursors are present in HBV patients, arguing against a complete deletion of precursors as the mechanism of induction of such tolerance. In addition, while HBV-specific precursors are not activated by the large amount of viral Ags expressed during the course of chronic infection, nevertheless they can be activated, at least to a certain extent, by deliberate immunization with a therapeutic construct.
In the present study, we have further characterized the immune responses following Theradigm-HBV immunization in both normal subjects and HBV patients. While proliferative responses in chronically infected HBV patients to the HTL epitope included in the construct appeared normal, the cytokine profile observed suggested the induction of a Th0/Th2 HTL responses rather than a Th1 response. Decreases in recall proliferative responses to whole TT were also observed, and decreased proliferative responses to TT in turn correlated with the outcome of Theradigm-HBV immunization. Altered lymphokine profiles in chronically infected HBV patients were detectable even in response to mitogenic stimulation of PBMC, further illustrating a generalized and previously unappreciated alteration of HTL responses associated with chronic HBV infection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Peptides were synthesized according to standard F-moc solid phase synthesis methods 17 . Lipidated HTL-CTL peptide constructs were designed with the HTL epitope (TT830-843, OVA323-336, or PADRE) at the amino terminus. A 3-alanine spacer separated the CTL epitope from TT830-843 and OVA323-336 but was not included in the PADRE-containing lipopeptide. Each amino acid coupling was followed by a capping cycle with 10% acetic anhydride:dimethylformamide (v/v) to facilitate subsequent purification. HTL-CTL peptides were then lipidated by coupling preformed symmetrical anhydride of palmitic acid to the amino terminus of the resin-bound lys-ser-ser-elongated peptide. The peptides and the protecting groups on the amino acids were cleaved using trifluoroacetic acid, ethanedithiol, water (9.5:2.5:2.5, v/v/v) or trifluoroacetic acid, thioanisole, phenol, ethanedithiol, water (10 ml:0.5 ml:0.75 g:0.25 ml:0.5 ml) depending upon the protecting group present on the amino acids.
Unlipidated peptides were purified using conventional HPLC methods. Lipopeptides were purified by repetitive washes with 50% acetic acid:water v/v, followed by two washes with H20 and lyophilization.
The purity and identity of all compounds were checked by analytical
HPLC and mass spectrometry. Purity was 
75% for lipidated peptides
and 90% for unlipidated peptides.
Human study population
Twenty-six healthy HLA-A2 individuals who tested seronegative for markers of HBV infection as determined by standard clinical laboratory assays were enrolled in a Phase I clinical trial as described previously 16 . The phase II clinical trial population consisted of 90 chronically HBV-infected HLA-A2 patients that were positive for hepatitis B surface Ag (HBsAg), hepatitis B e Ag (HBeAg), and HBV DNA ( J. Heathcote et al., manuscript in preparation). Both cohorts were immunized with the experimental therapeutic vaccine, Theradigm-HBV, which consists of a lipopeptide containing the TT830-843 HTL epitope covalently linked to the HBcAg 18–27 CTL epitope and palmitolylated at the N-terminus. Normal subjects received doses of either 0.05, 0.5, or 5 mg Theradigm-HBV; HBV patients received doses of either 0.05, 0.5, 5, 10, or 15 mg Theradigm-HBV. Subjects were immunized at 6-wk intervals and received up to four injections of Theradigm-HBV. Blood was drawn the second and fifth week following each injection. During the course of Theradigm-HBV immunization, HBV patients did not receive any other antiviral or immunomodulatory therapy.
Mice
B6D2F1 were obtained from the Jackson Laboratories (Bar Harbor, ME); 8- to 12-wk-old mice were used in all experiments. The HBV transgenic mice used in this study have been described previously 4, 5, 6, 7 . The 1.3.32 lineage of HBV transgenic mice transcribe the 3.5- and 2.1-kb HBV RNAs in their hepatocytes and express both HBeAg and HBsAg as well as replicative HBV DNA intermediates and complete virus particles. Mice from the lineage 1.3.32 were extensively backcrossed onto the C57BL/6 parental background and mated with BALB/c ByJ mice to generate H-2bxd F1 hybrids that have the appropriate genetic background necessary for recognition by H-2d-restricted, HBsAg-specific CTLs.
