Immunostimulatory activity of haptenated proteins

Noah W. Palm and Ruslan Medzhitov1

Howard Hughes Medical Institute and Department of Immunobiology, Yale University School of Medicine, 300 Cedar Street, New Haven, CT 06510

Communicated by Richard A. Flavell, Yale University School of Medicine, New Haven, CT, September 30, 2008 (received for review July 15, 2008) Antigen recognition alone is insufficient for the activation of In previous studies, we investigated the role of the TLR adaptive immune responses mediated by conventional lympho- signaling pathway in the induction of adaptive immune re- cytes. Additional signals that indicate the origin of the antigen are sponses, using mice deficient in the critical TLR signaling also required. These signals are generally provided by the innate adaptor Myeloid Differentiation primary response gene 88 immune system upon recognition of conserved microbial struc- (MyD88) (9, 10). To isolate the contribution of TLRs we used a tures by a variety of pattern recognition receptors (PRRs). The single defined TLR ligand, Lipopolysaccharide (LPS), as an Toll-like receptors (TLRs) are the best-characterized family of PRRs adjuvant, and the native proteins ovalbumin (OVA) or human and control the activation of adaptive immune responses to a serum albumin (HSA), as model antigens. incomplete Freund’s variety of immunizations and infections. However, recent studies adjuvant (IFA) and aluminum hydroxide, which contain low have questioned the role of TLRs in the induction of antibody levels of contaminating PRR ligands, were used simply for their responses and, thus, this issue has become controversial. In con- depot effect, which is important for soluble antigens. Using this ϩ trast to earlier studies supporting a role for TLRs in antibody system, we demonstrated that optimal CD4 T cell responses and responses, these studies used haptenated antigens rather than T-dependent antibody responses require intact TLR signaling; native antigens for immunization, but did not consider the poten- furthermore, we showed that optimal T-dependent antibody tial effect of antigen haptenation on immunogenicity. Here, we responses to OVA and HSA not only require TLR signaling in show that commonly used haptenated proteins, unlike native the antigen presenting dendritic cells, but also depend on TLR proteins, are inherently immunogenic. This immunogenicity is signaling in B cells (10). Notably, the contribution of TLRs to TLR-independent, but the T and B cell responses induced are antibody responses depended on the antibody isotype and was primarily hapten-specific, rather than protein-specific. Thus, al- restricted to IgM, IgG1 and IgG2 classes, whereas the IgE though haptens have immunostimulatory activity, it is distinct response was not dependent on TLRs (10, 11). The role of a B from classical adjuvants, which induce immune responses directed cell intrinsic TLR signal in antibody production was also dem- at the admixed antigens. Our results thus highlight an unappreci- onstrated in the human system (12) and in the murine system in ated and unique immunogenicity of haptenated proteins, and response to viral infection (13, 14) or virus-like particles (15), provide an experimental explanation for a seeming discrepancy and in autoimmunity (16–18). between published results. Despite a large body of evidence linking TLRs and adaptive immunity, 2 recent reports have questioned the role of TLRs in adjuvant ͉ antibody ͉ innate immunity ͉ Toll-like receptor antibody production based on analysis of antibody responses in MyD88 and TRIF (TIR-domain-containing adapter-inducing ␤ nnate immune recognition is mediated by several families of IFN- ) double-deficient mice or mice with a B cell-specific Ipattern recognition receptors (PRRs), including the Toll-like deficiency in MyD88 (19–21). The reasons for this discrepancy receptors (TLRs), NOD-like receptors (NLRs), Dectin-1 and are not clear, creating considerable confusion with regard to the involvement of TLRs in the control of antibody responses. related C-type lectin receptors, and retinoic acid inducible Studies producing conflicting data on the role of TLRs in gene-I (RIG-I) and melanoma differentiation-associated gene-5 antibody responses differ notably in the type of antigen used for (MDA-5) (1). Each family of PRRs is specialized to deal with immunization. Although studies supporting a role for TLRs (9, particular classes of pathogens. For example, Dectin-1 detects 10) used unmodified native protein antigens, studies that failed the fungal cell wall component ␤-glucan (2) and induces