CpG-carrier size and density affects dendritic cell signaling, subset-tropism and systemic immune polarization Public
LeLeux, Jardin Alexandra (2016)
Abstract
Microbial pathogens range in size, shape, as well as biochemical and molecular properties. This has led to the evolution of a variety of pathogen recognition receptors (PRRs) in mammalian immune cells that are responsible for sensing pathogen-associated molecular patterns (PAMPs) and initiating specific types of immune responses. However, the breadth of the PRR responses, especially how dendritic cells sense pathogen physical properties in conjunction with specific molecular patterns and translate that into unique immune responses, remains unknown. Here, we have developed pathogen-like particles (PLPs) that mimic physical properties of large viruses or bacteria to demonstrate that CpG-mediated dendritic cell signaling can be precisely modulated by varying PLP parameters, specifically size and adjuvant density. We demonstrate controlled tunability of DC programming, allowing directed maturation of distinct T cell phenotypes, antibody class switching and in vivo immune-polarization. Furthermore, we show, for the first time, that the surface-density of CpG on PLPs can finely control DC signaling by regulating the kinetics of NFκB transcription and STAT3 phosphorylation. These findings suggest that DCs sense physical aspects of pathogen-like materials, broadening the tools that can be used to modulate immunity, better understand innate immune response mechanisms, and develop new and improved vaccines.
Table of Contents
CHAPTER 1: OVERVIEW, HYPOTHESIS AND SPECIFIC AIMS 1
1.1 OVERVIEW........................................................................................................... 1
1.2 HYPOTHESIS........................................................................................................ 2
1.3 SPECIFIC AIMS.................................................................................................... 2
1.3.1 Aim 1: Determine how DCs of two peripheral subsets interact with PLPs in vitro. 2
1.3.2 Aim 2: Investigate the efficacy of PLP formulations screened in Aim 1 in vivo. 3
1.3.3 Aim 3: Evaluate mechanism that dictates formulation size-dependent DC programming. 3
1.4 OUTLINE.............................................................................................................. 4
CHAPTER 2: BACKGROUND AND SIGNIFICANCE, 5
2.1 IMMUNITY: A PRIMER..................................................................................... 6
2.1.1 Inducing a robust memory response: innate and adaptive immunity.............. 6
2.2 PARTICLES FOR DELIVERY OF ANTIGEN AND ADJUVANTS: PATHOGEN-LIKE PARTICLES AS VACCINES 23
2.2.1 Comparison of particle parameters............................................................... 23
2.3 FUTURE DIRECTIONS..................................................................................... 28
2.4 ABBREVIATIONS.............................................................................................. 29
CHAPTER 3: Aim 1.......................................................................... 31
3.1 METHODS.......................................................................................................... 34
3.1.1 Materials........................................................................................................ 34
3.1.2 Mice and primary cell isolation..................................................................... 34
3.1.3 Synthesis of micro- and nano- PLGA particles with polyethylenimine conjugation 35
3.1.4 CpG/OVA loading on PEI-PLGA particles.................................................. 36
3.1.5 Microscopy of PLPs..................................................................................... 37
3.1.6 Dendritic cell uptake...................................................................................... 37
3.1.7 Dendritic cell activation evaluation - flow cytometry.................................. 37
3.1.8 Dendritic cell activation evaluation - intracellular staining............................ 38
3.1.9 Dendritic cell activation evaluation - ELISA cytokine analysis.................... 38
3.1.10 Dendritic cell activation evaluation - Antigen presentation........................ 38
3.1.11 Statistical Analysis...................................................................................... 39
3.2 RESULTS............................................................................................................. 40
3.2.1 Characterization of pathogen-like particles (PLPs)....................................... 40
3.2.2 Development of DC subset cultures............................................................. 44
3.2.3 DCs show size preference in vitro................................................................ 50
3.2.4 DC activation is modulated in a PLP-size dependent manner....................... 54
3.2.5 PLP size-dependent DC programming influences T cell maturation............. 62
3.3 DISCUSSION....................................................................................................... 70
3.4 ABBREVIATIONS.............................................................................................. 72
CHAPTER 4: Aim 2.......................................................................... 74
4.1 METHODS.......................................................................................................... 75
4.1.1 Materials........................................................................................................ 75
4.1.2 Particle distribution and DC subset analysis of draining lymph nodes......... 75
4.1.3 Immunization protocol.................................................................................. 76
4.1.4 Immunohistochemistry staining.................................................................... 77
4.1.5 OVA-B16 Melanoma survival study............................................................ 77
4.1.6 Statistical Analysis........................................................................................ 77
4.2 RESULTS............................................................................................................. 78
4.2.1 Micro-PLPs traffic more efficiently to draining lymph nodes...................... 78
4.2.2 Migratory DCs carry CpG-PLPs to draining lymph nodes from skin......... 80
4.2.3 Immunological response to micro- and nano-PLPs....................................... 85
4.2.4 Therapeutic value of micro- or nano-PLPs against tumor challenge............ 100
4.3 DISCUSSION..................................................................................................... 103
4.4 ABBREVIATIONS............................................................................................ 105
CHAPTER 5: Aim 3........................................................................ 106
5.1 METHODS........................................................................................................ 109
5.1.1 Materials...................................................................................................... 109
5.1.2 Analysis of uptake preference by flow cytometry..................................... 109
5.1.3 TLR9-IRAK4 Proximity Ligation Assay.................................................... 110
5.1.4 NFkB Activation Kinetics........................................................................... 110
5.1.5 Phospho-STAT3 Kinetics........................................................................... 110
5.1.6 IRF 4/5/7/8 regulation.................................................................................. 110
5.1.7 Statistical Analysis...................................................................................... 111
5.2 RESULTS........................................................................................................... 112
5.2.1 Micro- and nano-PLPs are taken up with similar kinetics.......................... 112
5.2.2 TLR9 signalig is delayed in nano-PLP treated BMDCs.............................. 113
5.2.3 NFκB-mediated transcription is delayed in nano-PLP treated BMDCs........... 114
5.2.4 Nano-PLPs induce rapid phosphorylation of STAT3................................ 116
5.2.5 CpG-density play a critical role in modulating DC programming............... 117
5.2.6 IRF4 may play a role in PLP mediated DC programming........................... 120
5.3 DISCUSSION..................................................................................................... 126
5.4 ABBREVIATIONS............................................................................................ 129
CHAPTER 6: CONCLUSIONS AND FUTURE DIRECTIONS 130
6.1 CONCLUDING SUMMARY........................................................................... 130
6.2 FUTURE CONSIDERATIONS........................................................................ 135
APPENDIX..................................................................................... 138
A.1. NANO-PLPs DECREASE INDUCED EXPRESSION OF PDL2................. 138
A.1.1. MATERIALS AND METHODS................................................................. 140
A.1.1.1. Materials................................................................................................. 140
A.1.1.2. Quantification of spontaneously matured vs induced PDL2+ cells....... 140
A.1.1.3. Intracellular staining for immunomodulatory cytokines......................... 140
A.1.2. RESULTS...................................................................................................... 141
A.1.2.1. Quantification of spontaneously matured vs induced PDL2+ cells....... 141
A.1.2.2. Intracellular staining for immunomodulatory cytokines......................... 143
A.1.3. DISCUSSION................................................................................................ 145
REFERENCES................................................................................ 146
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