Human Macrophage Activation Programs Induced by Bacterial Pathogens

Human macrophage activation programs induced by bacterial pathogens

Gerard J. Nau*†‡, Joan F. L. Richmond*‡, Ann Schlesinger*‡, Ezra G. Jennings*§, Eric S. Lander*§, and Richard A. Young*§¶

*Whitehead Institute, 9 Cambridge Center, Cambridge, MA 02142; †Infectious Disease Unit, Massachusetts General Hospital, Boston, MA 02114; and §Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139

Communicated by Gerald R. Fink, Whitehead Institute for Biomedical Research, Cambridge, MA, December 5, 2001 (received for review October 2, 2001) Understanding the response of innate immune cells to pathogens bia, MD). Monocytes were cultured at a density of 2 ϫ 107 may provide insights to host defenses and the tactics used by cells͞10 ml of DMEM (Invitrogen) with 20% FCS (Intergen, pathogens to circumvent these defenses. We used DNA microar- Purchase, NY), 10% human serum (Nabi, Boca Raton, FL), and rays to explore the responses of human macrophages to a variety 50 ␮g͞ml gentamicin (Invitrogen) in Primaria T-25 flasks (Bec- of bacteria. Macrophages responded to a broad range of bacteria ton Dickinson) for 5 days at 37°C, 5% CO2. On days 5 and 7, half with a robust, shared pattern of gene expression. The shared of the media was removed and replaced with media lacking FCS. response includes genes encoding receptors, signal transduction Media on the cultured macrophages was changed to 5 ml of molecules, and transcription factors. This shared activation pro- DMEM with 1% human serum on day 9, 1 hour before exper- gram transforms the macrophage into a cell primed to interact with iments were begun. its environment and to mount an immune response. Further study revealed that the activation program is induced by bacterial com- Macrophage Exposure to Bacteria. Latex beads (0.8 ␮m; Sigma) ponents that are Toll-like receptor agonists, including lipopolysac- were washed with ethanol and added as described (6). Bacteria charide, lipoteichoic acid, muramyl dipeptide, and heat shock were diluted in media to 50 ␮l containing 108-109 bacteria, to give proteins. Pathogen-specific responses were also apparent in the macrophage expression profiles. Analysis of Mycobacterium tu- multiplicities of infection (MOIs) of 5:1 to 50:1, and added to berculosis-specific responses revealed inhibition of interleukin-12 cultures of macrophages. Dilutions were plated on agar plates, production, suggesting one means by which this organism survives and colonies were counted to confirm the accuracy of dilution host defenses. These results improve our understanding of mac- and viability of bacteria. This range of MOIs was selected rophage defenses, provide insights into mechanisms of pathogen- because preliminary experiments revealed that, although the esis, and suggest targets for therapeutic intervention. expression profiles elicited by different MOIs are largely similar, some genes with low levels of expression are not robustly detected at low mois (see Fig. 10, which is published as support- ost–pathogen relationships are characterized by the com- ing information on the PNAS web site, www.pnas.org). Media plex interplay between host defense mechanisms and at- H was removed at 4 h, and cells were washed with Hanks’ balanced tempts to circumvent these defenses by microorganisms (1, 2). salt solution (HBSS, Invitrogen) and fed with 5 ml of DMEM Macrophages play key roles in host defense by recognizing, ␮ ͞ engulfing, and killing microorganisms (3). Among the microor- with 1% human serum and 50 g ml gentamicin. Staphylococcus ganisms recognized by macrophages, bacteria are an important aureus strain ISP794, derived from strain 8325, and Listeria monocytogenes strain EGD were grown overnight in brain–heart and highly diverse class of human pathogens. Bacterial patho- ϫ gens that overcome host defenses ensure their ability to survive infusion broth, pelleted, and resuspended in 0.5 vol of 20% and propagate (4). Therefore, a thorough understanding of the glycerol to make frozen stocks that were thawed for the exper- normal host response to bacteria provides a foundation to iments. M. tuberculosis Erdman stain, obtained from Tubercu- understand bacterial tactics for evading these responses and thus losis Research Materials and Vaccine Testing Contract, and IMMUNOLOGY disease prevention. Mycobacterium bovis BCG (ATCC no. 35734) were grown in The extent to which macrophage responses to different bac- Middlebrook 7H9 broth with 0.5% glycerol, 0.05% Tween 80, teria are distinct or similar at the level of transcription is not and ADC enrichment (Becton Dickinson). A 6- to 7-day-old understood. Given the cellular components shared between culture was used for