Two or more TCRs and co-receptors need to be engaged simultaneously to initiate the T cell response, because only if multiple TCRs and co-receptors are brought together can appropriate biochemical signaling cascades be activated -Also, each T cell needs to engage antigen (i.e., MHC-peptide complexes) for a long period, at least several minutes, or multiple times to generate enough biochemical signals to initiate a response. Once these conditions are achieved, the T cell begins its activation program. The TCR recognizes antigens, but it is not able to transmit biochemical signals to the interior of the cell. -The TCR is noncovalently associated with a complex of three proteins that make up CD3 and with a homodimer of another signaling protein called the ζ chain. $$ The TCR, CD3, and ζ chain make up the TCR COMPLEX $$. -In the TCR complex, the function of antigen recognition is performed by the variable TCR α and β chains, whereas the CONSERVED SIGNALING FUNCTION is performed by the attached CD3 and ζ proteins.
On resting naive T cells, which are cells that have not previously recognized and been activated by antigen, the LFA-1 integrin is in a low-affinity state. If a T cell is exposed to chemokines produced as part of the innate immune response to infection, that T cell's LFA-1 molecules are converted to a high-affinity state and cluster together within minutes -Antigen recognition by a T cell also increases the affinity of that cell's LFA-1. Therefore, once a T cell sees antigen, it increases the strength of its binding to the APC presenting that antigen, providing a positive feedback loop. CD28 receptor, which is expressed on virtually all T cells **Signals from CD28 on T cells binding to B7 on APCs work together with signals generated by binding of the TCR and co-receptor to peptide-MHC complexes on the same APCs.
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CD28-mediated signaling is essential for initiating the responses of naive T cells; -in the absence of CD28-B7 interactions, engagement of the TCR alone is unable to activate the T cells. $$ The requirement for costimulation ensures that naive T lymphocytes are activated fully by microbial antigens, and not by harmless foreign substances, because, as stated previously, microbes stimulate the expression of B7 costimulators on APCs (harmless foreign substances DO NOT)%%.
And are involved in the recognition of peptide antigens presented on class II major histocompatibility complex (MHC) of antigen-presenting cells (APCs) through the T-cell receptor (TCR). Mounting evidence indicates that T-cell activation is incomplete when initiated by the TCR signal alone, and that the costimulatory CD28. In addition, we analyzed broad trends in phosphorylation data sets to uncover underlying mechanisms associated with T cell activation. We found that, upon stimulation of the TCR, phosphorylation events extensively targeted protein modules involved in all of the salient phenomena associated with T cell activation:.
*recognition of class I MHC-associated peptides and REQUIRES costimulation and/or helper T cells 1) An unusual feature of CD8+ T cell activation is that its initiation often requires that cytoplasmic antigen from one cell (e.g., a virus-infected cell) has to be CROSS-PRESENTED by dendritic cells. $$$$$$$When virus-infected cells are ingested by host dendritic cells and the viral antigens are cross-presented by the APCs, the same APC may present antigens from the cytosol in complexes with class I MHC molecules and from vesicles in complex with class II MHC molecules. Thus, BOTH CD8+ T cells and CD4+ T cells specific for viral antigens are activated near one another$$$$$$ 2) Another characteristic of CD8+ T cells is that their differentiation into CTLs may require the concomitant activation of CD4+ helper T cells.
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A protein kinase called Lck (that is non-covalently attached to the cytoplasmic tails of these co-receptors) ---> several transmembrane signaling proteins are associated with the TCR, including the CD3 and ζ chains. CD3 and ζ contain tyrosine-rich motifs, called immunoreceptor tyrosine-based activation motifs (ITAMs), that are critical for signaling. 1) Lck, which is carried near the TCR complex by the CD4 or CD8 molecules, phosphorylates tyrosine residues contained within the ITAMs of the ζ and CD3 proteins.
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2) The phosphorylated ITAMs of the ζ chain become docking sites for a tyrosine kinase called ZAP-70 (ζ-associated protein of 70 kD), which also is phosphorylated by Lck and thereby made enzymatically active. 3) The active ZAP-70 then phosphorylates various ADAPTER PROTEINS and ENZYMES, which assemble near the TCR complex and mediate additional signaling events. -->>> (3 pathways) 4) Three major signaling pathways linked to ζ chain phosphorylation and ZAP-70 are: -the calcium-NFAT pathway, -the Ras/Rac-MAP kinase pathway, -the PKC-NF-κB pathway. **NFAT isa transcription factor whose activation is dependent on Ca2+ ions.
1) The calcium-NFAT pathway is initiated by ZAP-70-mediated phosphorylation and activation of an enzyme called phospholipase Cγ (PLCγ), which catalyzes the hydrolysis of a plasma membrane inositol phospholipid called phosphatidylinositol 4,5-bisphosphate (PIP2). 2) One byproduct of PLCγ-mediated PIP2 breakdown, called inositol 1,4,5-triphosphate (IP3), stimulates release of Ca2+ ions from the endoplasmic reticulum, thereby raising the cytoplasmic Ca2+ concentration. 3) In response to the elevated calcium, a plasma membrane calcium channel is opened, leading to the influx of extracellular Ca2+ into the cell, which sustains the elevated Ca2+ concentration for hours.