phago- to find such a role (19–21) used antigens that were chemically cytosis and activation of the T helper-17 (Th17) arm of adaptive modified by haptenation—a procedure involving the conjugation immunity (3, 4). Nucleotide-binding oligomerization domain of multiple small chemical moieties to a carrier protein. Hap- containing 1 (NOD1) and NOD2 proteins detect peptidoglycan tenation is a widely used approach to study antibody responses. fragments in the cytosol and contribute to both innate and Antibody responses to haptenated proteins are T cell dependent adaptive responses (5). RIG-I and MDA-5 recognize viral RNA (22) and, thus, protein haptenation is thought to simply provide and trigger IFN production and antiviral immunity, including defined epitopes for the measurement of antibody titers and activation of cytotoxic T lymphocytes (CTLs) (6, 7). TLRs detect affinities without altering the requirements for B cell responses. a variety of molecular structures derived from bacterial, viral and However, we show here that certain haptenated proteins are fungal pathogens and lead to the activation of T and B cells (8). highly immunogenic, whereas native proteins are nonimmuno- Each of these families of PRRs is capable of activating the genic. Unlike native proteins mixed with adjuvant, which induced appropriate class of the adaptive immune response. Therefore, strong anti-protein responses, haptenated proteins induced strong the combination of immunostimulatory components (i.e., PRR hapten-specific responses, but weak protein-specific responses. This ligands) contained in a given pathogen determines the relative unique immunogenicity was TLR-independent, and CD4ϩ T cell requirement for any particular PRR in activation of adaptive and IgG1 responses to haptenated antigens were therefore largely immunity. Most pathogens contain ligands for more than 1 MyD88-independent. Haptenated proteins therefore possess a family of PRRs, which complicates analyses of the roles of different PRR families. However, analysis of the specific mech- anisms of activation of the adaptive immune response can be Author contributions: N.W.P. and R.M. designed research; N.W.P. performed research; facilitated by the use of model antigens and simple adjuvants, N.W.P. and R.M. analyzed data; and N.W.P. and R.M. wrote the paper. whereby targeting of a particular class of PRRs, using their The authors declare no conflict of interest. corresponding microbial ligands, provides an adjuvant activity 1To whom correspondence should be addressed. E-mail: [email protected]. and confers immunogenicity to otherwise nonimmunogenic This article contains supporting information online at www.pnas.org/cgi/content/full/ protein antigens. 0809403105/DCSupplemental.

4782–4787 ͉ PNAS ͉ March 24, 2009 ͉ vol. 106 ͉ no. 12 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0809403105 Downloaded by guest on September 24, 2021 HSA+LPS Alum DNP(29)-HSA Alum ABOVA+LPS IFA DNP(7)-OVA+LPS IFA HSA+LPS IFA DNP(29)-HSA IFA 40 60 125 15 75 150 100 45 30 50 75 100 30 10 50 20 25 50 15 25 5 IgG1 Anti-DNP ( µ g/ml) IgG1 Anti-HSA ( µ g/ml) IgG1 Anti-OVA ( µ g/ml) IgG1 Anti-OVA 0 IgG1 Anti-DNP ( µ g/ml) 0 0 0 10 WT MyD88-/- WT MyD88-/- WT MyD88-/- WT MyD88-/- 7.5 10 7.5 IgG1 Anti-HSA ( µ g/ml) 2.0 0 IgG1 Anti-DNP ( µ g/ml) 0 7.5 WT MyD88-/- WT MyD88-/- 5.0 1.5 5.0 5.0 1.0 Fig. 2. Haptenated proteins in aluminum hydroxide adjuvant induce robust 2.5 2.5 2.5 0.5 IgG1 responses in MyD88-deficient mice. WT C57BL/6 and MyD88-deficient IgG2c Anti-HSA ( µ g/ml) IgG2c Anti-OVA ( µ g/ml) IgG2c Anti-OVA IgG2c Anti-DNP ( µ g/ml) 0 IgG2c Anti-DNP ( µ g/ml) 0 0 0 mice were immunized i.p. with either HSA plus LPS or DNP(29)-HSA in alum. WT MyD88-/- WT MyD88-/- WT MyD88-/- WT MyD88-/- Serum HSA- or DNP-specific IgG1 was measured on day 14 after immunization Fig. 1. Haptenated proteins in IFA induce robust IgG1 responses in MyD88- by ELISA. deficient mice. Age matched MyD88-deficient and WT C57BL/6 mice were immunized in the footpad with OVA or DNP(7)-OVA plus LPS (A) or with low ؉ endotoxin HSA plus LPS or DNP(29)-HSA (B) emulsified in IFA. HSA-, OVA- or Haptenated Proteins Induce MyD88-Independent CD4 T Cell Re- DNP-specific serum antibody titers were measured by ELISA on day 14 after sponses. Antibody responses can be characterized as either immunization. T-dependent or T-independent based on the requirement for T cell help. Highly multivalent antigens and antigens that specif- ically induce innate immune pathways in B cells can induce unique, hapten-focused immunogenicity that affects the require- T-independent antibody