infection. Escherichia coli strain sd-4 (ATCC bacteria and the signaling pathway shared by Toll-like receptors no. 11143) was grown in Luria–Bertani medium with strepto- (TLR) that respond to these components (5), macrophages may mycin, and frozen stocks were created as described (6). The respond to all bacteria in a standard fashion. However, the Salmonella typhi (Quailes strain), S. typhimurium (ATCC no. diversity of bacteria and the differences in their pathogenesis 14028) and enterohemorrhagic E. coli O157:H7 (EHEC) were may also lead to pathogen-specific responses. For example, some provided as frozen stocks and thawed on the day of the microorganisms, like Mycobacterium tuberculosis, survive within experiment. macrophages (4) and might be expected to elicit specific changes in their host phagocytes. Macrophage Exposure to Bacteria Components. Macrophages were To improve our understanding of host defenses and patho- stimulated with isolated and purified bacterial components in genesis, we undertook a detailed, comparative examination of media containing 10% human serum over the entire 24-h time the transcriptional responses of macrophages to a variety of course. Endotoxin-free status was verified for all components, bacteria, including a number of pathogens. This experimental and quantitated for lipopolysaccharide (LPS) and heat shock approach allowed us to discover the themes that define the innate immune responses of macrophages to bacteria. The macrophage transcriptional responses also discriminate between Abbreviations: TLR, Toll-like receptor; MOI, multiplicity of infection; EHEC, enterohemor- different pathogens, and analysis of the response to M. tubercu- rhagic E. coli O157:H7; LPS, lipopolysaccharide; hsp, heat shock protein. losis yielded insights into the mechanism of pathogenesis. ‡G.J.N., J.F.L.R., and A.S. contributed equally to this work. ¶To whom reprint requests should be addressed. E-mail: [email protected]. Materials and Methods Ͼ The publication costs of this article were defrayed in part by page charge payment. This Macrophage Culture. Freshly elutriated human monocytes ( 95% article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. pure) were purchased from Advanced Biotechnologies (Colum- §1734 solely to indicate this fact.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.022649799 PNAS ͉ February 5, 2002 ͉ vol. 99 ͉ no. 3 ͉ 1503–1508 Downloaded by guest on September 29, 2021 proteins (hsps), by using the Associates of Cape Cod (Falmouth, (CCR6 and CCR7) and interleukins [IL-7 receptor (IL-7R) and MA) gel-clot assay at a sensitivity of 0.3 endotoxin units. LPS IL-15 receptor ␣ (IL-15RA)], and down-regulated molecules from E. coli (Sigma, L-2880) and Salmonella (Sigma, L-4774) necessary to respond to transforming growth factor ␤ (Table 1). were added at a final concentration of 1 ␮g͞ml. Lipoteichoic acid We defined this collection of genes as the macrophage activation (Sigma, L-2515) and muramyl dipeptide (Sigma, A-9519) were program because it represents signaling pathways and induced added at 10 ␮g͞ml and 100 ␮g͞ml, respectively. Formyl- functions that are triggered on exposure to Gram-negative methionine-leucine-phenylalanine (Sigma, F-3506) was added bacteria, Gram-positive bacteria, and mycobacteria. to 100 nM; protein A to 10 ␮g͞ml; and D-(ϩ)-mannose (Sigma, We next tested whether specific bacterial components could M-8296) to 25 mM. Staphylococcus protein A (539202) was elicit the activation program. Macrophages were cultured with purchased from Calbiochem. Recombinant mycobacterial hsps purified components specific for Gram-negative bacteria (LPS), were the kind gift of StressGen Biotechnologies (Victoria, BC, Gram-positive bacteria (lipoteichoic acid and protein A), and Canada) and were used at 10 ␮g͞ml. components shared by all three classes of bacteria [muramyl dipeptide, heat shock proteins (hsp65 and hsp70), formyl- cRNA Target Preparation and Array Hybridization. After incubation, methionine-leucine-phenylalanine, and mannosylated proteins the supernatant was recovered from the flasks, and RNA was (D-(ϩ)-mannose)]. LPS, lipoteichoic acid, muramyl dipeptide, harvested from the macrophages by using 2 ml of TriReagent and hsp65 and hsp70 induced the majority of the activation (Molecular Research Center, Cincinnati) according to the man- program (Fig. 1B). The other components failed to induce this ufacturer’s protocols. Total RNA was processed and hybridized set of gene expression changes (Fig. 1B), as did monophosphoryl to Hu6800 GeneChips according to standard Affymetrix (Santa lipid A (detoxified lipid A). Thus, some, but not all, bacterial Clara, CA) protocols (7). components are capable of inducing the activation program.