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4) Cytoplasmic Ca2+ binds a protein called calmodulin, and the Ca2+-calmodulin complex activates a phosphatase called CALCINEURIN. This enzyme removes phosphates from NFAT, which resides in the cytoplasm. 5) Once dephosphorylated, NFAT is able to migrate INTO THE NUCLEUS, where it binds to and activates the promoters of several genes, including the genes encoding the * T cell growth factor interleukin-2 (IL-2) and components of the IL-2 receptor*. 1) These pathways are initiated by ZAP-70-dependent phosphorylation and accumulation of adapter proteins at the plasma membrane, leading to the recruitment of Ras or Rac, and their activation by exchange of bound guanosine diphosphate (GDP) with GTP.
2) Ras•GTP and Rac•GTP initiate different enzyme cascades, leading to the activation of distinct MAP kinases. 3) The terminal MAP kinases in these pathways, called extracellular signal-regulated kinase (ERK) and c-Jun amino-terminal (N-terminal) kinase (JNK), promote the expression of a protein called c-Fos and the phosphorylation of another protein called c-Jun. 4) c-Fos and phosphorylated c-Jun combine to form the transcription factor AP-1 (activating protein-1), which enhances the transcription of several T cell genes. 1) PKC is activated by diacylglycerol (DAG), which, like IP3, is generated by phospholipase C-mediated hydrolysis of membrane inositol lipids. (ultimately from ZAP-70 process) 2) PKC acts via adapter proteins that are recruited to the TCR complex to activate NF-κB.
3) NF-κB exists in the cytoplasm of resting T cells in an inactive form, bound to an inhibitor called IκB. 4)TCR-induced signals, downstream of PKC, activate a kinase that phosphorylates IκB and targets it for destruction. 5) As a result, NF-κB is released and moves to the nucleus, where it promotes the transcription of several genes. **CD40 ligand (CD40L). -The CD40L gene is transcribed in CD4+ T cells in response to antigen recognition and costimulation, and the result is that CD40L is expressed on helper T cells after activation.
-->It binds to its receptor, CD40, which is expressed mainly on macrophages, B lymphocytes, and dendritic cells. -->Engagement of CD40 activates these cells, and thus CD40L is an important participant in the activation of macrophages and * B LYMPHOCYTES* by helper T cells ALSO: the interaction of CD40L on T cells with CD40 on DENDRITIC CELLS (APCs) stimulates the expression of costimulators on these APCs and the production of T cell-activating cytokines (IL-12; by the APCs themselves), thus providing a POSITIVE FEEDBACK (amplification) mechanism for APC-induced T cell activation. *stimulate phagocyte-mediated ingestion and killing of microbes, a key component of cell-mediated immunity -The most important cytokine produced by TH1 cells is interferon-γ (IFN-γ), so called because it was discovered as a cytokine that inhibited (or interfered with) viral infection.
Its 3 functions: 1) IFN-γ is a potent activator of MACROPHAGES - enhanced microbial killing 2) IFN-γ also stimulates the production of antibody isotypes that promote the phagocytosis of microbes, because these antibodies bind directly to phagocyte Fc receptors, and they ACTIVATE COMPLEMENT, generating products that bind to phagocyte complement receptors. 3)IFN-γ also stimulates the expression of class II MHC molecules and B7 costimulators on macrophages and dendritic cells, and this action of IFN-γ may serve to amplify T cell responses. *stimulate phagocyte-independent, EOSINOPHIL-MEDIATED immunity, which is especially effective against HELMINTHIC PARASITES -TH2 cells produce IL-4, which stimulates the production of IgE antibodies, -and IL-5, which activates eosinophils. -IgE activates mast cells and binds to eosinophils.
-In addition, some of the cytokines produced by TH2 cells, such as IL-4 and IL-13, promote the expulsion of parasites from mucosal organs and inhibit the entry of microbes by STIMULATING MUCUS SECRETION. This type of host defense sometimes is called 'barrier immunity' because it blocks the entry of microbes at mucosal barriers. **cytokines of TH2 cells also activate macrophages -Unlike TH1-mediated activation, which stimulates the ability of macrophages to kill ingested microbes, TH2-mediated macrophage activation enhances other functions, such as synthesis of extracellular matrix proteins involved in TISSUE REPAIR. -This type of response has been called 'alternative' macrophage activation.
1, Ning Wu 1, 1,2, Xin Liu 1, 1, Yifan Zhou 1, Xin Liu 3, Jiaoyan Huang 1, 3* and 1* • 1Institute for Immunology, Tsinghua-Peking Center for Life Sciences, Tsinghua University School of Medicine, Beijing, China • 2College of Veterinary Medicine, Shandong Agricultural University, Tai’an, Shandong, China • 3Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Tsinghua University School of Pharmaceutical Sciences, Beijing, China Dendritic cells (DCs) are highly specialized antigen-presenting cells that play crucial roles in innate and adaptive immunity. Previous studies suggested that Toll-like receptor (TLR) agonists could be used as potential adjuvants, as activation of TLRs can boost DC-induced immune responses. TLR2 agonists have been shown to enhance DC-mediated immune responses.
However, classical TLR2 agonists such as Pam3CSK4 are not stable enough in vivo, which limits their clinical applications. In this study, a novel structurally stable TLR2 agonist named SUP3 was designed.
