Measles in Immunized Infants , and T Cell Proliferation to Γ IL-12, IFN

IL-12, IFN-γ, and T Cell Proliferation to Measles in Immunized Infants Hayley A. Gans, Yvonne Maldonado, Linda L. Yasukawa, Judy Beeler, Susette Audet, Mary M. Rinki, Ross DeHovitz This information is current as and Ann M. Arvin of September 28, 2021. J Immunol 1999; 162:5569-5575; ; http://www.jimmunol.org/content/162/9/5569 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. IL-12, IFN-␥, and T Cell Proliferation to Measles in Immunized Infants1

Hayley A. Gans,2* Yvonne Maldonado,* Linda L. Yasukawa,* Judy Beeler,† Susette Audet,† Mary M. Rinki,* Ross DeHovitz,‡ and Ann M. Arvin*

Measles infection in infants is associated with severe complications, and secondary infections are attributed to generalized immunosuppression. Measles binding to its monocyte receptor down-regulates IL-12 which is expected to diminish Th1-like cytokine responses, including IFN-␥. Whether young infants can be immunized effectively against measles is an important public health of age (56 ؍ or 12 mo (n ,(46 ؍ n) 9 ,(60 ؍ issue. We evaluated Ag-specific IL-12, IFN-␥, and T cell responses of infants at 6 (n and 29 vaccinated adults. IL-12 and IFN-␥ release by PBMC stimulated with measles Ag increased significantly after measles immunization in infants. IL-12 and IFN-␥ concentrations were equivalent in younger and older infants, but IL-12 concentrations IL-12 production by monocytes was down-regulated by measles; the Downloaded from .(0.04 ؍ were significantly lower in infants than in adults (p addition of recombinant human IL-12 enhanced IFN-␥ production by PBMC stimulated with measles Ag, but infant T cells released significantly less IFN-␥ than adult T cells under this condition. Of particular interest, the presence of passive Abs to measles had no effect on the specific T cell proliferation or IFN-␥ production after measles stimulation. Cellular immunity to measles infection and vaccination may be limited in infants compared with adults as a result of less effective IFN-␥ and IL-12 production in response to measles Ags. These effects were not exaggerated in younger infants compared with effects in infants who were immunized at 12 mo. In summary, infant T cells were primed with measles Ag despite the presence of passive Abs, but their http://www.jimmunol.org/ adaptive immune responses were limited compared with those of adults. The Journal of Immunology, 1999, 162: 5569–5575.