responses. However, antibody re- ments for adaptive immune activation. Thus, studies questioning sponses to native proteins and haptenated proteins alike are T the role of TLRs in antibody responses reached their conclusions cell-dependent (22). We have shown previously that T cell because of the unappreciated effects of protein haptenation on responses to native antigen plus LPS emulsified in IFA are immunogenicity. This created a controversy in the field regarding TLR-dependent (refs. 9 and 24 and Fig. 3). We wished to the role of TLRs in antibody responses. Our data reconfirm the ϩ examine whether CD4 T cell responses to haptenated antigens importance of TLRs in antibody responses to native proteins plus are also TLR-signaling dependent. Therefore, we compared TLR ligands, provide a simple explanation for a seeming discrep- CD4ϩ T cell responses to immunizations with HSA plus LPS or ancy in datasets and highlight the unique immunogenicity of DNP-HSA in IFA in WT C57BL/6 and MyD88-deficient mice. haptenated antigens. As reported in refs. 9 and 10, CD4ϩ T cell responses to HSA plus Results LPS in MyD88-deficient mice were severely reduced as com- Haptenated Proteins in IFA Induce Robust IgG1 Responses in MyD88- pared with WT mice (Fig. 3 and refs. 9 and 10). In contrast, compared with WT mice, DNP-HSA induced a robust, albeit Deficient Mice. The most obvious difference between studies ϩ supporting a role for TLRs in antibody responses and those slightly reduced, CD4 T cell response in MyD88-deficient mice arguing against a role for TLRs is in the type of antigen used for (Fig. 3). immunization; studies that found a role for TLRs in antibody responses used native protein antigens or live pathogens, Haptenated Proteins Can Induce Adaptive Immune Responses in Mice whereas studies arguing against a role for TLRs used proteins Completely Deficient in TLR-Signaling. TLRs use both MyD88- and modified by haptenation. To determine whether this experimen- TRIF-dependent signaling pathways (8). TLR-dependent sig- tal difference was responsible for the discrepancy in datasets, we naling thus remains partially intact in MyD88-deficient mice. directly compared the role of TLRs in antibody responses to Therefore, we also examined the induction of adaptive immune haptenated versus native proteins in response to immunization responses to haptenated protein in MyD88 and TRIF double- with antigen emulsified in the depot adjuvant IFA. WT C57BL/6 deficient mice, which lack all known TLR-dependent signaling. and MyD88-deficient mice (23) were immunized with the hap- As was observed for MyD88-deficient mice, MyD88 and TRIF ϩ tenated proteins dinitrophenyl (DNP)-HSA (on average 29 double-deficient mice showed defects in their CD4 T cell and molecules of DNP per HSA) or DNP-OVA (on average 7 IgG1 responses to HSA and LPS, but exhibited robust responses molecules of DNP per OVA), or native HSA or OVA with or without LPS, and antibody titers in the serum were measured after 14 days. As we demonstrated in ref. 10, MyD88-deficient HSA+LPS mice immunized with native protein (either HSA or OVA) DNP-HSA exhibited reduced titers of antigen specific IgG1 and IgG2c as 150 WT 75 MyD88-/- compared with WT mice (Fig. 1 A and B). In contrast, DNP- 125 HSA (Fig. 1B) and DNP-OVA (Fig. 1A) induced robust IgG1 100 50 responses in MyD88-deficient mice, whereas IgG2c production 75 remained largely MyD88 dependent. 50 25 C.P.M. (x10^3) C.P.M. C.P.M. (x10^3) C.P.M. Haptenated Proteins in Aluminum Hydroxide Induce MyD88-Indepen- 25 0 0 dent IgG1 Responses. We next examined whether native versus 1 10 100 1000 1 10 100 1000 haptenated proteins in aluminum hydroxide (alum) exhibited Protein (µg/ml) Protein (µg/ml)

differential requirements for TLR signaling. We examined the ϩ IgG1 response to HSA plus LPS versus DNP-HSA in alum in WT Fig. 3. Haptenated proteins in IFA induce a CD4 T cell response in MyD88- deficient mice. MyD88-deficient and WT C57BL/6 mice were immunized in the C57BL/6 and MyD88-deficient mice. In line with what we ϩ footpad with either HSA and LPS or DNP (29)-HSA in IFA. CD4 T cells were observed for IFA, the IgG1 response to HSA plus LPS in alum isolated from draining lymph nodes on day 8 after immunization and restim- was defective in MyD88-deficient mice, whereas the IgG1 re- ulated in vitro with irradiated splenocytes as antigen presenting cells and sponse to DNP-HSA in alum was normal in MyD88-deficient titrating doses of antigen. Proliferation was measured by incorporation of IMMUNOLOGY mice (Fig. 2). 3H-Thymidine during the last 16 h of a 72-h stimulation (cpm).