Data Analysis. The data were analyzed by using a custom-built M. tuberculosis-Specific Macrophage Gene Expression Changes. To Oracle (Redwood Shores, CA) database, CHIPDB. Fluorescence identify the alterations in the host macrophage expression intensities were normalized to median array intensities for all profiles elicited by specific organisms, a difference index was conditions tested on cells from a single donor, floored at 50, and devised. This analysis focused on M. tuberculosis, E. coli, and S. fold-change was calculated relative to duplicate time-0 controls. aureus because multiple, independent time course experiments Data were considered significant when (i) expression changed by were performed for each of these organisms (Fig. 2A). By using at least 2-fold at two consecutive time points, or 10-fold (acti- this index, several differentially regulated genes with positive and vation program) or 3-fold (differential gene expression) at a negative effects on host defenses were identified. M. tuberculosis single time point, and (ii) increased gene expression included at poorly induced the expression of IL-12 p40 and IL-15, as least one present call (Affymetrix algorithm), or both zero time described in greater detail below. STAF50, a transcriptional points were present when gene expression decreased. Additional repressor of HIV LTR, and STAT2, a transcription factor critical details are available on the web site supporting this manuscript to responsiveness to interferons, and IL-10 receptor ␣ were at http:͞͞web.wi.mit.edu͞young͞pathogens. induced in macrophages by E. coli. Similarly, S. aureus induced a proinflammatory lysophospholipase, HU-K5, but also down- Results regulated several cytoskeletal components and up-regulated a Bacteria-Induced Macrophage Activation Program. Human macro- negative regulator of G-protein signaling. Thus, organism- phages derived from primary monocytes were exposed to bac- specific macrophage gene expression changes were discernable. teria and bacterial components, and the resulting expression Because of the impact of tuberculosis on world health, we levels of 6,800 genes were monitored over a 24-h time course by focused additional experiments on macrophages exposed to M. using high density DNA microarrays. The eight bacteria studied tuberculosis. These experiments were designed to test the dif- were drawn from three broad classes with different cellular ference index and evaluate clues to pathogenesis. It was striking components and pathogenesis: Gram-negative bacteria [E. coli, that the expression levels of genes encoding two proteins critical enterohemorrhagic E. coli O157:H7 (EHEC), Salmonella typhi, for host defense and adaptive immunity, IL-12 and IL-15, were and S. typhimurium], Gram-positive bacteria (S. aureus and L. differentially regulated. The induction of these genes was sig- monocytogenes), and mycobacteria (M. tuberculosis and M. bovis nificantly lower after exposure to M. tuberculosis than other bacillus Calmette–Gue´rin). bacteria (Fig. 2B). Although IL-12 met the criteria for inclusion Analysis of macrophage gene expression data showed that the in the macrophage activation program (Table 1), the difference expression of 977 genes significantly changed on exposure to one index disclosed substantial differences in the levels of IL-12 or more of the bacteria. Unsupervised hierarchical clustering of expression by macrophages exposed to M. tuberculosis and other these genes (8) revealed prominent groups of genes that had bacteria. As predicted by the microarray data, supernatants from similar changes in expression. Despite the diversity of the macrophages stimulated by M. tuberculosis contained less IL-12 bacteria studied, a shared transcriptional response was elicited, p40 than macrophages exposed to E. coli (Fig. 3A). Macrophages consisting of 132 genes induced and 59 repressed (Fig. 1A; the exposed to M. tuberculosis also secreted less IL-12 p70 and IL-15 entire data set is displayed in Fig. 4, which is published as protein than macrophages cultured with E. coli (not shown). supporting information on the PNAS web site). Latex beads Because the lower levels of IL-12 in macrophages exposed to failed to induce comparable expression changes (Fig. 1A). The M. tuberculosis could be due to active repression of IL-12 changes in gene expression induced after exposure to bacteria production by M. tuberculosis, a coculture experiment was per- contained, as expected, proinflammatory genes, including many formed. Macrophages were exposed to M. tuberculosis,toE. coli, cytokines and chemokines (Fig. 1A, Table 1). The majority of or to both simultaneously. Macrophages exposed to a mixture of this transcriptional response, however, is comprised of genes that M. tuberculosis and E. coli produced low levels of IL-12, com- are involved with the interaction between macrophages and their parable to macrophages exposed to M. tuberculosis alone and environment. A large number of receptors, signaling molecules, substantially less than macrophages exposed to E. coli alone (Fig. and transcription factors were differentially regulated by mac- 3A). Although M. tuberculosis inhibited the production of IL-12 rophages on exposure to bacteria, as were adhesion molecules, levels normally induced by E. coli, this inhibition did not extend genes involved in tissue remodeling, enzymes, and anti-apoptotic to the production of TNF-␣ (Fig. 3B). These findings indicate molecules. For example, macrophages up-regulated many of the that M. tuberculosis alters the macrophage activation program NF-␬B pathway members as well as receptors for chemokines and dominates the stimulatory effects of E. coli exposure.