easles is a leading cause of infant morbidity and mor- sles binding to CD46 results in down-regulation of IL-12 pro- tality in many countries (1–3). In recent outbreaks in duction. Based on the Th1/Th2 model of CD4ϩ T cell re- M the United States, the incidence and mortality rates of sponses, decreased IL-12 release would be expected to favor measles were highest among children Ͻ12 mo old (4). Infants are predominance of a Th2-like response, because IL-12 is a crucial at risk for measles pneumonia, suggesting an inadequate capacity early stimulus for Th1 clonal expansion. IL-12 stimulates rest- to limit viral dissemination, and are susceptible to secondary in- ␥ ing and activated T cells, induces the production of IFN- by T by guest on September 28, 2021 fections, indicating that measles induces a generalized immuno- cells, activates NK cells, and inhibits IL-4 production (25–31). suppression (1, 3, 5–7). Host responses assessed during and after Since IL-12 promotes the rapid induction of Ag-specific T cells measles infection, or immunization with live attenuated measles in the naive host, decreased IL-12 production could impair the vaccines, exhibit immunologic patterns consistent with a dimin- acquisition of adaptive immunity to measles and facilitate a ished Th1 response or a polarization toward Th2 activation (5, 6, generalized immunosuppression by failure to regulate IL-4 pro- 8–12). Delayed type hypersensitivity reactions to other Ags, such duction. The purpose of this study was to compare virus-spe- as Mycobacterium tuberculosis, mitogen-stimulated T cell prolif- cific T cell responses in infants and adults with vaccine-induced ␥ eration, and release of IL-2 and IFN- are diminished, and NK cell measles immunity, using assays for IL-12 production, T cell function is reduced (9–11, 13–19). In addition, nonspecific, spon- proliferation, and IFN-␥ release by PBMC stimulated with mea- taneous release of IL4 by circulating PBMC and total IgE concen- sles. Our hypothesis was that infants might have a limited Th1- trations are increased (11, 20). like response to measles compared with adults. Measles virus infects monocytes through binding to the The evaluation of adaptive immunity to measles vaccination CD46 surface protein, which acts as a measles-specific entry in infants who are younger than 12 mo has important clinical mediator (21–23). Karp et al. (24) have demonstrated that mea- relevance because it would be advantageous to achieve protective immunity as early as possible during the first year of life. Potential obstacles to immunization of younger infants against *Infectious Diseases Division, Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305; †Department of Health and Human Services, Di- measles include neutralization of the live attenuated vaccine vision of Viral Products, Office of Vaccine Research and Review, Center for Biolog- virus by measles Abs acquired transplacentally and immaturity ics Evaluation and Research, Food and Drug Administration, Rockville, MD 20867; of the immune system (32–39). We and others have shown that and ‡Department of Pediatrics, Palo Alto Medical Foundation, Palo Alto, CA 94301 passive Abs to measles interfere with vaccine-induced humoral Received for publication October 16, 1998. Accepted for publication February 9, 1999. immunity, but little is known about the effects of maternally The costs of publication of this article were defrayed in part by the payment of page derived measles Abs on induction of virus-specific cell-medi- charges. This article must therefore be hereby marked advertisement in accordance ated immunity (40–43). Infants whose mothers have measles with 18 U.S.C. Section 1734 solely to indicate this fact. vaccine-induced immunity lose passive Abs at a shorter interval 1 This work was supported by Grant AI37127 from the National Institute of Allergy after birth than those who receive higher concentrations of mea- and Infectious Diseases. sles Abs because their mothers have had natural measles (37, 2 Address correspondence and reprint requests to Dr. Hayley Gans, Stanford Univer- sity Medical Center 300 Pasteur Drive G312, Stanford, CA 94305-5208. E-mail ad- 42–46). In a recent study, we showed that the humoral response dress: [email protected] to measles vaccine was deficient in 6-mo-old infants compared

Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00 5570 IL-12, IFN-␥, AND T CELL PROLIFERATION TO MEASLES with 9 and 12-mo-old infants even when passively acquired Abs Cytokine production were not detectable (41). This further evaluation of measles- Supernatants from PBMC stimulated with measles Ag or uninfected cell specific T cell responses in infants was undertaken to assess control were collected from wells on days 4–8, stored at Ϫ70°C. Super- whether there are differences in proliferation, IL-12 or IFN-␥ natants were tested for the p40 and p70 subunit of human IL-12 using the responses when younger infants are immunized with measles ELISA ultrasensitive assay (Biosource, Camarillo, CA) and for IFN-␥ us- vaccine, and whether passive Abs affect these cell-mediated ing the ELISA method from Endogen (Cambridge, MA). Supernatants from PBMC stimulated with measles Ag or uninfected cell control, in the immune responses. presence or absence of rhIL-12 (added at 50 or 100 U/ml or 2.9 and 5.8 ng/ml, respectively), were collected on days 4–8, stored at Ϫ70°C, and tested for IFN-␥. The optimal concentrations of rhIL-12 were established Materials and Methods in preliminary assays and used for all subsequent tests; peak values from Study population either concentration were used for statistical analysis. Sensitivities of cytokine detection were defined by reference standards in each assay. The study subjects were 162 healthy infants, without intercurrent illnesses, who were 6 mo (n ϭ 60),9mo(n ϭ 46), or 12 mo old (n ϭ 56) and healthy Monocyte isolation and stimulation adults (n ϭ 29; age 20–40 yr). Infants were enrolled if they were 6 mo (range, ϩ3 wk), 9 mo (range, Ϯ3 wk), or 12 mo (range, ϩ3 wk). Children Monocytes were isolated by density gradient centrifugation from whole born before 36 wk gestation, whose birth weight was Ͻ2500 g, or who had blood from adult subjects and incubated with measles at a multiplicity of chronic underlying illnesses were excluded. Among the infants who were infection of 5, or with an uninfected Vero cell control, under nonadherent enrolled, 2 infants were withdrawn as participants before blood samples conditions. Monocytes were then enriched by adherence to culture flasks ϫ 5 were obtained and 26 infants were evaluated only before measles immu- and added to 96-well plates at concentrations of 2 10 /well in RPMI 1640 (Life Technologies), and 10% normal human sera (Sigma). Staphy- nization, including 7 who were 6 mo old, 4 who were 9 mo old, and 15 who Downloaded from were 12 mo old. Prevaccine specimens as well as samples taken 12 Ϯ 3wk lococcus aureus Cowan strain 1, 0.0075% (Calbiochem, La Jolla, CA), was after measles vaccination were available from 134 infants; because of lim- added to monocyte cultures after 60 h; after 24 h, supernatants were col- itations in the numbers of PBMC recovered, not all assays were performed lected and tested for the p40 and p70 subunit of human IL-12 using the in all infants. The study was approved by the Stanford University Com- ELISA ultrasensitive assay (Biosource). All cell reagents were LPS free to mittee for the Protection of Human Subjects and the Institutional Review the limits of detection of the Limulus amebocyte lysate pyrogen kit (1–2.3 Board of the Palo Alto Medical Foundation; written consent was obtained pg/ml) (BioWhittaker, Walkersville, MD). from parents or guardians and adult participants. No cases of measles were identified in our geographic area during the study period. Measles Ab assays http://www.jimmunol.org/ Infants 6 and 9 mo old were immunized with measles virus vaccine live Sera were obtained from infant participants on the day that vaccine was (Attenuvax (Merck, West Point, PA)) containing 1000 median tissue cul- given to determine whether passive Abs were present at the time of im- ture-infective doses (TCID50) of the U.S. reference measles virus. Twelve- munization. Sera were stored at Ϫ80°C and tested for measles-neutralizing month-old infants were immunized with M-M-R virus vaccine live (Merck; Ab using a modified plaque reduction neutralization (PRN) assay (47). The containing measles virus vaccine live, 1000 TCID50). Adults had received PRN assay was used because of its superiority in detection of low titers of at least one measles vaccination years before evaluation. measles Abs compared with commercial ELISA methods (48). Briefly, serial fourfold dilutions of heat-inactivated serum (1:4–1:4096) were mixed with an equal volume of a low passage strain of Edmonston measles T cell proliferation assay virus containing 25–35 PFU. Each serum dilution was incubated in dupli-