T cell proliferation assays
Human T cell proliferation assays were performed as described previously for the characterization of responses normal to volunteers immunized with Theradigm-HBV 11, 16 . Briefly, human PBMC were cultured at 1.5 x 105 cells/well in a 96-well flat-bottom plate in replicates of four to six wells for each condition. TT830-843 was added at 10 µg/ml, whole TT (Connaught, Swiftwater, PA) was added at 0.5 µg/ml, or PHA (Murex Biotech Limited, Dartford, England) was added at 4 µg/ml final concentration. After 7 days, 1 µCi of [3H]TdR (ICN, Irvine, CA) was added to each well of the PHA cultures and incubated for an additional 18 h. Human cultures stimulated with TT or TT830-843 were fed after 7 days with 100 µl of medium containing IL-2 at a final concentration of 10 U/ml. After an additional 2 days in culture, 1 µCi of [3H]TdR was added to each well and harvested after 18 h.
Proliferative responses in mice were determined as described previously 18 . Briefly, 1 x 106 murine splenocytes/well were plated in 96-well microtiter tissue culture plates in the presence of a log-dose peptide titration (100–0.01 µg/ml) of either PADRE or OVA323-336 peptides. After three days, 1 µCi of [3H]TdR was added to each well and incubated for an additional 18 h. Cells from both human and mouse assays were harvested onto glass filters (cell harvester 1295–001, LKB Wallac, Gaithersburg, MD), and [3H]TdR incorporation was measured (ß plate counter 1205, LKB Wallac).
Cytokine secretion assays
Frozen human PBMC samples were thawed in RPMI 1640, 2% human serum (Gemini Bioproducts, Calabasas, CA) containing 30 µg/ml DNase (Calbiochem, La Jolla, CA) and washed two times. Cells were resuspended in RPMI 1640 with 10% human serum and cultured at 1.5 x 106 cells/well in a 24-well plate. Cultures were stimulated with either 10 µg/ml TT830-843 or 4 µg/ml PHA. The cultures were incubated for 3 days, after which the supernatants were collected and stored at -80°C for subsequent analysis. Human cytokines were quantitated with human cytokine kits (Endogen, Woburn, MA) according to manufacturers’ protocols. The IL-12 detection kit recognizes both the p70 and p40 forms of IL-12.
For cytokine assays using murine cells, splenocytes from individual mice were treated to remove the RBC with RBC lysing buffer (Sigma, St. Louis, MO) and washed twice in RPMI, 10% FCS. Splenocytes were cultured at 4 x 106 splenocytes/well in a 24-well plate. Cultures were either stimulated with either 10 µg/ml of PADRE or OVA323-336. Culture supernatants were collected after 3 days. Cytokine secretion was measured using Endogen ELISA reagents (Endogen) according to manufacturers’ protocols.
Assays for murine CTL activity
Eleven to 14 days after immunization, 3 x 107 splenocytes, harvested from individual mice, were stimulated with 10.0 µg/ml HBV surface 28–39 peptide in upright 25 cm2 flasks containing 10 ml RPMI 1640 (Life Technologies, Grand Island, NY)-10% FCS. After 6 days, splenocytes from each flask were collected and assayed for cytolytic activity using a standard 4-h 51Cr-release assay using peptide-loaded P815 mastocytoma cells originally obtained from DBA/2 mice 19 . Target cells (3 x 106) were routinely labeled with 300 µCi 51Cr-sodium chromate (NEN Research Products, Boston, MA) for 60 min at 37°C, in the absence or presence of 2 µg/ml of the HBV surface 28–39 peptide. The percent release data was transformed into 30% lytic units (LU)/106 cells (LU30) 11 to more readily compare CTL responses observed in different samples. One LU corresponds to the number of effector cells required to induce 30% lysis of 104 51Cr-labeled target cells during a 4-h assay. Specific CTL activity is obtained by subtracting the LU30 obtained in the absence of the Ag from the LU30 obtained in the presence of Ag.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To characterize immune responses to Theradigm-HBV, PBMC from
chronically infected individuals were examined for their capacity to
proliferate in response to the universal HTL epitope, TT830-843, which
is a component of Theradigm-HBV. Proliferative responses were evaluated
both in terms of frequency of responding individuals (average
stimulation index (SI) 10) as well as magnitudes of the positive
responses. The results of this analysis are shown in Fig. 1
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A). In the 0.05-mg
dose group, 
10% of the individuals responded to the HTL epitope. As
dosing was increased, responder frequency increased to 60% in the
10-mg dose group. In contrast, responder frequency was reduced in the
15-mg dose group; this observation may be a consequence of the small