1504 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.022649799 Nau et al. Downloaded by guest on September 29, 2021 Fig. 1. Macrophage activation program elicited in response to a variety of bacteria and bacterial components. (A) The macrophage activation program. IMMUNOLOGY Macrophage gene expression was measured at 1, 2, 6, 12, and 24 h after the introduction of bacteria or latex beads. One hundred ninety-one genes met criteria for signiﬁcant changes (see Materials and Methods) on exposure to six of eight bacteria studied. The genes are ordered along the y axis by category, with the degree of change indicated by color intensity in the color bar. The display has been described in detail elsewhere (8). Minimal gene expression changes occurred in macrophages cultured in media over 24 h (Fig. 6, which is published as supporting information on the PNAS web site). Gene expression changes attributable to individual donors were excluded from this display (Fig. 7, which is published as supporting information on the PNAS web site). (B) A subset of bacterial components elicits much of the activation program (induced genes shown only). Control experiments demonstrated that residual endotoxin contamination of the recombinant hsps was not responsible for the extent of cytokine expression induced by hsps (Fig. 8, which is published as supporting information on the PNAS web site). The idea that the hsps were acting via TLR was supported by the observation that a speciﬁc TLR-4 antagonist, Rhodobacter sphaeroides lipid A (32), blocked cytokine production by macrophages exposed to E. coli, LPS, and hsp70 (Fig. 9, which is published as supporting information on the PNAS web site).

Discussion LPS for TLR4 (10, 11), lipoteichoic acid and muramyl dipeptide Studying host–pathogen interactions by using a parallel, com- for TLR2 (12, 13), and hsps for TLR2 and TLR4 (14, 15). The parative analysis of gene expression permitted the identification ability of hsps to elicit this response is likely to account for their of both shared and distinct gene expression responses in human potency as adjuvants in preclinical (16) and clinical vaccine trials macrophages. The shared macrophage activation program is (17). In contrast, the other components tested are not known to robust and is induced by Gram-positive bacteria, Gram-negative activate TLRs. These findings suggest that the activation pro- bacteria, and mycobacteria. The proinflammatory component of gram is due to signaling mediated by macrophage TLRs. the activation program may constitute a generic ‘‘alarm signal’’ Notably absent from the activation program were several that marshals antibacterial defenses. However, the majority of categories of genes commonly associated with macrophage the activation program is comprised of cell surface proteins and functions, such as genes involved in antigen presentation. How- signaling molecules engendering new functions in the macro- ever, we found that such genes are highly expressed in macrophage, suggesting a maturation process similar to that observed phages in the absence of bacterial stimulation. Comparison of with dendritic cells (9). gene expression profiles of unstimulated human macrophages to A subset of bacterial components is able to induce the unstimulated human fibroblasts revealed that cathepsins, activation program. These components are all agonists for TLRs: HLA-D genes, and others relevant to macrophage functions