Functional analysis showed that SUP3 induced much stronger antitumor response than Pam3CSK4 by promoting cytotoxic T lymphocytes activation in vivo. This effect was achieved through the following mechanisms: SUP3 strongly enhanced the ability of antigen cross-presentation by DCs and subsequent T cell activation. SUP3 upregulated the expression of costimulatory molecules on DCs and increased antigen deposition in draining lymph nodes. More interestingly, SUP3 induced less amount of pro-inflammatory cytokine production in vivo compared to other TLR agonists such as lipopolysaccharide. Taken together, SUP3 could serve as a novel promising immune adjuvant in vaccine development and immune modulations.
Specificity and activity of novel TLR2 agonist SUP3. (A) Schematic chemical structure of SUP3. (B) Surface expression of TLR2 in wild-type (WT) and Tlr2 −/− RAW 264.7 cell lines. Gray shadows represented isotype control and black lines represented TLR2. (C) TNFα production by RAW 264.7 cells upon stimulation with lipopolysaccharide (LPS) (1 µg/mL), Pam3CSK4 (Pam3 hereafter) (500 nM), or SUP3 (500 nM) for 8 h. (D) GMDCs were generated by culturing bone marrow cells with granulocyte-macrophage colony-stimulating factor and interleukin-4 for 7 days, then activated by LPS (20 ng/mL), Pam3 (200 nM), or SUP3 (200 nM) for 24 h.
The production of cytokines IL-6 and TNFα in culture supernatant was determined by ELISA. To validate the specificity of SUP3 for TLR2, we generated Tlr2 −/− RAW 264.7 cells with CRISPR-Cas9 system (Figure B).
Both Pam3 and SUP3 stimulation induced large amount of TNFα production by WT RAW cells, indicating that SUP3 could function similarly as Pam3 in triggering TLR2 response (Figure C). TLR2 deficiency abolished the effect of Pam3 and SUP3 on RAW cells, evidenced by no secretion of TNFα by Tlr2 −/− RAW cells upon challenge by Pam3 and SUP3 (Figure C). It demonstrated that TLR2 was the receptor for SUP3. To further determine the bioactivity of SUP3 as a TLR2 agonist, we determined the cytokine production by GMDCs upon TLR2 stimulation.
GMDCs were generated from bone marrow cells cultured in the presence of GM-CSF and interleukin-4 (, ). It has been demonstrated that GMDCs are potent pro-inflammatory cytokine producers upon TLR stimulation and GMDCs express TLR2 and respond to TLR2 ligands.
We found that SUP3 exhibit equal ability as Pam3 in activating GMDCs, as the production of IL-6 and TNFα were comparable by GMDCs upon stimulation by SUP3 and Pam3 (Figure D). Taken together, these data suggested that SUP3, the newly designed TLR agonist, engaged TLR2 specifically as well as Pam3.
SUP3 Induced More Vigorous CTL Responses Than Pam3 without Excessive Production of Inflammatory Cytokines In Vivo The consequence of immunization or vaccination is the acquirement of preventive or therapeutic effects on infections or tumors, via generating antigen-specific adaptive immune responses and memory. Elimination of tumors or intracellular infection requires antigen-specific CD8 + CTLs, the major executors in cellular immunity (, ). In mice, the differentiation and activation of CD8 + CTL is launched through antigen cross-presentation by CD8 + cDC (, ).
Some TLR agonists have been shown to enhance antigen cross-presentation by cDCs and the subsequent CD8 + T cell activation (,, –). To investigate whether SUP3 has an effect on CTL responses, we determined the activity of SUP3 in antigen-specific cellular immunity. Mice were immunized by intravenous injection of OVA coated splenocytes (OCS), as these cell-associated OVA antigens can be cross-presented efficiently by CD8 + cDC to CD8 + T cells ().
Seven days post-immunization, the percentages of OVA antigen-specific CD8 + T cells in spleens were determined by pentamer (SIINFEKL/H-2Kb) staining. The effects of various doses of SUP3 and Pam3 on the induction of CTL were determined and 2 nmol was selected as the optimal dose in the following assays (Figures S4A,B in Supplementary Material). Administration of SUP3 at a high dose of 10 nmol resulted in decreased CTL induction and enlarged spleens, whereas treatment with Pam3 at the same dose did not have the same effect (data now shown), indicating that SUP3 was much more potent than Pam3 and the mice could not tolerate such a high dose of agonists. We found that OCS formulated with SUP3 induced significantly more antigen-specific CD8 + T cells than that with Pam3 in vivo (Figures A,B). To further determine whether the enhanced CTL activation by SUP3 can lead to more vigorous antitumor activity, we immunized mice as described above and then challenged by intravenous inoculation of OVA-expressing B16 melanoma cells (B16-OVA) (). We found that mice immunized with OCS and SUP3 showed markedly reduced number of tumors in the lungs compared to those with OCS alone or OCS with Pam3 16 days after the challenge (Figures C,D).
It indicated that immunization with SUP3 conferred more efficient CTL responses, thus more effective protection against tumor formation. Taken together, these results demonstrated that SUP3 was far more effective than Pam3 in inducing CTL responses in vivo.
SUP3 induced more vigorous cytotoxic T lymphocytes (CTLs) responses than Pam3CSK4 without excessive production of inflammatory cytokines in vivo. (A,B) Mice were immunized by i.v. Injection of ovalbumin (OVA)-coated splenocytes (OCS) (2 × 10 7 cells per mouse) alone, OCS with SUP3 (2 nmol per mouse) or Pam3 (2 nmol per mouse) for 7 days. The percentages of pentamer positive cells (CD19 − CD3 + CD8 + SIINFEKL/H-2Kb-Pentamer +) in spleens, i.e., the OVA-specific CD8 + T cells, were determined by flow cytometry (A) and the statistic calculation is presented in (B).
