Multifunctional TH1 cells define a correlate of vaccine- mediated protection against Leishmania major Patricia A Darrah1, Dipti T Patel1, Paula M De Luca1, Ross W B Lindsay1, Dylan F Davey1, Barbara J Flynn1, S��ren T Hoff 2, Peter Andersen2, Steven G Reed3, Sheldon L Morris4, Mario Roederer5 & Robert A Seder1 CD4+ T cells have a crucial role in mediating protection against a variety of pathogens through production of specific cytokines. However, substantial heterogeneity in CD4+ T-cell cytokine responses has limited the ability to define an immune correlate of protection after vaccination. Here, using multiparameter flow cytometry to assess the immune responses after immunization, we show that the degree of protection against Leishmania major infection in mice is predicted by the frequency of CD4+ T cells simultaneously producing interferon-c, interleukin-2 and tumor necrosis factor. Notably, multifunctional effector cells generated by all vaccines tested are unique in their capacity to produce high amounts of interferon-c. These data show that the quality of a CD4+ T-cell cytokine response can be a crucial determinant in whether a vaccine is protective, and may provide a new and useful prospective immune correlate of protection for vaccines based on T-helper type 1 (TH1) cells. Vaccines have made a substantial global impact on morbidity and mortality of a variety of bacterial and viral infections. Nevertheless, there are no licensed vaccines that are protective against HIV, malaria or pulmonary tuberculosis infection. For these pathogens, the cellular immune response comprising CD4+ T cells, CD8+ T cells or both can be important in controlling infection and preventing or delaying the onset of disease. Thus vaccine development for these infections is focused on generating protective T-cell responses1. A key mechanism by which T cells mediate their effector function is through the production of various cytokines. However, due to the heterogeneity of T-cell cytokine responses generated by different vaccines, there are still no well defined immune correlates of protection for infections requiring T-cell responses. Therefore a crucial step in vaccine devel- opment requires improved understanding of the functional hetero- geneity of T-cell cytokine responses. After activation, naive CD4+ T cells can differentiate into functional subsets termed TH1 or TH2 cells2. TH1 responses are required to mediate protection against a variety of intracellular infections. Such responses consist of populations of cells that secrete interferon (IFN)-g, tumor necrosis factor (TNF) or interleukin (IL)-2 in various combinations3���6. Differences in the types of cytokines produced by individual cells have profound implications for their capacity to mediate effector function, be sustained as memory cells or both. In this regard, CD4+ T cells that secrete only IFN-g have limited capacity to develop into memory cells compared with IL-2- or IL-2- and IFN-g-producing cells6���8. This implies that, for example, vaccines eliciting a high frequency of single-positive IFN-g producing cells may be limited in their ability to provide durable protection. The premise that distinct populations of TH1 responses determine vaccine efficacy provides a conceptual framework to define an immune correlate of protection. Most vaccine studies for infections requiring TH1 responses mea- sure the frequency of IFN-g producing cells as the primary immune correlate of protection9. Although IFN-g is clearly necessary10���12, using it as a single immune parameter may not always be sufficient to predict protection13���16. TNF is another effector cytokine that can mediate control of intracellular infections17,18. Indeed, IFN-g and TNF synergize in their capacity to mediate killing of pathogens19,20. IL-2 has little direct effector function but strongly enhances the expansion of CD4+ and CD8+ T cells, leading to a more efficient effector response. Thus, we hypothesized that CD4+ T cells that are multi- functional and simultaneously produce IFN-g, TNF and IL-2 may provide optimal effector function and protection. The best technique for