Palm and Medzhitov PNAS ͉ March 24, 2009 ͉ vol. 106 ͉ no. 12 ͉ 4783 Downloaded by guest on September 24, 2021 HSA+LPS DNP(29)-HSA A B A B HSA HSA 60 4 120 7.5 10 60 10 HSA+LPS 20 DNP-HSA 100 3 7.5 15 40 80 5.0 7.5 45 2 60 5.0 30 5.0 10 20 40 2.5 1

C.P.M. (x10^3) C.P.M. 2.5 20 2.5 15 (x10^3) C.P.M. 5 IgG1 Anti-DNP ( µ g/ml) IgG1 Anti-HSA ( µ g/ml) IgG2c Anti-HSA ( µ g/ml) 0 0 IgG2c Anti-DNP ( µ g/ml) 0

0 IgG1 Anti-DNP ( µ g/ml) WT MyD88/ WT MyD88/ WT MyD88/ WT MyD88/ IgG1 Anti-HSA ( µ g/ml) 0 0 0 0 HSA HSA+ DNP- 110 100 1000 110 100 1000 TRIF TRIF TRIF TRIF µ LPS HSA HSA ( g/ml) DNP-HSA (µg/ml) MyD88/ C WT HSA+LPS TRIF 60 DNP-HSA 20 C DNP-HSA 75 6.0 150 50 HSA 15 40 4.5 100 30 10 50 20 3.0 C.P.M. (x10^3) C.P.M. C.P.M. (x10^3) C.P.M. 5 50 10 25 C.P.M. (x10^3) C.P.M. IgG1 ( µ g/ml) 1.5 IgG2c ( µ g/ml) 0 0 1 10 100 1000 1 10 100 1000 0 Protein (ug/ml) Protein (ug/ml) 0 0 110 100 1000 Protein (µg/ml) Fig. 4. Haptenated proteins induce robust adaptive responses in MyD88 and TRIF double-deficient mice. MyD88 and TRIF double-deficient and WT C57BL/6 DNP specificHSA specific DNP specificHSA specific mice were immunized with HSA plus LPS (A and C) or DNP(29)-HSA (B and C) Fig. 5. Protein haptenation imparts a hapten-focused immunogenicity on a in IFA in the footpad. OVA-specific (A) or DNP-specific (B) serum antibody titers nonimmunogenic protein. (A) WT C57BL/6 mice were immunized i.p. with were measured by ELISA on day 14 after immunization. (C) CD4ϩ T cells were low-endotoxin HSA, HSA plus LPS, or low-endotoxin DNP(29)-HSA in alum. isolated from the draining lymph nodes of immunized MyD88 and TRIF Serum DNP-specific or HSA-specific IgG1 and IgG2c titers were measured by double-deficient and WT C57BL/6 mice on day 8 after immunization and ELISA on day 14 after immunization. (B) WT C57BL/6 mice were immunized in restimulated with titrating doses of antigen and irradiated splenocytes for 3 the footpad with HSA, HSA plus LPS, or low-endotoxin DNP(29)-HSA in IFA and days. T cell proliferation was measured by [3H]thymidine incorporation during day 8 CD4ϩ T cell responses to titrating doses of either DNP-HSA or HSA were the last 16 h of culture (cpm). measured by [3H]thymidine incorporation for the last 12–16 h of a 72-h restimulation (cpm). (C) WT C57BL/6 mice were immunized in the footpad with DNP(29)-HSA in IFA. On day 14 after immunization, DNP-specific and HSA- to DNP-HSA, again with the only exception being the IgG2c specific IgG1 and IgG2c titers were measured by ELISA. On day 8 after immu- response, which remained largely MyD88- and TRIF-dependent nization, CD4ϩ T cells were isolated from draining lymph nodes and restimu- under both conditions (Fig. 4). lated with titrating doses of either DNP-HSA or HSA and irradiated splenocytes. Proliferation was measured by [3H]thymidine incorporation for Haptenation Confers a Hapten-Specific Immunogenicity on Nonimmu- the last 12–16 h of a 72-h stimulation. nogenic Protein Antigens. The protein antigen HSA, when purified to be devoid of contaminating TLR ligands, fails to induce a robust adaptive immune response when combined with a suitably Discussion TLR-ligand free depot adjuvant such as alum (Fig. 5 A and B and The use of haptenated proteins in immunology dates back to the refs. 9 and 10). Addition of the TLR4 ligand LPS to TLR-ligand early 20th century when Karl Landsteiner performed his pio- free HSA is sufficient to induce robust CD4ϩ T cell and antibody neering work on the specificity of serological responses (26). responses to HSA (Fig. 5 A and B and refs. 9 and 10). We Haptenated proteins have since been widely used to provide therefore asked whether haptenation of a pure (TLR-ligand defined epitopes for the measurement of antibody titers and free) protein antigen would also be sufficient to induce adaptive affinities. Although protein-haptenation is thought to do little immunity. We first examined the antibody response to DNP- more than create epitopes for B cell recognition, we show here conjugated, endotoxin-free HSA in alum. DNP-HSA in alum, that certain haptenated proteins can induce adaptive immune unlike endotoxin-free HSA, induced a robust IgG1 response responses under conditions where native proteins fail to induce (Fig. 