Nau et al. PNAS ͉ February 5, 2002 ͉ vol. 99 ͉ no. 3 ͉ 1505 Downloaded by guest on September 29, 2021 Table 1. Genes of the activation program Induced Repressed

Cytokines Signaling Proliferation Anti-Inflammatory Miscellaneous GCSF CNK BTG1 GCHFR PURA GMCSF DUSP1 BTG3 CAT MYO1E IL12B DUSP2 TNFRSF9 LTA4H VCL IL1RN DUSP5 TNFSF9 Pro-Inflammatory EVI2B IL6 EBI2 Tissue remodeling͞clotting CD14 ADFP IL6 GBP1 DAF ALOX5 MNDA PBEF HCK MMP1 MCP1 STAB1 ProIL1B INHBA MMP10 Adhesion TGFBI TNFA JAG1 MMP14 PECAM1 ESTs Chemokines KYNU SERPINB2 SPARC KIAA0022 IL8 LIMK2 SERPINB8 Receptors Unknown IL8 MAP2K3 Stress response RARA AD000092 IP10 MAP3K4 GADD45A TNFRSF1A D87075 MCP1 MINOR HSPA1A ENG U79288 MGSA NAF1 SOD2 L77730 P311 MIP1A NFKB1 SOD2 CD36L1 HG2090-HT2090 MIP1B PDE4B Transporters CD163 HG2090-HT2090 MIP2A PPP3CC ATP2B1 TGFBR2 HG2090-HT2090 MIP2B PTPN1 NRAMP2 CSFIR RANTES TRAF1 SLC7A5 MRC1 Adhesion TRIP10 Enzymes CD32 CD44 Transcription ADA P2RX1 CD44 DSCR1 AMPD3 Signaling ICAM1 ELF4 Beta-1,4-galatosyl transferase SRPK2 IFITM1 ETS2 BF SLA LAMB3 IRF1 CKB MERTK NINJ1 IRLB GJB2 DAB2 TNFAIP6 JUNB GLCLR Transcription Receptors MRF-1 HSD11B1 SF3A3 ADORA2A NFKBIA INDO EGR2 CCR6 NFKBIE MTF1 NFATC3 CCR7 NFKB p50 Miscellaneous MXI1 CCRL2 STAT4 ADM FOS DTR STAT5A ARHH Hbrm EBI3 TSC22 B4-2 Transporters HM74 XBP1 BRCA2 SLC29A1 IL15RA ZFP36 CD83 Tissue remodeling͞Clotting IL7R Pro-inflammatory GEM UPA LDLR COX2 GOS FGL2 P2RX7 COX2 GYPC TBXAS1 P2XR COX2 H2AFO Enzymes PLAUR GCH1 HIVEP2 SGSH PVR PTX3 ISG15 PPP2R5C SLAM Anti-apoptotic ISG20 IDH2 TNFRSF5 BIRC2 MACMARKS LPL TXN BIRC3 MIG2 MPI BIRC3 MX1 PYGL CFLAR RCN1 HSD17B4 IER3 SDC4 RNASE6 TNFAIP3 SNL GALC TNFAIP2 GLCLC TSSC3 RNASE1 ESTs ME1 KIAA0105 KIAA0172

The genes of the activation program are listed in the order displayed in Fig. 1 and are categorized by functional class. Several genes are repeated because they appear more than once on the array.

were preferentially expressed in the macrophages (Fig. 5, which repressed IL-12 production. Such a pathogen-specific response is published as supporting information on the PNAS web site). suggests that signaling pathways other than those mediated by The parallel, comparative analysis of gene expression also TLR are active, leading to distinctive changes in gene expression. revealed clues to M. tuberculosis pathogenesis. This organism The poor induction of IL-12 by M. tuberculosis is consistent with induced little IL-12 and IL-15 relative to other bacteria, and observations of macrophage responses to M. tuberculosis and to

1506 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.022649799 Nau et al. Downloaded by guest on September 29, 2021 Fig. 3. M. tuberculosis induced repression of IL-12 protein production. (A) M. tuberculosis induced less IL-12 protein than E. coli and suppressed IL-12 production induced by E. coli. IL-12 p40 accumulation in supernatants of macrophages 12 h after exposure to either M. tuberculosis, E. coli, or both bacteria was measured by ELISA (R & D Systems). Additional experiments demonstrated that this response was reproducible and not donor dependent. TNF-␣ ELISA was performed on supernatants from the same cultures after 4 h (peak expression of this cytokine preceding the wash step). Values are means Ϯ SD of replicate ELISA measurements. Neither IL-12 nor TNF-␣ was detected in macrophages treated with media alone.