assessing multiple functions of T cells simultaneously is multiparameter flow cytometry21. By assessing all combinations of IFN-g, TNF and IL-2 at the single-cell level, one can define the quality of the CD4+ T-cell cytokine response. In the present study we used an experimental infection model in which different vaccines elicited qualitatively distinct TH1 responses and conferred varying degrees of protection. Our results showed that the frequency of multifunctional TH1 cells simultaneously secreting IFN-g, TNF and IL-2 correlated best with protection. Notably, such cells seem specialized to secrete high amounts of IFN-g and TNF. These data provide fundamental insight into how distinct populations of Received 22 February accepted 17 April published online 10 June 2007 doi:10.1038/nm1592 1Cellular Immunology Section, Vaccine Research Center, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), 40 Convent Drive, Bethesda, Maryland 20892, USA. 2Department of Infectious Disease Immunology, Statens Serum Institute, Artillerivej 5, DK-2300 Copenhagen S, Denmark. 3Infectious Disease Research Institute, 1124 Columbia Street, Seattle, Washington 98104, USA. 4Laboratory of Mycobacterial Diseases and Cellular Immunology, Center for Biologics Evaluation and Research, Food and Drug Administration, 29 Lincoln Drive, Bethesda, MD 20892, USA. 5ImmunoTechnology Section, Vaccine Research Center, NIAID, NIH, 40 Convent Drive, Bethesda, Maryland 20892, USA. Correspondence should be addressed to R.A.S. (rseder@mail.nih.gov). NATURE MEDICINE VOLUME 13 [ NUMBER 7 [ JULY 2007 843 ARTICLES �� 200 7 Nature Publishing Group http://www.nature.com/naturemedicine

cytokine-producing cells are optimized for effector function and demonstrate a correlate of protection for vaccines targeting T-cell responses based on the quality of the response. RESULTS Vaccine-elicited protection against Leishmania major Models of L. major infection have been extensively used to understand the generation and maintenance of TH1 responses in the context of both natural infection and vaccine-elicited protection22. Infection of C57BL/6 mice mimics human disease, with the development of cutaneous lesions that are resolved by the host TH1 response through effector cytokines such as IFN-g and TNF. To study the functional heterogeneity of primary TH1 responses generated in vivo and to define immune correlates of protection, we used a variety of vaccine formulations in the L. major model. We compared live vaccination, the gold standard for life-long protection in mice23,24 and humans25 or a recombinant leishmanial protein (MML) and a toll-like receptor (TLR) ligand (CpG), another formulation that confers protection in mice26,27 to replication-defective adenovirus that expresses MML (MML-ADV). Although not yet tested in the L. major model, adenoviral vaccines28,29 elicit strong T-cell responses and have poten- tially broad application for use in HIV30, malaria31 and tuberculosis32. As a measure of protection, we compared lesion development and parasite burden after intradermal ear challenge with live L. major 1 month after immunization. The greatest degree of protection was observed in mice given the live and MML+CpG vaccines (Fig. 1a). Notably, we observed an inverse correlation between the dose of intramuscular (i.m.) MML-ADV and the level of protection although all doses of MML-ADV resulted in smaller lesions compared with those in unvaccinated mice, low-dose MML-ADV (107) protected mice to the greatest degree (Fig. 1a). In a separate experiment, we observed a similar inverse correlation when MML-ADV was adminis- tered subcutaneously (s.q. Fig. 1b), and the s.q. route consistently elicited better protection than the i.m. route at the same dose. The enhanced protection by s.q. immunization may reflect induction of skin-homing T cells or differences in the frequency or type of antigen-presenting cells by this route. A key finding is that less protection was observed with high-dose MML-ADV immunization, irrespective of route. The requirement for TH1 cells in mediating protection in vivo was assessed in several ways. First, vaccine-elicited