5A). Endotoxin-free HSA in IFA also fails to induce such responses; thus, haptenation does more than simply create robust CD4ϩ T cell responses (Fig. 5B and ref. 9). Therefore, epitopes for antigen receptor recognition. we examined the CD4ϩ T cell response to endotoxin-free In this study, we compared the innate requirements for DNP-HSA in IFA. Endotoxin-free DNP-HSA in IFA, unlike induction of adaptive immune responses to either native or endotoxin-free HSA in IFA, induced a robust CD4ϩ T cell haptenated proteins. We found that TLR-signaling was required response (Fig. 5B). for optimal IgG1, IgG2c and CD4ϩ T cell responses to immu- Notably, unlike HSA plus LPS, DNP-HSA in IFA did not nizations with the native proteins HSA and OVA plus LPS induce robust Ab responses or CD4ϩ T cell responses to emulsified in the depot adjuvant IFA, whereas TLR signaling- nonhaptenated HSA. Instead, immunizations with the hapte- deficient mice exhibited robust IgG1 and CD4ϩ T cell responses nated protein antigen DNP-HSA induced high-titers of DNP- to immunizations with the haptenated proteins DNP-OVA and specific antibodies, and low titers of HSA-specific antibodies DNP-HSA emulsified in IFA. IgG2c responses remained largely (Fig. 5C). In addition, CD4ϩ T cell responses induced by MyD88-dependent regardless of the antigen used for immuni- DNP-HSA were directed primarily toward dinitrophenylated zation. Haptenated proteins induced robust IgG1 responses in peptides as DNP-HSA elicited a strong CD4ϩ T cell restimula- MyD88-deficient mice in response to immunizations performed tion response whereas HSA elicited a very weak restimulation in multiple common adjuvants, including both IFA and alum, response in mice previously immunized with haptenated protein suggesting that this phenomenon is independent of the activity (Fig. 5C). Shimizu et al. (25) recently reported similar results of the depot adjuvant used, but instead depends on the qualities using the hapten oxazalone. Thus, haptenated proteins do not act of haptenated versus native proteins. The ability of haptenated like classical PRR stimulating adjuvants, such as TLR-ligands, proteins to induce robust MyD88-independent adaptive re- which induce responses to the proteins with which they are sponses was shared by multiple haptenated protein preparations, mixed, but instead induce qualitatively different responses that suggesting that this phenomenon is not specific to a particular are predominantly hapten-specific. haptenated protein preparation. Finally, haptenation converted

4784 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0809403105 Palm and Medzhitov Downloaded by guest on September 24, 2021 nonimmunogenic, PAMP-free HSA into a highly immunogenic bypass the requirement for TLRs in the induction of CD4ϩ T cell antigen that induced strong anti-hapten responses, but weak and IgG1 responses, but that the addition of PAMPs can still anti-protein responses. potentiate or otherwise alter adaptive immune responses to Our data thus show that haptenated proteins are immuno- haptenated proteins, such as class switching to IgG2c. genic, whereas native proteins are nonimmunogenic. This im- Haptens were defined by Landsteiner as small molecules that munogenicity is TLR-independent, but results in qualitatively become immunogenic only after association with a carrier different T and B cell responses as compared with immuniza- protein (26). Landsteiner and others, through empirical exam- tions with native proteins plus TLR ligands—although immuni- ination of various reactive chemicals conjugated to a variety of zations with protein plus TLR ligands induce T and B cell peptides and proteins, identified a number of highly immuno- responses to the protein with which they are mixed, haptenated genic (as well as many less immunogenic) hapten-carrier con- proteins induce predominantly hapten-specific T and B cell jugates. These conjugates became antigens of choice for immu- responses and only very weak protein-specific responses (25). nologists largely because of their uniquely potent immunogenic Thus, the nature of the immunogenicity of haptenated proteins activity (29). Thus, the selection of specific haptenated proteins appears to differ from the