PBMC were separated from whole blood by Ficoll-Hypaque gradient and cate in 24-well plates with Vero cell monolayers for 1 h and 45 min at 36°C by guest on September 28, 2021 5 added to 96-well microtiter plates at concentrations of 3.0 ϫ 10 /well in in 5% CO2. The PRN titer was defined as the serum dilution that reduced RPMI 1640 (Life Technologies, Gaithersburg, MD), and 10% normal hu- the plaque number by 50%. Titers Ͻ1:4 were considered negative and were man sera (Sigma, St. Louis, MO). Measles Ag, prepared from lysates of assigned a value of 1 for statistical analysis. Seroconversion was defined as Vero cells inoculated with Attenuvax measles vaccine (more attenuated a fourfold rise in Ab titer after levels before vaccination were corrected for Enders’ strain, Merck) or an uninfected cell control were added at dilutions decay over three half-lives. of 1:16 and 1:32 to triplicate wells for testing infant PBMC and in qua- druplicate wells for adults. Adult PBMC were also incubated with Ag and Statistical analysis control at a 1:64 dilution. Preliminary studies were performed with mul- Comparison of T cell and cytokine responses in the same patient were tiple measles Ag dilutions (range, 1:16–1:512); dilutions of 1:16 and 1:32 evaluated by Student’s paired t test, whereas comparisons between patients stimulated T cells from all positive donors. The highest stimulation index 3 were analyzed using a Student’s unpaired t test. ANOVA was performed to (SI) from either concentration was used for statistical analysis, since sub- discern differences in the means between more than two groups. The ␹2 and jects respond to Ag concentrations differently. The measles Ag used in Fisher exact tests were used to compare the number of vaccinees in each cohort these studies was not inactivated because pilot experiments documented no with proliferation responses. Values of p Յ 0.05 were considered significant. differences when T cells were stimulated with live or inactivated measles Ag. Recombinant human IL-12 (rhIL-12), with a bioactivity of 7.7 ϫ 107 U/ml, was generously provided by Genetics Institute (Cambridge, MA). In Results some experiments, rhIL-12 was added to PBMC stimulated with measles Measles-specific T cell proliferation responses in relation to age Ag or uninfected cell control at 20, 40, and 60 U/ml or 1.2, 2.4, and 3.5 at immunization ng/ml, respectively. Preliminary studies were performed with multiple concentrations of rhIL-12, based on published experience and consultation T cell proliferation after stimulation of PBMC with measles Ag with the manufacturer; additional concentrations above and below these was evaluated in 119 infants who were 6 mo (n ϭ 49), 9 mo (n ϭ standards were also tested. The optimal rhIL-12 concentrations were used 36), or 12 mo (n ϭ 34) old and in 29 adults (Fig. 1A). T cell for subsequent assays. T cell proliferation was measured by adding 3 ␮ responses to measles were detected after immunization in all of the [ H]thymidine after 5 days (1.25 Ci/ml) for 6–18 h. The SI was calcu- Ϯ Ϯ lated as the mean cpm in measles Ag-stimulated wells divided by the mean infant age groups; the mean SI SE increased from 2.0 0.2 to cpm in control wells. The highest SI from either concentration of measles 6.1 Ϯ 0.8 in 6-mo-old ( p ϭ 0.001), from 2.1 Ϯ 0.3 to 6.6 Ϯ 1.6 Ag in the presence or absence of rhIL-12 was used for statistical analysis. in 9-mo-old ( p ϭ 0.01), and from 1.4 Ϯ 0.1 to 5.6 Ϯ 0.9 in ϩ An SI of 3.0 or higher was considered positive, based on the mean SI SD 12-mo-old ( p ϭ 0.0002) infants. The mean SI after vaccination did of responses in infants before measles vaccination. The mean cpm in back- ground wells were compared between infant groups to assure that no sig- not differ by age cohort. In addition, there were no age-related nificant age-related differences existed that might influence SI results. differences among the infants when the percentage who had de- PBMC were also stimulated with PHA, 0.01 mg/ml (Difco, Detroit, MI). tectable T cell proliferation to measles, defined as SI Ͼ 3.0, was compared. Responses were detected in 71% of 6-mo-old, 69% of 9-mo-old, and 62% of 12-mo-old infants. The percentage of adults Ͼ 3 Abbreviations used in this paper: SI, stimulation index; rhIL-12, recombinant human with SI 3.0 was 86%, which was not significantly higher than IL-12; PRN, plaque reduction neutralization. the 68% in infants. However, the mean SI in vaccinated infants The Journal of Immunology 5571