Representative data from three independent experiments with three mice in each group were presented. (C,D) Mice were immunized as in (A) for 30 days, then inoculated with OVA-expressing B16 melanoma cells (B16-OVA) (5 × 10 5 cells per mouse) by i.v. Injection and analyzed for metastatic tumor formation in the lung 16 days after inoculation. Data from two independent experiments with three mice in each group were shown. Representative tumor formations in the lung from each group were displayed in (C), and the number of metastatic tumors was calculated in (D). (E) Mice were i.v.
Injected with SUP3 (2 nmol per mouse), Pam3 (2 nmol per mouse), or lipopolysaccharide (LPS) (1 µg per mouse) for 1 h, the serum was collected, and the levels of different cytokines were determined by ELISA. “n.d.” indicates “not detected.” * p.
SUP3 facilitated cross-presentation of ovalbumin (OVA) antigen by CD8 + conventional dendritic cells (cDCs) in vitro. (A–C) Purified splenic CD8 + cDCs were stimulated with or without Pam3 (200 nM) or SUP3 (200 nM) for 4 h, then pulsed by OVA protein (20 µg/mL) for another 4 h. Cells were washed and cocultured with carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled OT-I CD8 + T cells for 60 h and analyzed by flow cytometry.
(A) The dividing OT-I cells were measured by reduced level of CFSE-labeling. Representative data from three independent experiments with triplicated samples for each group was shown.
“No OVA” (gray shadow) represented negative control that untreated DCs cocultured with OT-I. “Indicated” (solid line) represented DCs pulsed by peptide SIINFEKL (the first), or OVA proteins (the rest) which were also pretreated by PBS, Pam3, or SUP3, respectively. (B) Absolute numbers of proliferated OT-I T cells were calculated. (C) Interferon-γ from the supernatant of coculture was determined by ELISA 24 h later. Pooled data of three independent experiments with triplicated samples for each group are shown. (D,E) Purified splenic CD8 + cDCs were pretreated by Pam3 (200 nM), SUP3 (200 nM), or PBS for 4 h, then incubated with OVA-Alexa Fluor (AF) 488 (5 µg/mL) at 37°C or on ice (negative control) for 30 min. The cells were then washed with cold PBS and analyzed by flow cytometry (D).
The mean fluorescence intensities (MFI) of AF488 were shown in (E). Exogenous antigens are internalized, degraded into short peptides, uploaded onto MHC-I and finally transported to cell surface for recognition by cognate T cells (–). The first step of antigen presentation is internalization of extracellular materials. To clarify whether antigen uptake process is affected by TLR2 engagement, we determined the endocytosis of fluorescence-conjugated soluble OVA (OVA-Alexa Fluor 488, OVA-AF488) by CD8 + cDCs. The results showed that the uptake of OVA by CD8 + cDCs was not influenced by TLR2 activation (Figures D,E), indicating that enhanced antigen presentation by CD8 + cDCs was not due to facilitated antigen uptake process. SUP3 Upregulated Expression of Costimulatory Molecules on CD8 + cDCs and Induced Low Level Production of Inflammatory Cytokines Costimulatory molecules, which serve as the second signal for T cell activation, can be efficiently upregulated upon TLR activation. To investigate whether SUP3 affected the expression of costimulatory molecules on CD8 + cDCs, we stimulated purified DCs by SUP3 or Pam3 in vitro and stained with antibodies to costimulatory molecules as well as MHC molecules.
We found that both SUP3 and Pam3 induced substantial upregulation of CD40, CD80, CD86, and MHC-II on CD8 + cDCs, but not MHC-I (Figure A), demonstrating that SUP3 induced effective costimulation as well as Pam3 in vitro. We then tested whether SUP3 had similar effects on splenic CD8 + cDCs in vivo. Upon in vivo administration of SUP3 or Pam3 via intravenous (i.v.) route, the expressions of CD40 and CD86 increased on splenic CD8 + cDCs from both SUP3- or Pam3-treated mice, indicating the ability of SUP3 to promote DC activation in vivo (Figure B). SUP3 stimulated maturation of CD8 + conventional dendritic cells (cDCs). (A) Purified CD8 + cDCs were treated with SUP3 (200 nM) or Pam3 (200 nM) in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) (20 ng/mL) for 24 h. The surface expression of costimulatory and major histocompatibility complex (MHC) molecules were then analyzed by flow cytometry.
Representative data from three independent experiments were shown. “Isotype” represented relative isotype antibody staining of Pam3 or SUP3 stimulated cells. (B) SUP3 (2 nmol per mouse), Pam3 (2 nmol per mouse), or PBS were i.v.
Injected into mice for 1 h. Splenic CD8 + cDCs were enriched, and surface expression of CD40 and CD86 was analyzed by flow cytometry. Representative data from two independent experiments with three mice for each group were shown.
