Efficient immunogenic peptide antigen delivery to dendritic cells using an ESCRT-mediated extracellular vesicle formation method

Abstract Afferent lymph is transported throughout lymph nodes (LNs) by the conduit system. Whereas this conduit network is dense in the T-cell zone, it is sparse in B-cell follicles. In this study, we show that this differential organization emerges during lymph node development. Neonatal LNs lack B follicles, but have a developed T-cell zone and a dense conduit network. As new T and B cells enter the developing LN, the conduit network density is maintained in the T, but not the B zone, leading to a profound remodeling of the follicular network that nevertheless maintains its connectivity. In adults, the residual follicular conduits transport soluble antigen to deep regions, where follicular dendritic cells are abundant and appear to replace the fibroblastic reticular cells that enwrap conduits in the T zone. This strategic location correlates with the capacity of the follicular dendritic cells to capture antigen even in the absence of antigen-specific antibodies. Together, these results describe how the stromal organization of the T and B regions of LNs diverges during development, giving rise to distinct antigen transport and delivery modes in the 2 compartments.

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Response of naive antigen-specific CD4+ T cells in vitro: characteristics and antigen-presenting cell requirements.

Journal of Experimental Medicine ◽

10.1084/jem.176.5.1431 ◽

1992 ◽

Vol 176 (5) ◽

pp. 1431-1437 ◽

Cited By ~ 156

Author(s):

M Croft ◽

D D Duncan ◽

S L Swain

Keyword(s):

T Cells ◽

Dendritic Cells ◽

T Cell ◽

Cd4 T Cells ◽

Cd4 Cells ◽

Peptide Antigen ◽

Naive T Cells ◽

Naïve T Cells ◽

Antigen Presenting

Because of the low frequency of T cells for any particular soluble protein antigen in unprimed animals, the requirements for naive T cell responses in specific antigens have not been clearly delineated and they have been difficult to study in vitro. We have taken advantage of mice transgenic for the V beta 3/V alpha 11 T cell receptor (TCR), which can recognize a peptide of cytochrome c presented by IEk. 85-90% of CD4+ T cells in these mice express the transgenic TCR, and we show that almost all such V beta 3/V alpha 11 receptor-positive cells have a phenotype characteristic of naive T cells, including expression of high levels of CD45RB, high levels of L-selectin (Mel-14), low levels of CD44 (Pgp-1), and secretion of interleukin 2 (IL-2) as the major cytokine. Naive T cells, separated on the basis of CD45RB high expression, gave vigorous responses (proliferation and IL-2 secretion) to peptide antigen presented in vitro by a mixed antigen-presenting cell population. At least 50% of the T cell population appeared to respond, as assessed by blast transformation, entry into G1, and expression of increased levels of CD44 by 24 h. Significant contributions to the response by contaminating memory CD4+ cells were ruled out by demonstrating that the majority of the CD45RB low, L-selectin low, CD44 high cells did not express the V beta 3/V alpha 11 TCR and responded poorly to antigen. We find that proliferation and IL-2 secretion of the naive CD4 cells is minimal when resting B cells present peptide antigen, and that both splenic and bone marrow-derived macrophages are weak stimulators. Naive T cells did respond well to high numbers of activated B cells. However, dendritic cells were the most potent stimulators of proliferation and IL-2 secretion at low cell numbers, and were far superior inducers of IL-2 at higher numbers. These studies establish that naive CD4 T cells can respond vigorously to soluble antigen and indicate that maximal stimulation can be achieved by presentation of antigen on dendritic cells. This model should prove very useful in further investigations of activation requirements and functional characteristics of naive helper T cells.

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In vitroAssay for Screening of Optimal Targets for Antigen-Delivery to Murine Dendritic Cells

Scandinavian Journal of Immunology ◽

10.1111/sji.12365 ◽

2015 ◽

Vol 82 (6) ◽

pp. 498-505 ◽

Cited By ~ 2

Author(s):

L. H. Pugholm ◽

K. Varming ◽

R. Agger

Keyword(s):

Dendritic Cells ◽

Antigen Delivery

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Induction of CD4+ T cell–dependent antitumor immunity by TAT-mediated tumor antigen delivery into dendritic cells

Journal of Clinical Investigation ◽

10.1172/jci200215399 ◽

2002 ◽

Vol 109 (11) ◽

pp. 1463-1470 ◽

Cited By ~ 61

Author(s):

Helen Y. Wang ◽

Tihui Fu ◽

Gang Wang ◽

Gang Zeng ◽

Donna M. Perry-Lalley ◽

...

Keyword(s):

Dendritic Cells ◽

T Cell ◽

Tumor Antigen ◽

Antitumor Immunity ◽

Cd4 T Cell ◽

Antigen Delivery

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Unleashing Tumour-Dendritic Cells to Fight Cancer by Tackling Their Three A’s: Abundance, Activation and Antigen-Delivery

Cancers ◽

10.3390/cancers11050670 ◽

2019 ◽

Vol 11 (5) ◽

pp. 670 ◽

Cited By ~ 3

Author(s):

Aleksandar Murgaski ◽

Pauline M. R. Bardet ◽

Sana M. Arnouk ◽

Emile J. Clappaert ◽

Damya Laoui

Keyword(s):

Dendritic Cells ◽

T Cell ◽

T Cell Responses ◽

Tumour Microenvironment ◽

Antigen Delivery ◽

Cancer Treatments ◽

Delivery Methods ◽

Cell Responses ◽

Full Capacity ◽

Specific Antigens

Recent advances in cancer immunotherapy have mainly focused on re-activating T-cell responses against cancer cells. However, both priming and activation of effector T-cell responses against cancer-specific antigens require cross-talk with dendritic cells (DCs), which are responsible for the capturing, processing and presentation of tumour-(neo)antigens to T cells. DCs consequently constitute an essential target in efforts to generate therapeutic immunity against cancer. This review will discuss recent research that is unlocking the cancer-fighting potential of tumour-infiltrating DCs. First, the complexity of DCs in the tumour microenvironment regarding the different subsets and the difficulty of translating mouse data into equivalent human data will be briefly touched upon. Mainly, possible solutions to problems currently faced in DC-based cancer treatments will be discussed, including their infiltration into tumours, activation strategies, and antigen delivery methods. In this way, we hope to put together a broad picture of potential synergistic therapies that could be implemented to harness the full capacity of tumour-infiltrating DCs to stimulate anti-tumour immune responses in patients.