protection was com- pletely abrogated upon depletion of CD4+ T cells at the time of infection (data not shown). Next, depletion of IFN-g or, to a lesser extent, TNF at the time of infection (Fig. 1c) abolished vaccine- mediated protection. Hence, CD4+ T cells, IFN-g and TNF were required for vaccine-mediated protection in this model. Functional heterogeneity among TH1 responses Based on the requirement for IFN-g and role for TNF as effector cytokines that mediate protection, we assessed the frequency of antigen-specific IFN-g-, IL-2- and TNF-producing CD4+ T cells at time of infection (Fig. 2a). One month after vaccination there was no difference between vaccine groups in the frequency of IFN-g-produ- cing cells in the spleen as assessed by intracellular cytokine staining (Fig. 2a) or enzyme-linked immunosorbent spot (ELISPOT) analysis (Fig. 2b). Note that the frequency of antigen-specific responses in live- vaccinated mice is under-represented, as all vaccine groups were restimulated with MML protein using a complete spectrum of leishmanial antigens, cytokine responses after live vaccination are B15���20% (data not shown). Collectively, these data show that the total frequency of antigen-specific IFN-g+ cells was not predictive of vaccine-elicited protection. Furthermore, protective efficacy was not related to differences in the breadth of the CD4+ T-cell response among the different vaccine formulations or to enhanced production of IL-4 or IL-13 in mice immunized with high-dose MML-ADV. We also looked for a potential role for IL-10 and regulatory T cells, but these did not seem to demonstrably affect the protection after high- dose MML-ADV vaccination (Supplementary Fig. 1 online). Based on the importance of IFN-g and TNF in mediating protec- tion (Fig. 1c), we undertook a more comprehensive functional analysis of cytokine-producing CD4+ T cells. Using multiparameter flow cytometry, seven distinct populations of cytokine-producing cells can be delineated at the single-cell level based on any combination of IFN-g, IL-2 or TNF (Supplementary Fig. 2 online). The relative frequency of these distinct populations defines the quality of the TH1 response. Thus for IFN-g, the total frequency of IFN-g-producing cells encompasses four distinct populations: IFN-g single-positive, Weeks after challenge 2 1 3 4 5 6 7 12 10 8 6 4 2 0 2 4 6 Lesion size (mm) Lesion size (mm) Lesion size (mm) Parasite number (log 10 ) Parasite number (log 10 ) Parasite number (log 10 ) 1 2 3 4 5 6 7 8 8 6 4 2 0 1 2 3 4 6 4 2 0 1 2 3 4 8 7 6 5 4 3 2 1 * * *�� *�� *�� *�� *�� *����� *����� *����� *��� �� *�� *�� *�� PBS Ctrl ADV 1011 i.m. 109 i.m. 107 i.m. MML+CpG Live MML-ADV PBS 1010 i.m. 106 i.m. 1010 s.q. 106 s.q. MML+CpG MML MML-ADV # # # # MML +CpG 106 s.q. 106 i.m. 1010 i.m. 1010 s.q. MML PBS MML +CpG 109 i.m. 107 i.m. 1011 i.m. Live PBS Anti-TNF Anti-IFN-�� No antibody 107 s.q. MML-ADV PBS Treatment Vaccine a b c Ctrl ADV MML-ADV MML-ADV PBS 107 s.q. MML-ADV 107 s.q. + anti-IFN-�� 107 s.q. + anti-IFN Figure 1 Vaccine-elicited protection against L. major. Mice were challenged in the ear with live L. major after immunization with a variety of vaccine formulations. Left panels, ear lesion diameter over time (mean �� s.e.m., n Z 12). Right panels, number of parasites per ear in each vaccine group (lines are geometric means). (a) MML-ADV (i.m.) dose dependence compared with negative (PBS, control adenovirus (Ctrl ADV)) and positive (MML+CpG, live parasite) controls. (b) Comparison of i.m. versus s.q. at high (1010) or low (106) MML-ADV dose. (c) Requirement of IFN-g or TNF for mediation of vaccine-elicited protection. Mice, vaccinated with 107 MML-ADV, received antibodies to IFN-g or TNF at the time of challenge. Shown are representative data from three of nine experiments. Symbols indicate statistical differences for lesion sizes or endpoint parasite numbers, comparing with PBS (*P o 0.04), with 1011 or 1010 i.m. (yP o 0.03), with 1010 s.q. (zP r 0.02) or with 107 s.q. (#P r 0.002). ARTICLES 844 VOLUME 13 [ NUMBER 7 [ JULY 2007 NATURE MEDICINE �� 200 7 Nature Publishing Group http://www.nature.com/naturemedicine