was 6.1 Ϯ 0.66, which was signiﬁcantly lower than the mean SI of 11.3 Ϯ 1.9 in adults ( p ϭ 0.002) (Fig. 1A).

IL-12 production to measles in relation to age at immunization IL-12 production elicited by measles Ag was evaluated in 67 infants, including 37 who were 6 mo old, 16 who were 9 mo, and 14 who were 12 mo old, and in 13 adults. Mean IL-12 concentrations (ϮSE) before vaccination were 6.2 Ϯ 2.4, 3.0 Ϯ 1.1, and 7.0 Ϯ 3.8 pg/ml compared with levels after vaccination of 21.3 Ϯ 7.5, 17.9 Ϯ 7.2, and 13.2 Ϯ 5.9 pg/ml, in the 6-, 9-, and 12-mo-old infants, respectively (Fig. 1B). When the infants were evaluated as one group, there was a signiﬁcant increase in IL-12 levels after compared with before measles vaccine ( p ϭ 0.01). No signiﬁcant differences were seen when IL-12 responses were compared between the infant groups but the mean IL-12 concentration after vaccination of infants was 19.6 Ϯ 4.4, which was statistically Ϯ lower than the mean IL-12 concentration of 41.4 11.7 pg/ml Downloaded from measured in adults ( p ϭ 0.04) (Fig. 1B).

IFN-␥ production to measles in relation to age at immunization IFN-␥ concentrations were measured in 65 infants who were 6 mo

(n ϭ 30),9mo(n ϭ 23), or 12 mo (n ϭ 12) old and in 13 adults. http://www.jimmunol.org/ An Ag-speciﬁc response was observed in each infant age group (Fig. 1C). IFN-␥ concentrations (ϮSE) before and after vaccination were 21.1 Ϯ 5.5 pg/ml vs 197.7 Ϯ 59.7 pg/ml ( p ϭ 0.002), 38.9 Ϯ 16 pg/ml vs 304.3 Ϯ 100.7 pg/ml ( p ϭ 0.01), and 42.6 Ϯ 13.7 pg/ml vs 110.5 Ϯ 27.8 pg/ml ( p ϭ 0.04) in the 6-, 9-, and 12-mo-old infants, respectively. No signiﬁcant differences were found when IFN-␥ concentrations were compared among the infant groups or when the mean of 213.2 Ϯ 43.4 pg/ml for all infants was compared with the mean IFN-␥ concentration of 404.7 Ϯ by guest on September 28, 2021 124.7 pg/ml measured in adults (Fig. 1C).