immunogenicity conferred by PRR as immunogens of choice may parallel the selection of many ligands. Because haptenated proteins possess a unique immu- components of adjuvants that were chosen empirically for their nogenicity and induce qualitatively different responses as com- immunogenicity. pared with native proteins, their use can complicate studies of The mechanism behind the immunogenicity of haptenated adjuvant requirements by occluding the activities of immuno- proteins remains unclear. However, a number of possible mech- genic components of an adjuvant. Furthermore, comparisons of anisms can be imagined. One possibility is that haptenated studies using native proteins and studies using haptenated pro- proteins stimulate adaptive immunity primarily through efficient teins must be made with the unique immunogenicity of hapte- triggering of the Ag receptors, and thereby bypass the require- nated proteins in mind. This consideration is especially impor- ment for innate instruction. One way in which haptenated tant for B cells, which have a remarkable ability to integrate proteins could trigger antibody production is through their multiple signals that collectively determine the efficiency of multivalent nature. Indeed, highly multivalent antigens, such as antibody production (27). T-independent type 2 (TI-2) antigens, bypass the requirement In a recent report, Gavin et al. immunized MyD88 and TRIF for T cell help through efficient cross-linking of the B cell double-deficient mice with trinitrophenyl (TNP;16)-keyhole lim- receptor. TI-1 antigens, in contrast, bypass the requirement for pet hemocyanin (KLH) in multiple adjuvants and concluded that T cell help through activation of innate immune receptors. TLRs play no role in the control of adaptive immunity (19, 20). However, haptenated proteins appear to represent neither such Meyer-Bahlburg et al. (21) examined the role of TLRs on B cells, scenario; instead, B cell responses to haptenated proteins remain using nitrophenyl (NP;37)-chicken gamma globulin (CGG) in T cell-dependent (22). Furthermore, T cell responses are pre- alum, and concluded that TLRs on B cells amplify early antibody sumably impervious to the effects of valency, yet we observed responses, but are not required for robust IgG responses. These robust T cell responses to haptenated proteins in MyD88- conclusions were based on the incorrect assumption that the deficient animals. Finally, B cell responses to physiologically requirements for antibody responses to haptenated and native relevant multivalent antigens, such as polyoma virus (14), influ- proteins are identical. However, our data show that haptenated enza virus (13) and viral like particles (15), are largely MyD88- proteins possess a unique, TLR-independent immunogenicity dependent. Therefore, it seems unlikely that valency alone that can obscure adjuvant requirements. Thus, the conclusions accounts for the immunogenicity of haptenated proteins. made by Gavin et al. (19) and by Meyer-Bahlburg et al. (21) are Differences in the affinities, avidities, and precursor frequen- only applicable to haptenated proteins and cannot be general- cies of antigen specific cells can also affect the requirements for ized to native proteins or natural infections. initiation of adaptive immunity. Because haptenated proteins Common adjuvants have 2 types of activities, both of which are induce mainly anti-hapten responses, and protein plus LPS required to induce a robust adaptive immune response to soluble induces strong anti-protein responses, it is not possible to proteins. One is a depot activity that prevents the dispersion of compare the effect of haptenation on lymphocyte responses to soluble antigens and promotes their uptake by antigen present- identical epitopes. Differences in antigen specific lymphocyte ing cells, and the other is an immunostimulatory activity that precursor frequency or antigen receptor affinities or avidities engages the innate immune system through the triggering of could therefore contribute to some of the differences in immu- PRRs. In the case of Complete Freund’s Adjuvant, for example, nogenicity and specificity we observe between native and hap- mineral oil acts largely to provide a