Fig. 2. Differences in expression profiles distinguish between bacteria. (A) Differential gene expression in macrophages exposed to M. tuberculosis, E. of normal mice to clear a mycobacterial infection (24, 28). coli,orS. aureus. The difference index represents how expression levels Second, exogenous IL-12 therapy has rescued two patients, one induced by the three bacteria differ from the average expression level with a pulmonary Mycobacterium abscessus infection and one induced by all bacteria studied: large values are assigned to genes whose with disseminated M. tuberculosis infection, whose infections expression levels exhibit the greatest differences in specific bacterial in- were refractory to antibiotics and supplemental IFN-␥ (29, 30). fections. Multiple time course experiments were used (M. tuberculosis, two Our findings support the idea that both IL-12 and IL-15 could repeats; E. coli, three repeats; and S. aureus, two repeats). For every gene be useful therapies for clinical tuberculosis, suggested by the within each profile, the responses of macrophages to each of the bacterial animal models (24, 28, 31). species in question were compared with the average response to all This study of host–pathogen interactions has provided insights bacterial species studied. Statistically significant differences were identi- into host defenses and pathogen-specific manipulations of those fied by using Student’s t test (P Ͻ 0.005). The difference index was calcu- defenses that should have practical applications. Understanding ϭ Ϫ lated as follows: D.I. log2 (fold-change of gene X in infection A) log2 the macrophage activation program, and the bacterial compo- (avg fold-change of gene X in all infections) . (B) M. tuberculosis induced nents that elicit it, will be useful in designing vaccines and lower levels of macrophage IL-12 and IL-15 gene expression than the cytokine therapies that engage the innate immune system in a average expression measured across all data sets. The average fold-change targeted fashion. The expression data described here should Œ values observed after exposure to M. tuberculosis ( ) are displayed for the provide a foundation for further studying the pathogenesis of time course. In comparison, the average fold-change values across all data these and other infectious agents such as fungi, viruses, and sets was significantly greater (■). parasites.

We thank John Wyrick for help with array procedures and data analysis recombinant Mycobacterium smegmatis transformed with the and John Barnett for database management and support. We also thank 19-kDa antigen of M. tuberculosis (18, 19). The effect of M. Lee Mizzen and StressGen Biotechnologies for their donation of re- tuberculosis on macrophage IL-12 production appears similar to combinant heat shock proteins and Christine Ladd for technical assis- IMMUNOLOGY the inhibition of IL-12 production observed with Leishmania tance. Virulent M. tuberculosis Erdman strain was provided by the major and Histoplasma capsulatum, two other intracellular Tuberculosis Research Materials and Testing Contract, National Insti- pathogens (20, 21). This finding may represent convergent tutes of Health (NIH), National Institute of Allergy and Infectious Diseases, NO1 AI-753230 at Colorado State University. Other bacteria evolution of organisms selected for their ability to survive within were kind gifts of Dr. David Hooper and Dr. Dilek Ince (S. aureus), Dr. macrophages. Stephen Calderwood (EHEC), and Dr. Elizabeth Hohmann (Salmonel- IL-12 plays a fundamental role in generating T helper 1 (Th1) lae) of Massachusetts General Hospital, and Dr. Douglas Drevets of the immune responses (22) and is critical for host resistance to University of Oklahoma Health Sciences Center (L. monocytogenes tuberculosis infection in mice and in humans (23–27). Repres- EGD). We thank Dr. Douglas Golenbock of University of Massachusetts sion of IL-12 production may enhance the survival of M. Medical School, and Dr. Nilofer Qureshi of University of Missouri- Kansas City School of Medicine, for providing Rhodobacter sphaeroides tuberculosis against the innate immune response or the devel- lipid A. This work was supported by funds from NIH; Corning, Inc.; oping adaptive immune response. This notion is supported by Affymetrix, Inc.; Millennium Pharmaceuticals, Inc.; and Bristol-Myers two observations. First, supplemental IL-12 enhances the ability Squibb Company.

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