Effect of passive Abs to measles on measles-specific T cell proliferation and cytokine responses in infants The presence of passive Abs did not affect the frequency with which T cell proliferation to measles was elicited after vaccination of infants (Fig. 2A). Twenty-six of 42 infants who were 6 mo old had passive Abs, compared with 15 of 35 infants 9 mo old and 0 of 36 infants 12 mo old. No significant differences were detected when the mean SI of infants with passive Abs within each age cohort was compared with the mean SI in infants with no detectable Abs. When data for all age groups were combined, the mean SI of infants who had passive Abs was 5.7 Ϯ 0.92 compared with 5.8 Ϯ 1.1 in those who had none ( p ϭ 1.0). Cohorting 6- and 9-mo-old infants according to titer of passive Ab present before vaccination, at both 1:25 and 1:80, did not correlate with decreas- ing T cell responses, as has been described with humoral immune FIGURE 1. T cell proliferation, IL-12, and IFN-␥ production before and responses in infants (49, 50). 12 wk after measles immunization. A, SI (calculated as the mean cpm in Passive Abs had no adverse effect on measles-induced IL-12 measles Ag-stimulated wells divided by the mean cpm in control wells) to release by PBMC after vaccination of infants (Fig. 2B). The mean measles Ag before (f) and 12 wk after (o) measles vaccination in infants IL-12 concentration was 29.2 Ϯ 9.5 in 17 infants who had passive who were 6, 9, or 12 mo of age at time of immunization and in vaccinated Abs compared with 9.2 Ϯ 1.9 among 22 infants who had none adults. Error bars represent SEs. A positive SI is defined as 3 or greater. B, ( p ϭ 0.03). f o IL-12 concentration (pg/ml) before ( ) and 12 wk after ( ) measles im- Passive Abs had no significant effect on IFN-␥ production in munization in infants and in vaccinated adults. Infants were 6, 9, or 12 mo of age at time of immunization. Error bars represent SEs. C, IFN-␥ con- response to measles Ag after immunization (Fig. 2C). Of 16 in- ␥ centration (picograms/ml) before (f) and 12 wk after (o) measles immu- fants who had passive Abs, the mean IFN- concentration was nization in infants and in vaccinated adults. Infants were 6, 9, or 12 mo of 105.9 Ϯ 36.5 compared with 235.1 Ϯ 62.6 among 32 infants who age at time of immunization. Error bars represent SEs. had none ( p ϭ 0.2). 5572 IL-12, IFN-␥, AND T CELL PROLIFERATION TO MEASLES Downloaded from FIGURE 3. T cell proliferative responses to PHA of infants before and 12 wk after measles immunization and of vaccinated adults. Shown are the mean cpm after stimulation with PHA (0.01 mg/ml) in infants before (f) and 12 wk after (o) measles immunization and vaccinated adults. Infants were 6, 9, or 12 mo of age at time of vaccination. Error bars represent SEs. http://www.jimmunol.org/

Effect of measles immunization on mitogen-induced T cell proliferation in relation to age Infants were tested for T cell proliferation to PHA responses just before and 3 mo after vaccination. The mean cpm Ϯ SE in PHA- stimulated cultures of PBMC were not statistically different among the infant groups or when measured before immunization and 3 mo later (data not shown). The mean cpm for all infants tested after immunization was 43,500 Ϯ 2,900, which was signiﬁcantly lower by guest on September 28, 2021 than the mean cpm of 58,000 Ϯ 6,700 observed in adults ( p ϭ 0.03) (Fig. 3).

Measles virus-induced suppression of IL-12 production by monocytes The experiment described by Karp et al. was repeated to conﬁrm that the measles virus used to prepare measles Ag for T cell proliferation and cytokine assays had the capacity to suppress IL-12 production by monocytes to concentrations equivalent to those measured in control wells. Furthermore, addition of S. aureus Cowan strain 1 stimulated IL-12 production in control Vero cell cultures but had no effect on human monocytes cultured with measles Ag (data not shown).