depot, whereas heat-killed tenated proteins. The process of haptenation also likely dena- mycobacteria act to trigger the innate immune system (28). A tures protein antigens, thus promoting their aggregation. This possible explanation for the TLR-independent adjuvant effect could also contribute to the altered immunogenicity of hapte- reported by Gavin et al. (19) is simply the depot effect of the nated antigens and lead to a loss of native protein epitopes for adjuvants, which is naturally not mediated by TLRs or any other antibody recognition. PRRs, combined with the inherent immunogenicity of the Another possible explanation for the unique, TLR- haptenated antigens used in these experiments. independent immunogenicity of haptenated proteins is that they Although IgG1 responses to haptenated proteins appeared engage an alternative innate pathway to adaptive immunity. relatively unchanged between WT and MyD88-deficient mice, Indeed, the T cell response to DNP-HSA is IL-6-dependent (Fig. this does not necessarily mean that TLR ligands do not alter S1), implicating innate control of adaptive responses to hapte- adaptive responses to haptenated proteins. Indeed, CD4ϩ T cell nated proteins. Both TLR-dependent and TLR-independent responses to haptenated proteins remained partially MyD88- paths to adaptive immunity have been described (3, 7, 9–11, dependent; further, in contrast to the report by Gavin et al. (19), 30–32). Haptenated proteins may mimic, either in structure or we observed that IgG2c production in response to haptenated activity, naturally encountered ligands that trigger adaptive proteins was largely MyD88-dependent. Notably, B cell-intrinsic immunity through a TLR-independent innate pathway. Indeed, TLR signaling has been shown to be required for IgG2a/c reactive haptens are known to stimulate the innate immune production in multiple systems, including in response to native system during the induction of T cell-dependent contact hyper- proteins (10), haptenated proteins (this article and ref. 21), virus sensitivity (CHS) (33). The inflammatory cytokine Interleu-

like particles (15), influenza virus (13), and polyoma virus (14). kin-1␤ (IL-1␤) is critical for CHS (34). IL-1␤ family members IMMUNOLOGY We conclude that haptenated proteins, under certain conditions, require processing by Caspase-1 for their activation and secre-

Palm and Medzhitov PNAS ͉ March 24, 2009 ͉ vol. 106 ͉ no. 12 ͉ 4785 Downloaded by guest on September 24, 2021 tion. Caspase-1 activation occurs in a protein complex, referred for immunizations using native protein plus TLR ligands (9, 10) to as an inflammasome, containing a Nod-like receptor, such as and for numerous pathogens of multiple classes (13, 30). How- NALP3, the adaptor protein ASC, and Caspase-1 itself (35). ever, conditions under which TLRs are less important or com- Recently, NALP3 and ASC, through Caspase-1 and IL-1␤, were pletely dispensable for the induction of adaptive immunity also shown to be critical for CHS (36, 37). Furthermore, NALP3 certainly also exist (3, 7, 31, 32), and the full spectrum of and Caspase-1 were also recently shown to be critical for an immunogenic signals and receptors that can lead to adaptive immunostimulatory activity of alum (38–41). Therefore, hapte- immunity remains to be determined. Our data suggest that nated proteins might similarly induce immunity through haptenated proteins represent one such immunogenic signal. Caspase-1 and IL-1␤. However, we found that both CD4ϩ T cell and IgG responses to DNP-HSA appeared normal in Caspase-1 Materials and Methods deficient mice (Fig. S2) and NALP3-deficient mice (Fig. S3). Mice. All mice were bred and maintained at the Yale University School of Thus, if haptenated proteins induce adaptive immunity through Medicine and were used in accordance with Yale Animal Research and Care the induction of an alternate innate immune pathway, it must be guidelines. ASC-deficient mice were made by J. Bertin and A. Coyle and were distinct from the NALP3, Caspase-1, IL-1␤ dependent pathway a kind gift of Millenium Pharmaceuticals through R.A.F.. All mice were back- that is involved in CHS. We did, however, observe partial defects crossed to the C57BL/6 background at