sample size or might reflect the induction of tolerance at the highest
dose tested. As in the case of responder frequencies, the magnitude of
the TT830-843-specific proliferative responses in the HBV patients was
found to be similar to the magnitude of responses observed in normal
subjects (Fig. 1B). Relationship between HTL and CTL responses
The results of previous clinical studies in normal subjects
suggested that HTL responses are important in the development of CTL
responses to Theradigm-HBV 11, 16 . To determine whether this was
also the case in patients chronically infected with HBV, the
correlation between HTL and CTL responses was analyzed. As observed in
normal subjects, HBV patients that developed HTL responses to TT830-843
were more likely to also develop HBcAg 18–27-specific CTL responses
(Table I). This data indicates that in
individuals chronically infected with HBV, the development of an HTL
response following Theradigm-HBV immunization is strongly associated
with the development of a CTL response.
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Cytokine profile of specific HTL responses in normal and chronically infected Theradigm-HBV recipients
To investigate these issues further, we characterized the cytokine profile elaborated in response to stimulation with the TT830-843 peptide. Preliminary experiments determined that lymphokine levels in the culture supernatants of both normal subjects and chronically infected HBV patients peaked after 3 days (data not shown). Based on these results, PBMC from normal subjects and chronically infected Theradigm-HBV recipients were stimulated for 3 days with TT830-843 peptide, culture supernatants were then collected, and cytokine secretion was measured by ELISA. Only patients who demonstrated a positive proliferative response were selected for this study because preliminary experiments indicated the patients who failed to mount a proliferative response also failed to secrete any of the cytokines under study (data not shown). Normal HTL responders were also tested as a basis of comparison.
Very similar levels of IL-2 secretion were observed in both populations
(Fig. 2). In contrast, secretion of the
Th1-associated cytokines IL-12 and IFN- were considerably reduced in
comparison to normal subjects (Fig. 2). Secretion of the Th2-associated
cytokine IL-5 appeared to be elevated, although not significantly (Fig. 2). Similar trends were observed when supernatants were collected at
either earlier or later time points, suggesting that the differences
were not simply due to an alteration in the kinetics of lymphokine
production (data not shown).
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Decreased preimmunization proliferative responses to the recall Ag TT are associated with hyporesponsiveness to Theradigm-HBV immunization
Next, we examined whether these differences were limited to
responses to the Theradigm-HBV immunogen or if a more general
alteration in the HTL function might be detectable. To this end, we
examined the proliferative responses of PBMC derived from normal
subjects and HBV patients following stimulation with TT protein. We
found that in comparison to PBMC from normal subjects, PBMC from
chronically infected HBV patients proliferate less frequently to TT
stimulation (Table II). In
addition, the magnitude of the proliferative responses also appeared to
be somewhat reduced. Taken together, these results suggested a general
alteration of HTL immune function in chronically infected HBV patients.
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). Taken together, these data suggest
that a pre-existing nonspecific alteration of HTL function maybe
related to the hyporesponsiveness of chronically infected HBV patients
to Theradigm-HBV immunization.
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Similar experiments were performed using PHA as a mitogenic
stimulus. PBMC derived from HBV patients exhibited the same altered
cytokine profile following 3 days of PHA stimulation as observed
following TT830-843 stimulation (Fig. 3).
While IL-2 secretion from PBMC of chronically infected individuals was
similar that observed from PBMC of normal subjects, IL-12 and IFN-
secretion were markedly reduced. In addition, a significant increase in
secretion of IL-5 was also detected. To determine whether the altered
pattern cytokine secretion was generally related to liver disease,
similar experiments were performed using PBMC isolated from HCV
patients. The altered cytokine pattern characterized in HBV patients
was not evident in HCV patients. In contrast to HBV patients, HCV
patients secreted normal amounts of IL-5 and IL-12 (data not shown).