Effects of rhIL-12 on measles-speciﬁc T cell proliferation and IFN-␥ production T cell proliferation was measured in 11 vaccinated adults after stimulation with measles Ag alone and with the addition of rhIL- 12. All the adults had a positive SI to measles (SI Ͼ 3), with a mean SI Ϯ SE of 14.1 Ϯ 2.9. No difference in measles-induced T FIGURE 2. T cell proliferation and IL-12 and IFN-␥ production in the cell proliferation was demonstrated when rhIL-12 was added. The Ϯ ϭ presence and absence of passive Abs. A, SI to measles Ag 12 wk after mean SI was 7.4 1.5 ( p 0.06). measles vaccination of infants in the presence (f) and absence (o)of IFN-␥ release by T cells from 6 adults was measured at Days 1, passive Abs. Infants were 6, 9, or 12 mo of age at time of immunization and 3, 5, and 7 after incubation with measles Ag alone, measles and represent the same infant group as reported in Fig. 1. B, IL-12 concentra- rhIL-12, or rhIL-12 alone. IFN-␥ concentrations were higher when tion (picograms/ml) in infants 12 wk after measles immunization in the PBMC were stimulated with measles in the presence of rhIL-12 presence (f) and absence (o) of passive Abs. C, IFN-␥ concentration compared with measles Ag alone ( p ϭ 0.02) or rhIL-12 alone (picograms/ml) in infants 12 wk after measles immunization in the pres- ( p ϭ 0.0003) (Fig. 4). The peak difference was observed on Day f o ence ( ) and absence ( ) of passive Abs. 7, with mean IFN-␥ concentrations of 1634.12 Ϯ 196.0 in the measles-stimulated wells with rhIL-12 added. The Journal of Immunology 5573

responses seen in neonates may extend beyond the first year, but when immune maturation takes place is not known and is likely to vary depending on the antigenic stimulus (33, 55, 56). Animal experiments indicate that newborn responses to Ags are associated with diminished levels of TNF-␣ and IFN-␥ production and are shifted toward a Th2-type cytokine pattern (34, 35). Since measles infection and measles vaccine have been associated with sponta- neous IL-4 release by circulating PBMC and other Th2-like responses, immunization of young infants could enhance their predominance relative to the induction of antiviral Th1-like responses (6, 9, 10, 12). This issue is of practical importance because protection of younger infants against serious or life-threatening measles would be beneficial in geographic areas where measles re- mains endemic (1, 2). Our experiments addressed these questions with a comparative analysis of cellular immunity elicited by measles immunization of infants at 6, 9, and 12 mo of age. We found that T cell recognition of measles Ag was elicited in 71, 69, and 62%, respectively, and no age-related decreases in IL-12 or IFN-␥ ␥ FIGURE 4. IFN- production in infants before and 12 wk after measles production among younger infants were detected. However, mea- Downloaded from vaccination in the presence and absence of IL-12. Shown is the IFN-␥ sles-specific T cell proliferation and IL-12 responses of infants concentration (picograms/ml) in infants before (f) and 12 wk after (o) measles immunization in the presence and absence of IL-12 (50 or 100 were significantly lower than those of adults with vaccine-induced U/ml). Infants were 6, 9, or 12 mo of age at time of immunization. Error immunity to measles. bars represent SEs. IL-12 is critical for the induction of IFN-␥, a major Th1 T cell cytokine which is involved in the clonal expansion of Ag-specific

