Analysis of the skin of mice humanized for the immune system

Analysis of the skin of mice humanized for the immune system A cc ep te d A rt ic le This article has been accepted for publication and undergone full peer review but has not been through the copyedit[.]

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may

PROF BEHAZINE COMBADIERE (Orcid ID : 0000-0002-2353-4406)

Received Date : 21-Jul-2016

Revised Date : 17-Feb-2017

Accepted Date : 23-Feb-2017

Article type : Letter to the Editors

Analysis of the skin

of mice humanized for the immune system

1

Sorbonne Universités, UPMC University Paris 06, UMR_S CR7, Centre d’Immunologie et des Maladies Infectieuses- Paris (CIMI-Paris), F-75013, Paris, France

2INSERM U1135, CIMI-Paris, F-75013, Paris, France

3

Service de chirurgie plastique, reconstructrice, esthétique, centre de brûlées, Hôpital Saint-Louis, Assistance Publique Hôpitaux de Paris (AP-HP), Paris, France

4

Service de dermatologie et allergologie, Hôpital Tenon, Assistance Publique Hôpitaux de Paris (AP-HP), Paris, France;

*

Corresponding author: Dr Béhazine Combadière, Centre d’Immunologie et des Maladies

Infectieuses CIMI-Paris, 91 Boulevard de l'Hôpital, 75013 Paris, France Phone: +33140779888, Fax: +33140779734, E-mail: behazine.combadiere@upmc.fr

Abbreviations: APC, antigen presenting cells; CLA, cutaneous lymphocyte antigen ; cDC,

conventional dendritic cells; hHPC, human hematopoietic progenitor cells; HIS, human

immune system; MVA, Modified Vaccinia Ankara; pDC, plasmacytoid dendritic cells

Keywords: intradermal vaccination, HIS mice, skin T cells, route of administration, antigen

presenting cells

Abstract (96 words / 100 words max)

Development of new immunotherapeutic strategies relies on the ability to activate the right cells at the right place and at the right moment, and on the capacity of these cells to home to the right organ(s) Skin delivery has shown high potency for immunotherapeutics

administration However, an adequate in vivo model of human skin immunity is still a critical

bottleneck We demonstrated here that the skin of HIS mice is colonized by human hematopoietic cells, mainly human T cells and that complementation with human APC at the vaccination site allowed the induction of an immune response

Background

The skin, the largest organ of the human body, is the first line of protection against pathogens, physical and chemical injuries It is a major immunological organ with an important density of antigen-presenting cells (APC) (1), able to capture pathogens through endocytic pathways and exhibiting cross-presentation ability In addition, the skin tissue contains a large pool of T cells, mostly memory T cells expressing the skin-homing marker CLA (cutaneous lymphocyte antigen) and displaying long-term immune protection capacity (2, 3) These are features highly desirable for induction of vaccination and tissue maintenance

allows for the generation of a potent CD8 T cell response and the induction of a humoral immune response in the mucosa (4) However, the lack of an appropriate small animal model

to study human skin immunity in vivo remains a major roadblock, which has been addressed

by constructing mice humanized with human skin graft (5) However, this mouse model is technically challenging to routinely establish Alternatives exist with mice humanized for the human immune system (HIS) (6-8)

Questions addressed

HIS mice represent an attractive tool to study ontogeny of cellular components of the human

immune system in vivo However, little is known about the skin compartment in these

animals Here, we investigated colonization of the skin of HIS mice by human hematopoietic cells in order to establish a preclinical model to investigate vaccination and immunotherapeutics application via the skin route

Experimental design

HLA-A2 transgenic NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(HLA-A2.1)1Enge/SzJ) mice were humanized (NSG-HLA-A2-HIS) with a single intra-hepatic injection of 0.5x105 -1.5x105 HLA-A2+ CD34+ human hematopoietic progenitor cells (hHPC) into sub-lethally irradiated newborn NSG-HLA-A2 mice (< 5 days old) This led to a high level engraftment

of a human immune system with long-term maintenance capacity in vivo NSG-HLA-A2-HIS

mice were used as they demonstrated improved T cell functionality after infection/immunization

Results

We first evaluated by flow cytometry the level of human hematopoietic cell (hCD45+) engraftment in the skin as compared to various organs of NSG-HLA-A2-HIS mice We observed a high level of hCD45+ engraftment in the spleen, liver, lymph node (Figure 1a), and thymus of the animals (data not shown) The lung and bone marrow exhibited good to intermediate level of human hCD45+ cells engraftment (Figure 1a), whereas the vagina and intestine (data not shown) were moderately colonized by human hematopoietic cells This data were confirmed by histology (supplementary Figure 1) In comparison, the skin from the flank and the ear was moderately engrafted with human CD45+ cells, which were mostly located in the dermis (Figure 1b&c) The engraftment efficiency of human cells in the skin was time-dependent, with increasing human cell density in the skin of older animals (23-26 weeks old, Figure 1c) as compared to younger ones (12-13 weeks old, Figure 1b)