(C) Purified CD8 + cDCs were treated with SUP3 (200 nM) or Pam3 (200 nM) for indicated periods (0~120 min), and western blot analysis was performed with the cell lysate for phosphorylated p65 (p-p65), IκBα, p-ERK, ERK, p-JNK, JNK, p-p38, and p38. Blot of p38 was also regarded as loading control. (D) Purified CD8 + cDCs were treated with CpG 1668 (20 nM), SUP3 (200 nM), or Pam3 (200 nM) in the presence of GM-CSF (20 ng/mL). Supernatants were harvested 24 and 48 h post-stimulation, and cytokines were measured by ELISA. Representative data from two independent experiments with triplicated samples for each group were shown.
Toll-like receptor stimulation is required for functional maturation of DCs, which is essential for T cell activation and initiation of adaptive immunity. Antigen recognition without danger signals like TLR ligands leads to tolerance but not antigen processing for subsequent presentation (, ). It is well established that TLR-induced activation of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling events contribute to activation of responding cells. To determine whether SUP3 could activate classical Toll-dependent signaling pathways in CD8 + cDCs, we treated purified CD8 + DCs by TLR2 agonists at various time points (0–120 min) and detected the expression of the relevant signaling molecules and their phosphorylated forms by immunoblotting. We found SUP3 stimulation induced phosphorylation of p65 and degradation of IκBα and enhanced phosphorylation of ERK, JNK, and p38 in CD8 + cDCs (Figure C), indicating that similar to Pam3, SUP3 was able to induce activation of NF-κB and MAPK pathways. Taken together, SUP3 could induce activation of canonical TLR signaling events in CD8 + cDCs.
In addition to costimulation, secretion of cytokines by DCs is another major feature of DC activation upon TLR engagement. Cytokines like bioactive IL-12p70 predominately produced by CD8 + cDCs drive functional differentiation of activated T cells and provide the third signal for DC-induced T cell activation ().
As SUP3 could facilitate CD8 + T cell activation (Figures A–C), we therefore examined whether SUP3 could induce inflammatory cytokines production by CD8 + cDCs. We activated purified CD8 + cDCs by SUP3, Pam3, or CpG in vitro and quantified the amounts of cytokines in the supernatant by ELISA. Interestingly, in contrast to CpG stimulation which induced large amount of IL-12p70, IL-6, and TNFα production by CD8 + cDCs, SUP3 and Pam3 induced no IL-12p70 and only low levels of IL-6 and TNFα (Figure D). These results demonstrated that SUP3 acted as a mild agonist in inflammatory responses. Collectively, our results demonstrated that SUP3 acted as an efficient enhancer of antigen cross-presentation by CD8 + cDCs through upregulating surface costimulatory molecules and promoting activation of NF-κB and MAPK signaling pathways, without inducing large amount of inflammatory cytokines. Thus, SUP3 fulfilled the properties of a good adjuvant with strong immunostimulatory capacity for T cell activation and minimal induction of inflammatory response. SUP3 Enhanced CD4 + T Cell Activation and Production of Antibodies against TD Antigens In addition to cellular immunity, the other arm of adaptive immunity is humoral immunity mediated by antibodies.
Production of TD antigen-specific antibodies requires the assistance of Th2 subtypes, which is mainly induced by CD11b + cDCs (,, ). Exogenous antigens are routinely presented via MHC-II pathway by CD11b + cDCs and ultimately recognized by CD4 + T cells for helper T cell differentiation ().
CD11b + cDCs expressed comparable levels of TLR2 to that of CD8 + cDCs (Figure S5A in Supplementary Material). We predicted that SUP3 might enhance the functions of CD11b + cDCs thus Th2-mediated production of TD antigen-specific antibodies. To test this hypothesis, we immunized mice via intraperitoneal (i.p.) injection of TD antigen NP-KLH with or without TLR agonists. Formulation with alum was regarded as positive control in this setting, as alum is always used as an efficient adjuvant in antibody induction. Serum antibody titers were determined every week post-immunization. As expected, SUP3 induced higher titers of TD antigen-specific antibodies including IgM, total IgG and high affinity IgG than Pam3 did (Figure A). This result suggested that SUP3 remained functioning for a longer time than Pam3 in vivo, thus resulting in higher levels of antibody production.
SUP3 enhanced antigen presentation ability of CD11b + conventional dendritic cells (cDCs) and production of antibodies against thymus-dependent (TD) antigens. (A) Mice were immunized by TD antigen nitrophenyl-keyhole limpet hemocyanin (NP-KLH) (5 µg per mouse) with or without SUP3 (2 nmol per mouse), Pam3 (2 nmol per mouse), or alum (3 µg per mouse) as adjuvants, respectively. Serum IgM (nitrophenyl 30mer-captured, NP30-captured), total IgG (NP30-captured), and high affinity IgG (nitrophenyl 8mer-captured, NP8-captured) specific to NP at indicated time points were examined by ELISA. (B,C) Carboxyfluorescein diacetate succinimidyl ester-labeled OT-II CD4 + T cells (2 × 10 6 cells per mouse) were transferred into mice 1 day prior immunization. Then the recipients were subcutaneously (s.c.) injected with ovalbumin (OVA) alone (100 µg per mouse), or OVA (100 µg per mouse) mixed with SUP3 (2 nmol per mouse) or Pam3 (2 nmol per mouse) at groin. Three days after immunization, division of OT-II CD4 + T cells from inguinal lymph nodes were analyzed by flow cytometry (B).
Absolute numbers of proliferated OT-II CD4 + T cells were calculated in (C). Pooled data were from two independent experiments with three mice in each group.