T cells (25–31, 57, 58). Earlier studies have shown that measles- http://www.jimmunol.org/ specific IFN-␥ release and T cell-proliferative responses are lower When 15 vaccinated infants were tested, the addition of rhIL-12 after measles infection or immunization than those induced by to PBMC stimulated with measles Ag also resulted in a significant other viruses (6, 59, 60). Diminished IL-12 production, associated increase in IFN-␥ production by T cells (Fig. 5). The mean IFN-␥ with the direct binding of measles to its monocyte receptor, may concentrations in measles-stimulated wells were 151.8 Ϯ 55.6 account for these differences (24). Our hypothesis was that infants pg/ml compared with 747.3 Ϯ 180.2 in the presence of rhIL-12 and may be particularly susceptible to the effects of low IL-12 produc- measles Ag ( p ϭ 0.01) (Fig. 5). The peak IFN-␥ concentration after Ϯ tion to measles Ag because their T cells may be inherently less rhIL-12 and measles stimulation in children was 747.3 180.2 which ␥ Ϯ efficient at IFN- gene transcription (34, 54–56, 61). First, we was significantly lower than the peak of 1634.2 196 from adult by guest on September 28, 2021 confirmed the block of IL-12 production by monocytes exposed to PBMC stimulated with measles and rhIL-12 ( p ϭ 0.01). high concentrations of measles. Second, we demonstrated that the Measles-specific humoral immune responses addition of rhIL-12 to PBMC cultures along with measles Ag was associated with a dramatic increase in IFN-␥ release by T cells Measles-neutralizing Ab titers before vaccination were 13 (95% from adults with vaccine-induced measles immunity. Finally, we confidence interval, 7–26), 4 (95% confidence interval, 2–8), and 1 (95% confidence interval, 1–1) in the 6-, 9-, and 12-mo-old infant, respectively. Twelve weeks after measles vaccination, neutralizing Ab titers rose to 76 (95% confidence interval, 37–156), 353 (95% confidence interval, 164–756), and 1023 (95% confidence interval, 756-1704) in the 6-, 9-, and 12 mo-old-infants, respectively (6 vs 9 mo, p ϭ 0.0002; 6 vs 12 mo, p ϭ 0.0001; 9 vs 12 mo, p ϭ 0.01). As previously reported, in the absence of detectable passive Abs, seroconversion and neutralizing Ab titers of 6-mo-old infants were statistically lower than those of 9- or 12-mo-old infants. There was no statistical difference in the humoral immune responses between 9- and 12-mo-old infants who lacked detectable passive Abs (data not shown).

Discussion The morbidity and mortality rates caused by infectious diseases, including measles, are highest among infants and young children, suggesting that a maturation of immune responses occurs during this developmental phase (1, 51–53). Although cell-mediated immunity is critical in controlling viral infections, information about the capacity of infants to respond to specific viruses or viral vaccines is limited. Deficiencies in Ag-induced T cell proliferation FIGURE 5. IFN-␥ production to measles over time in vaccinated adults ␥ and cytokine production, particularly IFN- , have been docu- in the presence and absence of IL-12. Shown is the IFN-␥ concentration mented in infants with herpes simplex infections during the first (picograms/ml) on odd days 1–7 in vaccinated adults in the presence of few weeks of life (52, 54). Descriptions of the age-dependent im- measles (Œ), IL-12 (f), and measles with IL-12 (50 or 100 U/ml) (F). munogenicity of certain vaccines suggests that the limited immune Error bars represent SEs. 5574 IL-12, IFN-␥, AND T CELL PROLIFERATION TO MEASLES showed that whereas IL-12 also enhanced IFN-␥ production by References infant T cells stimulated with measles Ag, the IFN-␥ concentra- 1. Aaby, P., J. Bukh, D. Kronborg, I. M. Lisse, and M. C. da Silva. 1990. Delayed tions were significantly lower than those produced by T cells from excess mortality after exposure to measles during the first six mos of life. immune adults under these conditions. Taken together, these ob- Am. J. Epidemiol. 132:211. 2. Aaby, P., J. Clements, and V. Orinda. 1991. Mortality from measles: measuring servations suggest that despite some measles-specific induction of the impact. In Expanded Programme on Immunization. World Health Organiza- IL-12 release, the quantities of IL-12 made may not be sufficient to tion, ed. World Health Organization, Geneva. induce concentrations of IFN-␥ high enough to promote the max- 3. Clements, C. J., and F. T. Cutts. 1995. The epidemiology of measles: thirty years of vaccination. Curr. Top. Microbiol. Immunol. 191:13. imal expansion of infant T cells that recognize measles Ags. The 4. Gindler, J. S., W. L. Atkinson, L. E. Markowitz, and S. S. Hutchins. 1992. increased susceptibility of infants to severe measles is likely to be Epidemiology of measles in the United States in 1989 and 1990. Pediatr. Infect. multifactorial and may be mediated in part by lower IL-12 re- Dis. J. 11:841. 5. Griffin, D. E. 1995. Immune responses during measles virus infection. Curr. Top. sponses than with adults, associated with a more limited capacity Microbiol. Immunol. 191:117. to produce IFN-␥. 6. Griffin, D. E., B. J. Ward, and L. M. Esolen. 1994. Pathogenesis of measles virus infection: an hypothesis for altered immune responses. J. Infect. Dis. 170(Suppl. An increased susceptibility to secondary infections is an impor- 1):S24. tant reason for the high rates of infant morbidity and mortality 7. Wakeham, P. F. 1978. Severe measles in Afghanistan. J. Trop. Pediatr. Environ. associated with measles (2, 3). These complications are attributed Child Health 24:87. 8. Griffin, D. 1991. Immunologic abnormalities accompanying acute and chronic to generalized immunosuppression caused by measles infection viral infections. Rev. Infect. Dis. 13(Suppl. 1):S129. (6). Immunization with live attenuated measles vaccine has been 9. Griffin, D. E., and B. J. Ward. 1993. Differential CD4 T cell activation in measles. followed by a transient decrease in mitogen-induced T cell prolif- J. Infect. Dis. 168:275. 10. Ward, B. J., R. T. Johnson, A. Vaisberg, E. Jauregui, and D. E. Griffin. 1991. Downloaded from eration for a few weeks after vaccination (16, 17). We found no Cytokine production in vitro and the lymphoproliferative defect of natural mea- suppression of proliferation to PHA when the responses of infants sles virus infection. Clin. Immunol. Immunopathol. 61:236. 11. Ward, B. J., and D. E. Griffin. 1993. Changes in cytokine production after measles tested just before immunization were compared with those mea- virus vaccination: predominant production of IL-4 suggests induction of a Th2 sured 3 mo later. There was no evidence that generalized immu- response. Clin. Immunol. Immunopathol. 67:171. nosuppression persisted for this time interval regardless of the age 12. Smedman, L., A. Joki, A. Jose da Silva, M. Troye-Blomberg, B. Aronsson, and P. Perlmann. 1994. Immunosuppression after measles vaccination. Acta Paediatr. at measles immunization; PHA responses of 6-mo-old infants were