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Internalization and endosomal degradation of receptor-bound antigens regulate the efficiency of cross presentation by human dendritic cells

Blood ◽

10.1182/blood-2012-01-402370 ◽

2012 ◽

Vol 120 (10) ◽

pp. 2011-2020 ◽

Cited By ~ 119

Author(s):

Bithi Chatterjee ◽

Anna Smed-Sörensen ◽

Lillian Cohn ◽

Cécile Chalouni ◽

Richard Vandlen ◽

...

Keyword(s):

Dendritic Cells ◽

Vaccine Development ◽

Mannose Receptor ◽

Antigen Delivery ◽

Cross Presentation ◽

Late Endosomes ◽

Early Endosomes ◽

Antibody Conjugates ◽

Human Dendritic Cells ◽

Endocytic Compartments

Abstract Dendritic cells (DCs) can capture extracellular antigens and load resultant peptides on to MHC class I molecules, a process termed cross presentation. The mechanisms of cross presentation remain incompletely understood, particularly in primary human DCs. One unknown is the extent to which antigen delivery to distinct endocytic compartments determines cross presentation efficiency, possibly by influencing antigen egress to the cytosol. We addressed the problem directly and quantitatively by comparing the cross presentation of identical antigens conjugated with antibodies against different DC receptors that are targeted to early or late endosomes at distinct efficiencies. In human BDCA1+ and monocyte-derived DCs, CD40 and mannose receptor targeted antibody conjugates to early endosomes, whereas DEC205 targeted antigen primarily to late compartments. Surprisingly, the receptor least efficient at internalization, CD40, was the most efficient at cross presentation. This did not reflect DC activation by CD40, but rather its relatively poor uptake or intra-endosomal degradation compared with mannose receptor or DEC205. Thus, although both early and late endosomes appear to support cross presentation in human DCs, internalization efficiency, especially to late compartments, may be a negative predictor of activity when selecting receptors for vaccine development.

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Preparation of protein loaded poly(d,l-lactide-co-glycolide) microparticles for the antigen delivery to dendritic cells using a static micromixer

European Journal of Pharmaceutics and Biopharmaceutics ◽

10.1016/j.ejpb.2005.08.015 ◽

2006 ◽

Vol 62 (3) ◽

pp. 247-253 ◽

Cited By ~ 14

Author(s):

C WISCHKE ◽

D LORENZEN ◽

J ZIMMERMANN ◽

H BORCHERT

Keyword(s):

Dendritic Cells ◽

Antigen Delivery

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Modification of host dendritic cells by microchimerism-derived extracellular vesicles generates split tolerance

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1618364114 ◽

2017 ◽

Vol 114 (5) ◽

pp. 1099-1104 ◽

Cited By ~ 23

Author(s):

William Bracamonte-Baran ◽

Jonathan Florentin ◽

Ying Zhou ◽

Ewa Jankowska-Gan ◽

W. John Haynes ◽

...

Keyword(s):

T Cells ◽

Dendritic Cells ◽

Cd4 T Cells ◽

Extracellular Vesicle ◽

Membrane Microdomains ◽

Transplant Tolerance ◽

Split Tolerance ◽

Cross Dressing

Maternal microchimerism (MMc) has been associated with development of allospecific transplant tolerance, antitumor immunity, and cross-generational reproductive fitness, but its mode of action is unknown. We found in a murine model that MMc caused exposure to the noninherited maternal antigens in all offspring, but in some, MMc magnitude was enough to cause membrane alloantigen acquisition (mAAQ; “cross-dressing”) of host dendritic cells (DCs). Extracellular vesicle (EV)-enriched serum fractions from mAAQ+, but not from non-mAAQ, mice reproduced the DC cross-dressing phenomenon in vitro. In vivo, mAAQ was associated with increased expression of immune modulators PD-L1 (programmed death-ligand 1) and CD86 by myeloid DCs (mDCs) and decreased presentation of allopeptide+self-MHC complexes, along with increased PD-L1, on plasmacytoid DCs (pDCs). Remarkably, both serum EV-enriched fractions and membrane microdomains containing the acquired MHC alloantigens included CD86, but completely excluded PD-L1. In contrast, EV-enriched fractions and microdomains containing allopeptide+self-MHC did not exclude PD-L1. Adoptive transfer of allospecific transgenic CD4 T cells revealed a “split tolerance” status in mAAQ+mice: T cells recognizing intact acquired MHC alloantigens proliferated, whereas those responding to allopeptide+self-MHC did not. Using isolated pDCs and mDCs for in vitro culture with allopeptide+self-MHC–specific CD4 T cells, we could replicate their normal activation in non-mAAQ mice, and PD-L1–dependent anergy in mAAQ+hosts. We propose that EVs provide a physiologic link between microchimerism and split tolerance, with implications for tumor immunity, transplantation, autoimmunity, and reproductive success.

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