least 7 times and were used at 8–14 in T cell and Ab responses to haptenated proteins in mice weeks of age, and MyD88-deficient mice were backcrossed to the C57Bl/6 deficient for the inflammasome adaptor ASC (Fig. S4). These background 10 times. defects were specific for responses to haptenated proteins be- cause responses to native protein and LPS in ASC-deficient mice Reagents and Antibodies. HSA (low endotoxin), OVA, LPS and IFA were were normal. ASC-dependent, Caspase-1 independent activities, purchased from Sigma–Aldrich. Haptenated proteins of the indicated substi- tution ratios were purchased from Biosearch Technologies. Imject alum (alu- including the induction of critical inflammatory cytokines such minum hydroxide) was purchased from Pierce. Antibodies for ELISAs were as IL-6, have also been reported by others (42, 43). purchased from Southern Biotechnology. The cytosolic PRRs NOD1 and NOD2, which signal through the protein kinase Receptor-interacting protein 2 (RIP2), are Immunizations. Mice were immunized in the rear footpads with 50–100 ␮gof also involved in control of adaptive immunity (31, 44). How- protein or haptenated protein with or without 5–10 ␮g of LPS in PBS emulsi- ever, MyD88 and RIP2 double-deficient mice showed no fied 1:1 with IFA. Immunizations with 100 ␮g of protein or haptenated protein defect in antibody responses to haptenated proteins (Fig. S5). with or without 10 ␮g of LPS in alum were performed i.p. The Receptor for Advanced Glycation Endproducts (RAGE), which is implicated in inflammation and control of adaptive Enzyme Linked Immunosorbent Assay. Antigen specific ELISAs were performed immunity, also could potentially be involved in adaptive on sera isolated from immunized mice on day 14. Nunc MaxiSorp 96-well responses to haptenated proteins (45, 46), but we did not plates were coated with native protein to measure anti-protein responses or observe any defects in T cell responses to DNP-HSA in a haptenated protein not used for immunization (DNP (31)-BSA) to measure RAGE-deficient mice (Fig. S6). anti-hapten responses. For each serum sample eight 3-fold serial dilutions Regardless of the mechanism by which haptenated proteins starting at 1:100 were probed. Antibodies were detected using isotype specific induce TLR-independent adaptive responses, it is clear that biotinylated antibodies, Streptavidin-Horseradish Peroxidase (E bioscience) and O-phenyldiamine (Sigma). haptenated proteins differ from native proteins in multiple ways, including the nature of the responses they induce and the innate T Cell Proliferation Assay. CD4ϩ T cells from immunized mice were purified requirements for induction of those responses. from popliteal and inguinal lymph nodes on day 7–10 after immunization, Both antigen recognition and innate immune instruction are using MACS anti-CD4 beads (L3T4; Miltenyi Biotec) and an AutoMACS sorter. required for the activation of adaptive immune responses me- Purified T cells (Ͼ95% CD4ϩ;1ϫ 105) were cultured in 96-well plates with diated by conventional lymphocytes. Several innate immune irradiated (1,000 rad) wild-type splenocytes (3 ϫ 105) and titrating doses of pathways that are sufficient to induce an adaptive immune antigen for 72–84 h. Proliferation was measured by [3H]thymidine incorpo- response are currently known. The requirement for each of these ration during the last 12–16 h of incubation. pathways in the induction of adaptive immunity naturally de- pends on the combination of immunostimulatory components in ACKNOWLEDGMENTS. We thank D. Gray, A. Unni, C. Pasare, D. Schenten, D. a particular pathogen or the immunostimulatory components Stetson, and I. Brodsky for useful discussions and/or for critical review of this manuscript; F. Sutterwalla (University of Iowa, Iowa City) and R.A.F. for the included in an immunization. Adaptive immune responses are NALP3-deficient mice; R.A.F. for the Caspase-1 deficient mice; and R.A.F., John TLR-dependent when TLR ligands are the main innate immu- Bertin, and Anthony Coyle for the ASC-deficient mice. R.M. is an investigator nogenic components of adjuvants or pathogens; this is the case of the Howard Hughes Medical Institute.

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