83:164. http://www.jimmunol.org/ equivalent to those of infants who were 9 and 12 mo old. 13. Zweiman, B., D. Pappagianis, H. Maibach, and E. A. Hildreth. 1971. Effect of measles immunization on tuberculin hypersensitivity and in vitro lymphocyte Interference by passively acquired measles Abs with the immu- reactivity. Int. Arch. Allergy 40:834. nogenicity of measles vaccine has been a deterrent to immuniza- 14. Tamashiro, V. G., H. H. Perez, and D. E. Griffin. 1987. Prospective study of the tion of infants younger than 12 mo (4, 37, 42, 43, 62–64). In magnitude and duration of changes in tuberculin reactivity during uncomplicated and complicated measles. Pediatr. Infect. Dis. J. 6:451. addition to documenting the effects of passive Abs on active in- 15. Starr, H., and S. Berkovich. 1964. Effects of measles, ␥-globulin-modified mea- duction of neutralizing Abs in infants immunized at 6 and 9 mo, sles and vaccine measles on the tuberculin test. N. Engl. J. Med. 270:386. 16. Hirsch, R. 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T helper type 1 development of naive CD4 T cells requires the coordinate allow an approach to measles immunization that is more appro- action of interleukin-12 and interferon-␥ and is inhibited by transforming growth priate for the immune system of young infants. factor-␤. Eur. J. Immunol. 24:793. 28. Trinchieri, G. 1997. Cytokines acting on or secreted by macrophages during intracellular infection (IL-10, IL-12, IFN-␥). Curr. Opin. Immunol. 9:17. 29. Scharton-Kersten, T., L. C. Afonso, M. Wysocka, G. Trinchieri, and P. Scott. 1995. IL-12 is required for natural killer cell activation and subsequent T helper Acknowledgments 1 cell development in experimental leishmaniasis. J. Immunol. 154:5320. We thank the families, pediatricians, nursing staff, and laboratory staff of 30. Heufler, C., F. Koch, U. Stanzl, G. Topar, M. Wysocka, G. Trinchieri, A. Enk, R. M. Steinman, N. Romani, and G. Schuler. 1996. Interleukin-12 is produced by the Palo Alto Medical Foundation for their assistance with this study and dendritic cells and mediates T helper 1 development as well as interferon-␥ pro- the Stanford University students for their laboratory assistance. duction by T helper 1 cells. Eur. J. Immunol. 26:659. The Journal of Immunology 5575

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