The human cells observed in the skin of the animals as well as in other mucosal tissues (e.g lung, vagina) were mainly human T cells (Figure 2a and b), and only few human APC,

in contrast to human skin explants (supplementary Figure 2) The human T cells present in the skin were composed for two third of human CD4+ T cells and one third of human CD8+ T cells, which were both predominantly of memory phenotype (Figure 2c), similarly to what is observed in human skin explants (supplementary Figure 2) and the HIS mice lymphoid organs (supplementary Figure 3) A high fraction (∼80%) of these human T cells expressed CLA allowing them to reside in the skin (supplementary Figure 3) Interestingly, human CD4+ regulatory T cells (CD25++FoxP3+) were also identified in the skin of the animals at a frequency similar to the one observed in the spleen (Figure 2c)

We next immunized NSG-HLA-A2-HIS mice, intradermally, with MVA (Modified Vaccinia Ankara, 1pfu/cell) or PBS control No human T cell response was detected by IFNγ

compartment in HIS mice and their critical role in the induction of T-cell responses, we speculated that hAPC density of at the site of administration (i.e the skin) is potentially too low to optimally trigger T cells We thus tested the capacity of human DC complementation

in the skin of the NSG-HLA-A2-HIS mice (9), deriving autologous human cDC from human CD14+ cells isolated from the same cord blood than the hHPC used to reconstitute NSG-HLA-A2-HIS mice Autologous umbilical cord blood CD14+ monocytes were isolated by MACS and cultured for 3 days in presence of 50 ng/mL hGM-CSF and 5 ng/mL hIL-4 Autologous human monocyte-derived DC were loaded for 2h at 37°C with MVA or PBS Next, NSG-HLA-A2-HIS mice were immunized intradermally in the flank with 0,5.106 matched human CD14+-derived MVA- or PBS-loaded DC loaded A human T-cell response was observed 7 days after immunization by IFNγ ELISPOT in the spleen of the MVA-vaccinated animals (Figure 2d), with significantly more responder animals (3/5) than in the control group (0/5) (Chi-2 test, p=0.019)

Conclusions

We demonstrated here that the skin of HIS mice is colonized by human hematopoietic cells, mainly human T cells expressing CLA and exhibiting a memory phenotype Complementation with human APC at the vaccination site allowed the induction of an immune response This highlighted the potential of the HIS mice has an innovative preclinical model for human skin immunity as well as for prospective analysis of vaccination via skin route, and notably DC therapies As such, this will also generate knowledge on understanding the early events following immunotherapeutic administration and support

demands for in vivo biomodeling of the interaction of immunotherapeutic with the human

immune system

Acknowledgments

MC, MP, AJH, MD, AS performed research; DB, MM provided human materials MC, BC designed the research, analysed data, wrote the manuscript Dr Centlivre was supported by a FRM post-doctoral fellowship (SPF20121226281), Dr Combadière received EU-FP7 CUTHIVAC (n°241904) and FRM fundings We thank Dr Morosan for animal facility and Dormeur Foundation, Vaduz for providing AID EliSpot Reader and Cryostat HM550 apparatus, Dr Verrier for providing MVA

Conflict of interests

The authors declare they have no conflict of interests

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Figure Legend

Figure 1 Colonization of the skin of humanized mice by human cells (a) The frequency

of human CD45+ cells was determined for NSG-HLA-A2-HIS mice (n=4) in various organs

by flow cytometry Each symbol corresponds to an individual animal BM: bone marrow, LN: lymph node The paired t test analysis is presented in the table (ns: not statistically significant, *<0.05, **<0.01, ***<0.001) (b) Representative 5 μm flank skin cryosections labeled with DAPI (blue) and anti-human CD45 (red) of 2 HLA-A2Tg-NSG-HIS mice of 14 weeks old and (c) 2 HLA-A2Tg-NSG-HIS mice of 26 weeks old Magnification x10 The arrows indicate the rare human CD45+ cells present in the skin of 14-weeks old animals e: epidermis, d: dermis

Figure 2 Human cells present in the skin of humanized mice and vaccination via

cells (CD3+), pDC (BDCA2+HLADR+), monocytes (Mφ, CD14+

CD11c+HLA-DR+), conventional DC (cDC, CD11c+CD14-HLADR+) – in the skin of NSG-HLA-A2-HIS mice (26-30 weeks old, n=4) was determined by flow cytometry Each symbol corresponds to an individual animal ***P<0.001 (paired t test) (b) Representative 5 μm flank skin cryosection

of NSG-HLA-A2-HIS mouse of 26 weeks old labeled with DAPI (white), anti-human CD3 (blue) and anti-human HLA-DR (red) Magnification x20 e: epidermis, d: dermis (c) Analysis of the human CD3+ cells in the skin of NSG-HLA-A2-HIS mice (n=5) by flow cytometry The expression of CD45RA defined the nạve phenotype of the human T cells (d) IFNγ ELISPOT analysis of MVA-specific human T cells responses in the spleen of NSG-HLA-A2-HIS mice (32 weeks old) on day 7 after intradermal injection of 5.105 autologous human monocyte-derived DC loaded with MVA (1pfu/cell) or PBS in the flank of the animals Total splenocytes of MVA- or PBS-immunized NSG-HIS mice were incubated with

medium (med) or MVA for 40 hours and analyzed by ELISPOT assay The graph represents the number of spot forming unit (SFU) counted in the MVA stimulated well minus the ones counted in the medium stimulated well and according to the frequency of human CD45+ cells

in the spleen of each vaccinated animals *P=0.019 (Chi-square test)

Accepted

Accepted