“No OVA” (gray shadow) represented negative control that untreated DCs cocultured with CSFE-labeled OT-II. “Indicated” (solid line) represented DCs pulsed by peptide OVA323 (the first), or OVA proteins (the rest) which were also pretreated by PBS, Pam3, or SUP3, respectively.
(D,E) Purified splenic CD11b + cDCs were stimulated by Pam3 (200 nM) or SUP3 (200 nM) for 4 h then pulsed by OVA (20 µg/mL) for another 4 h. Cells were then thoroughly washed and cocultured with CFSE-stained OT-II CD4 + T cells for 4 days and analyzed by flow cytometry. (D) The division of OT-II cells was measured by reduced level of CFSE-labeling. (E) Absolute numbers of proliferated T cells from (D) were calculated. (F) SUP3 (2 nmol per mouse), Pam3 (2 nmol per mouse), or PBS were i.v. Injected into mice for 1 h, and splenic CD11b + cDCs were enriched and analyzed for CD86 expression by flow cytometry. “Isotype” represented relative isotype antibody staining of Pam3 or SUP3 stimulated cells.
(G) Purified CD11b + cDCs were activated by SUP3 (200 nM), Pam3 (200 nM), or CpG (20 nM) in granulocyte-macrophage colony-stimulating factor (20 ng/mL) containing medium for 24 h. The cytokines from supernatant were determined by ELISA (* p. As mentioned above, CD4 + Th2 cells polarized by CD11b + cDCs are critical in TD antigen-specific antibodies production. Thus, SUP3 might enhance TD antibody production via facilitating CD4 + T cell activation in vivo. To test this hypothesis, purified CD4 + OT-II T cells from spleens of OVA-specific TCR transgenic mice were labeled by CFSE and transferred into WT mice for 1 day, followed by subcutaneous (s.c.) immunization with soluble OVA proteins with TLR agonists for another 3 days. Treatment with SUP3 induced more proliferation of OT-II CD4 + T cells than that with Pam3 in draining lymph nodes (Figures B,C), demonstrating the ability of SUP3 to induce stronger CD4 + T cell activation in vivo, thus leading to enhanced TD antibodies production.
SUP3 Facilitated Antigen Presentation by CD11b + cDCs In Vitro As CD4 + T cell activation is enhanced upon SUP3 treatment, we speculated that SUP3 functioned through improving functions of CD11b + cDCs. To test this speculation, we evaluated OVA antigen presentation by SUP3 pretreated CD11b + cDCs to OT-II CD4 + T cells. An enhanced OT-II CD4 + T cell proliferation was observed upon stimulation of CD11b + cDCs by SUP3 in vitro (Figures D,E), indicating that SUP3 strengthened antigen presentation ability of CD11b + cDCs. The maturation of CD11b + cDC is marked by upregulation of costimulatory molecules. Intravenous administration of SUP3 and Pam3, both induced maturation of splenic CD11b + cDC indicated by CD86 upregulation (Figure F).
Furthermore, similar to CD8 + cDCs, CD11b + cDCs produced lower level of IL-6, TNFα, and MIP1α upon stimulation by SUP3 and Pam3, implying a weak ability of SUP3 in inducing inflammatory responses (Figure G). Collectively, these results demonstrated that SUP3 facilitated antigen presentation by CD11b + cDCs and subsequent activation of OT-II CD4 + T cells, which led to higher levels of TD antibody production in vivo.
These observations again demonstrated the physiological advantages of SUP3 over Pam3 as an immune adjuvant. SUP3 Enhanced Antigen Deposition in APCs in Draining Lymph Nodes As immune adjuvants enhance immunogenicity of simultaneously administrated antigens via increasing antigen deposition in lymph nodes, and prolonging antigen presentation (, ), we proposed that SUP3 might prolong the duration of antigen deposition in draining lymph nodes.
To test this, we s.c. Immunized mice with OVA-AF647 at groin, or co-administrated with either SUP3 or Pam3 for 24 h. Antigen deposition in various immune cell populations from inguinal lymph nodes was then assessed. After immunization of OVA with SUP3, increased numbers of antigen-containing CD8 + cDCs, CD11b + cDCs, macrophages, and monocytes were observed compared to those with OVA alone or OVA with Pam3 (Figures A,B). Moreover, the total number of cells containing antigens increased more upon administration of SUP3 over Pam3 (Figure B). This finding indicated that SUP3 facilitated antigen deposition in cDCs, macrophages and monocytes in draining lymph nodes, thus contributed to enhanced T cell activation. SUP3 enhanced antigen deposition in draining lymph nodes.
Mice were immunized at groin by s.c. Injection of ovalbumin (OVA)-Alexa Fluor 647 (AF647) (25 µg per mouse) with or without SUP3 (2 nmol per mouse) or Pam3 (2 nmol per mouse) for 24 h. Inguinal lymph nodes were isolated, and immune cells were analyzed for levels of AF647 fluorescence by flow cytometry (A).
The number of OVA-AF647 positive cells was calculated in (B). Representative data were from two independent experiments with three mice in each group. SUP3 Directly Promoted Stronger B Cell Activation In Vitro B lymphocytes can function as APCs that directly respond to invading pathogens through recognition via innate sensors. In addition to myeloid cells including DCs, macrophages, and monocytes, B cells are only lymphocytes positive for TLR2 (Figure S5A in Supplementary Material). TLR ligation on B cells drives fast IgM production to eliminate pathogens before the formation of effective antibody-mediated adaptive immunity, which requires 5–7 days to develop (). The direct TLR triggered IgM production by B cells, as a thymus independent (TI) pathway, compensates the delayed formation of humoral immunity to control pathogens.