These data confirm that chronic HBV infection is associated with an
altered balance in the secretion of Th1/Th2 lymphokines. Furthermore,
these data demonstrate that this phenomenon is not limited to
Theradigm-HBV-specific responses but extends to the bulk of the various
T cells responsive to PHA stimulation.
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The data presented above suggest that the use of a more potent HTL epitope might lead to increased effectiveness of lipopeptides, especially in regard to their capacity to overcome T cell tolerance at the CTL level. To examine the relative immunogenicity of constructs containing the different HTL epitopes, we used the HBV transgenic mouse strain, 1.3.32. These mice are transgenic for the entire HBV genome, express high levels of viral Ags, and have been shown to be an useful animal model for studying the role the immune response plays in controlling HBV infection 4, 5, 6, 7 . These mice were immunized with lipopeptides that contained the dominant Ld-restricted HBV surface 28–39 CTL epitope 20 and one of two different HTL epitopes. One construct incorporated the naturally occurring OVA323-336 HTL epitope 18 . The other construct incorporated the nonnatural PADRE HTL epitope, which has been optimized for HTL function. PADRE is a synthetic HTL epitope engineered by introducing anchor residues for different DR motifs into a polyalanine backbone. The resulting peptide binds a variety of DR molecules as well as certain mouse class II alleles, including I-Ab, I-Ed, and I-Ek 18 .
As shown in Fig. 4, the PADRE-containing
lipopeptide elicited more vigorous responses in terms of proliferation
(Fig. 4A) and IL-2 secretion (Fig. 4B) than did
the lipopeptide containing the OVA-derived HTL epitope. Little
difference was noted in comparing the IL-2 production and proliferative
responses obtained with 1.3.32 mice and control mice.
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secretion was somewhat
reduced in comparison to control mice immunized with the same construct
(Fig. 4C). In contrast, immunization with the
PADRE-containing lipopeptide induced higher levels of IFN- release
in both strains of mice. PADRE was in fact found to induce higher
levels of IFN- secretion in cultures derived from 1.3.32 transgenic
mice than in cultures from control mice (Fig. 4C). In
conclusion, these results suggest that the PADRE lipopeptide induced a
more vigorous and characteristically Th1-like (as judged from IFN-
release) response than the lipopeptide incorporating the conventional
murine OVA323-336 HTL epitope. Lipopeptides incorporating the PADRE HTL epitope break tolerance at the CTL level
Next, the CTL responses to HBV surface 28-39 peptide
(HBVenv28) induced in either 1.3.32 mice or control
mice by injection with the OVA323-336 or PADRE-containing
lipopeptides were evaluated. The results are shown in Fig. 5. In the case of the
OVA323-336-containing lipopeptide, 90% (9 of 10) of the normal mice
responded with an average CTL response of 10.3 LU. As expected, 1.3.32
mice responded poorly, likely because of tolerance at the CTL level.
Specifically, only 30% (3 of 10) of the 1.3.32 mice responded with an
average CTL response of 4.3 LU. More vigorous responses were induced
using the lipopeptide construct incorporating the PADRE HTL epitope.
All of the control mice (10 of 10) immunized with this construct
responded with average CTL responses of 34.8 LU. More strikingly,
vigorous responses were also seen in 1.3.32 mice; 93% (13 of 14) of
the 1.3.32 mice exhibited CTL responses averaging 28 LU. Furthermore,
the CTL induced in 1.3.32 mice using the PADRE-containing construct
were capable of recognizing P815 cells that endogenously express the
HBsAg (data not shown). These results demonstrate that PADRE constructs
can be used to break tolerance at the CTL level.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present data has at least two important implications: 1) with regard to the role of HTL in support of CTL generation as well as for the pathogenesis of chronic HBV infection, and 2) in regards to the development of therapeutic vaccines for chronic viral infection and cancer.
The data presented here illustrate a generalized and previously
unappreciated alteration in HTL function associated with chronic HBV
infection. Secretion of the Th1-associated lymphokines IFN- and
IL-12 appears to be impaired in favor of increased production of
the Th0/Th2-associated lymphokine IL-5. This qualitative alteration of
HTL responses does not appear to be Ag specific as it is detected after
mitogenic stimulation of PBMC. Several immunological parameters were
normal including proliferation, IL-2 secretion, and CD4/CD8 ratios and
the recall CTL response to the dominant influenza matrix peptide 58–66
(data not shown), consistent with the lack of clinical signs of general
immune suppression in patients affected by chronic HBV infection.