To evaluate B cell response to SUP3, we analyzed the expression of activation markers and production of IgM upon treatment by SUP3 in vitro. Engagement of TLR2 by SUP3 and Pam3 induced marked upregulation of CD25, CD40, and CD86 on B lymphocytes (Figure A), implying a direct effect of SUP3 on B cell activation.
In addition, more antigen-independent production of IgM was induced by SUP3 than that induced by Pam3 (Figure B), indicating that SUP3 could induce a stronger and longer lasting B cell response than Pam3. This finding is in line with the better stability of SUP3 compared to Pam3. Our results demonstrated that TLR2 agonists could enhance B cell function not only in T cell-dependent but also in T-independent manners. SUP3 promoted stronger B cell activation in vitro.
Purified splenic B cells (2 × 10 6 cells/mL) were treated with SUP3 (200 nM), Pam3 (200 nM), or lipopolysaccharide (LPS) (1.5 µg/mL), respectively. (A) Surface CD25, CD40, and CD86 were stained 24 h post-stimulation. (B) Supernatants were harvested 3 or 5 days post-stimulation, and total IgM in the supernatant was determined by ELISA. Representative data was from two independent experiments with triplicated wells in each group (** p. Discussion Our newly designed TLR2 agonist SUP3 is structurally similar to classical TLR2 ligand Pam3. The chemical modification by replacing oxygen with nitrogen in Pam3 stabilizes the covalent bonds between glycerol backbone and palmitic acid chains.
In addition, glycine instead of lysine reduces the chiral center and further simplifies the structure. These improvements overcome current disadvantages of Pam3, such as weak stability and structural complexity.
Meanwhile, the specificity and affinity to TLR2 and bioactivity to promote TLR2-dependent cytokines are retained. Moreover, the peptide components of SUP3 make it easy and convenient to be conjugated with antigenic peptides. This type of conjugation could easily confer adjuvant activity to the conjugated antigens and induce markedly enhanced immune responses toward antigens without additional adjuvant formulations (,,, ). Intriguingly, this special feature is not shared with any other TLR agonists except those of TLR2. Adjuvants of vaccines are defined as components capable of enhancing and shaping antigen-specific immune responses (). Ideal adjuvants should possess properties including safety and effectiveness with defined and well-controlled functions (). An effective adjuvant should help to reduce required dose of antigens or the number of immunizations and have significant improvement in antibody- as well as cell-mediated immune responses ().
Although both SUP3 and Pam3 possess these features of an ideal adjuvant, SUP3 is chemically more stable and structurally simpler than Pam3. SUP3 also exhibited clear advantages compared to Pam3 in biological activities, suggesting that SUP3 could be a good substitution of Pam3 in vaccinations and cancer treatment. Recognition of microbial or viral components by PRRs on DCs elicits immediate host response and ultimately long-lasting adaptive immunity (). DCs are one of the first encounters for non-self components, such as pathogens and tumor antigens. The initial responses of DCs to these pathogens determine the shape of adaptive immunity.
Thus DCs occupy an indispensable position in orchestrating immune responses to eliminate pathogens and tumors. Consequently, immune modulations targeting DCs could be an effective strategy to harness immune system to endow protective immunity.
Cancer immunotherapy with DCs is favorable due to fewer side effects, although with limited benefit observed from recent studies (). Enhancement of adaptive immunity is a key parameter of adjuvants in the context of vaccines.
Shaping adaptive immunity always requires several days of time. In this study, we observed that more antigen-specific CTLs were generated in the presence of SUP3 than that with Pam3 during the induction of effector T cell differentiation by OCS in vivo. Consistent with this observation, the presence of SUP3 induced much stronger protective response against tumor challenge, as revealed by significantly reduced tumor formation in the lungs. Those effects of SUP3 could be achieved through enhancing antigen cross-presentation by CD8 + cDCs, which are responsible for CTL induction and subsequent clearance of tumor cells.
SUP3 significantly enhanced the surface expression of costimulatory molecules on cDCs, providing one of the essential signals for T cell activation. Intriguingly, SUP3 enhanced antigen cross-presentation by CD8 + cDCs was accompanied by a low to moderate levels of inflammatory cytokines, which is different from effects of many other TLR agonists used as adjuvant such as LPS. This functional feature of SUP3 can be considered as an advantage for a good adjuvant, as over production of inflammatory cytokines could be detrimental. Altogether, SUP3 can serve as a potential adjuvant in therapeutic or prophylactic vaccines.
Antigen-specific antibody-mediated humoral immunity is the other arm of adaptive immunity. Indeed, SUP3 not only enhances CTL-mediated cellular immunity but also facilitates the production of various types of specific antibodies to TD antigens. As mentioned previously, TD antibody production requires effector CD4 + T cells activated through MHC-II antigen presentation pathway mainly promoted by CD11b + cDCs.
Addition of SUP3 enhanced antigen presentation ability of CD11b + cDCs in in vitro assays and administration of SUP3 enhanced CD4 + T cell activation in vivo. Thus, SUP3 induced more antibody production than Pam3 in T-dependent manner through enhancing antigen presentation by CD11b + cDCs. Moreover, an enhanced TI antibody production by B cells was also observed after SUP3 administration. B cells are capable of recognizing and responding to pathogens directly via innate immune receptors like TLR. TLR activation of B cells could ultimately result in IgM secretion, which rapidly neutralizes pathogens, namely the TI antibody response ().
In this study, SUP3 directly promoted IgM production by B cells, suggesting its effects on both innate and adaptive immunity. Overall, SUP3 enhanced cellular immunity as well as TD and TI antibody production. This functional property of SUP3 made it a promising ideal adjuvant in immunotherapy and vaccination.
The more vigorous immune stimulatory activities of SUP3 compared to Pam3 in CTL induction, rejection of B16-OVA tumors, CD4 + T cell activation, and the production of TD antibodies could all attribute to its better stability and sustainable activity over Pam3 in vivo. Thus, SUP3 could act as an effective adjuvant in immunization. An ideal adjuvant should have minimum or even none adverse effects. Generally, TLR agonists are potent immune stimuli capable of inducing large amount of inflammatory cytokines, which may lead to cytokine storm to damage the hosts before inducing protective immune responses. Our study and previous studies showed that engagement of TLR2 by triacyl lipopeptides did not promote severe inflammatory responses, which would be beneficial for its clinical applications (,, ).
Ligation of TLR2 also activated PI3K–Akt–β-catenin axis to induce IL-10 expression, which is a regulatory cytokine to suppress inflammation (). TLR2 agonists cloud also induce rapid degradation of IRAK1, which is an essential component of inflammatory response (). These effects might contribute to the weaker inflammatory properties of TLR2 agonists. In addition, TLR2 ligation signal via adaptor MyD88 to promote downstream events, whereas LPS recognition by TLR4 can activate both MyD88 and TRIF adaptors mediated pathways (, ). Thus, LPS could induce stronger inflammatory responses via two pathways than that induced by TLR2 agonists. Meanwhile, this immune regulatory effect did not attenuate T cell activation by DCs (). Again, SUP3 could be considered as a novel effective immune adjuvant.
Adjuvants are not only applied in vaccination but also in cancer immunotherapy. CTLs induced by DCs are major knights to combat tumors. Enhanced DC activation will lead to vigorous antitumor cytotoxicity by CTLs. Our studies proved that SUP3 conferred more protective immunity from tumor metastasis to the lungs. Tumors escape from immune attack by diminishing CTL responses. PD-1/PD-L1 is important check-point molecules during immune responses, which suppress antitumor immune responses. Blocking and eliminating their inhibitory effects by neutralizing antibodies significantly enhanced antitumor immune responses and had been used in clinical trials for cancer therapies (–).
However, this treatment is not always effective for some patients due to various reasons. A combination of an effective immune adjuvant together with anti-PD-1/PD-L1 antibodies may improve the efficacy of the treatment. Our synthetic TLR2 agonist SUP3 could be an outstanding candidate for this purpose. Further tests are needed to determine the effectiveness of this strategy. Overall, in this study, we developed a novel TLR2 agonist SUP3, which is more stable than Pam3 and could enhance DC functions without inducing excessive inflammation, the most favorable property of an ideal adjuvant. As DCs are essential initiators of antigen-specific immune responses, the enhanced activation and function of DCs by TLR2 agonists should improve immune responses to pathogens or tumors. Our study suggested that the new TLR2 agonist SUP3 could be a promising adjuvant in vaccination and immune modulations.
Author Contributions The study was designed by XG, NW, YS, JH, XBL, and LW. XG, XL (4th author), TW, YZ, XL (7th author) and JH preformed experiments and acquired the data. XG, NW, YS, XL (7th author), JH, XBL, and LW analyzed and interpreted the data. Manuscript was written by XG, NW, YS, XBL, and LW. This study was supervised by LW and XBL.
Conflict of Interest Statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Acknowledgments We thank Dr. Wanli Liu and Ms. Xuejiao Dong (Tsinghua University) for assistance in investigation of antibody responses, Dr. Yun-Cai Liu (Tsinghua University) for providing B16-OVA cells, and Dr. Gang Liu (Tsinghua University) for analysis of HEK-Blue hTLR2 assay. Funding This work was supported by a National Major Scientific Research Program by Ministry of Science and Technology of People’s Republic of China (No.
2015CB943200), a Key Project Grant from National Natural Science Foundation of China (No. 31330027), and Tsinghua-Peking Center for Life Sciences. Supplementary Material The Supplementary Material for this article can be found online. Abbreviations CFSE, carboxyfluorescein diacetate, succinimidyl ester; CTL, cytotoxic T lymphocyte; DC, dendritic cell; GM-CSF, granulocyte-macrophage colony-stimulating factor; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; MHC, major histocompatibility complex; NP-KLH, nitrophenyl-keyhole limpet hemocyanin; OCS, OVA-coated splenocytes; OVA, ovalbumin; PBS, phosphate buffered saline; TD antigen, thymus-dependent antigen. Reviewed by:, University of Duisburg-Essen, Germany, University of Zurich, Switzerland Copyright: © 2017 Guo, Wu, Shang, Liu, Wu, Zhou, Liu, Huang, Liao and Wu.
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*Correspondence: Xuebin Liao,; Li Wu.