Our data are consistent with studies that analyzed HBV-specific HTL
function 21 . Bertoletti and colleagues reported that in chronic HBV
infection the majority of the liver infiltrating T cells are of Th0
type and are capable of secreting both IFN- and IL-5. In contrast,
in acute self-limiting hepatitis T cells appear to be predominantly of
the Th1 type 22 .
High levels of circulating viral Ags might favor the induction of Ag-specific tolerance and/or shifts in the balance of Th1/Th2 responses. Differential engagement of Th1 vs Th2 responses have been addressed in studies by Milich 23 , which suggest that Th1 and Th2 subsets of T cells might differ in their sensitivity to tolerance induction 24, 25, 26 . Dendritic cells can be conditioned by IL-10 to prime preferentially for Th2 responses 27, 28, 29, 30 . Therefore, a bias toward the Th2 phenotype could be initially established in HBV-specific responses as a result of high levels of circulating Ags but eventually lead to a nonspecific Th2 bias resulting from altered APC function 31 . This hypothesis would be consistent with the important role IL-12 plays in the clearance of HBV infection 32 . Alterations of the HTL response described herein might not be limited to chronic HBV infection and could be associated with other chronic viral diseases and cancer 33, 34, 35 , for which alterations of the immune responsiveness have also been described.
The experiments performed in HBV transgenic mice demonstrate that immunization with lipopeptides containing the mouse Ld-restricted HBV surface 28–39 CTL epitope, and a potent HTL epitope can break tolerance at the level of HBV-specific CTL responses. This data should be interpreted with the knowledge that the transgenic mice may only incompletely mimic the disease pathogenesis in humans. HBV transgenic mice do not display the altered lymphokine pattern in response to mitogen that we observed in infected humans, and the CTL specificities elicited in transgenic mice are low affinity and consequently have limited histological impact (data not shown). Despite these caveats, the data nonetheless suggest that use of potent T cell help that elicits a more markedly Th1-type lymphokine profile may play an important role in overcoming nonresponsiveness at the CTL level.
Finally, the results presented in this report have implications in
terms of the further development of immunotherapy for chronic HBV
infection. Based on our results, the combination of Theradigm-HBV with
other nonspecific immunostimulants such as IFN- or IL-12, which
might reverse the HTL alteration, should be considered. Alternatively,
combination therapy with antiviral compounds such as acyclovir 36 or
lamivudine 37 , which might remove these suppressive effects by
lowering the viral load, also represents an attractive clinical
strategy. Recent studies have shown that lamivudine treatment can
restore T cell responsiveness in patients with chronic HBV 38 . Lastly
the potent PADRE HTL epitope, which is considerably more immunogenic in
human T cells than the TT830-843 epitope, should also be included in
the next generation of epitope-based HBV immunotherapeutic.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: HBV, hepatitis B virus; HTL helper T lymphocyte; HBcAg, hepatitis B core Ag; TT, tetanus toxoid; DR, HLA-DR; PADRE, Pan DR epitope; HBeAg, hepatitis B e Ag; HBsAg, hepatitis B surface Ag; LU, lytic unit; SI, stimulation index.
Received for publication July 14, 1998. Accepted for publication November 18, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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/ß downregulate hepatitis B virus gene expression in vivo by tumor necrosis factor dependent and independent pathways. J. Virol. 68:1265. negatively regulates hepatitis B virus gene expression in transgenic mice. J. Virol. 66:3955. globulins in adult mice induces antigen-specific unresponsiveness of T helper type 1 but not type 2 lymphocytes. J. Exp. Med. 175:9. producing (Th1) cells. J. Immunol. 148:3687.[Abstract]
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cpm); this is defined as the incorporation of
[3H]TdR in the presence of Ag minus the incorporation of
[3H]TdR in the absence of Ag. Average responses from four
individual B6D2F1 or 1.3.32 transgenic mice are presented.
B and C, The IL-2 and IFN-