Cross priming of the melanoma antigen, Melan A, by skin ... Katie Intercalated Degree Prize winner...Cross-priming of the melanoma antigen, Melan-A, by skin dendritic cells Introduction

Cross-priming of the

melanoma antigen, Melan-A,

by skin dendritic cells

Katie Harriette Best

Supervisor: Dr Muzlifah Haniffa

Human Dendritic Cell Laboratory

Institute of Cellular Medicine

Masters by Research 2013-2014

Word Count: 951 excluding title page, acknowledgements and references.

Cross-priming of the melanoma antigen, Melan-A, by

skin dendritic cells

Introduction

Cutaneous melanoma is the most dangerous and aggressive form of skin cancer, characterised

by the transformation and invasive growth of melanocytes1. The incidence rate of melanoma

is rising worldwide2-3

.

Radical excision of the primary tumour is currently the only curative treatment option4,

however surgery is rarely an option for patients with metastatic disease. The prognostic

outlook for metastatic disease is bleak, with two-year overall survival rates of 10%5.

Melanoma has poor sensitivity to traditional treatment methods involving chemotherapy and

radiation therapy. Furthermore, recently approved immunotherapies vemurafenib and

ipilimumab have proven to be of limited, objective clinical use due to resistance and poor

response respectively1,6-7

. The latter is partly due to paucity in knowledge about the biological

function of the antigen presenting cells (APCs) responsible for processing melanoma-

associated antigens and generating a cytotoxic anti-melanoma immune response.

Dendritic cells (DCs) constitute a heterogenous population8 of professional APCs, with a

pivotal role in linking the innate and adaptive immune system. DCs are found in peripheral

tissues such as the skin, where they sample the local microenvironment and migrate to the

draining lymph nodes in order to interact with T cells and initiate immune responses. DCs

harbour the unique capacity to induce de novo antigen-specific cytotoxic T lymphocyte

(CTL) responses by the phenomenon of cross-priming, see Figure 1.

The human immune system is capable of mounting a cytotoxic response against melanoma9,

and thus enhancing specific tumour-killing immune responses against melanoma presents as

an attractive therapeutic strategy. To date, several melanoma-associated antigens have been

identified and the strikingly immunodominant10

, well characterised melanoma-associated

antigen, Melan-A, has generated considerable clinical interest with regards to developing an

anti-melanoma vaccine11

.

As critical regulators of immune responses, DCs are of significant importance in designing

rational anti-cancer vaccines12-13

. Very little is known about how human skin DCs process

cancer antigens to generate tumour killing responses and whether this can be clinically

translated to advance cancer therapy. Demonstrating the ability of skin DCs to elicit a

cytotoxic anti-melanoma response will facilitate the development of novel anti-melanoma

immunotherapies through direct targeting of cross-priming DC subsets.

The aim of this study was to test the ability of human skin APCs to cross-prime naïve CD8+

T cells against the melanoma-associated antigen, Melan-A, in vitro.

Figure 1. Diagram of cross-priming. Migratory skin DCs uptake Melan-A antigen in the

periphery and traffic to the draining lymph node. With the help of CD4+ T cells, DCs are

licensed to present Melan-A antigenic peptide via their major histocompatibility complex

(MHC) class I molecule to naïve CD8+ T cells. The naïve CD8

+ T cells are cross-primed against

Melan-A and enter the circulation as Melan-A-specific effector cytotoxic T lymphocytes (CTLs).

Methods

To test the ability of human skin APCs to cross-prime naïve CD8+

T cells against the

melanoma-associated antigen, Melan-A, I co-cultured naïve CD8+

T cells from HLA-A*02+

peripheral blood stem cells with allogeneic HLA-A*02+

skin APCs loaded with 25µg/ml

Melan-A protein overnight, for 9 days. For a positive control, skin APCs were loaded with

Melan-A short peptide. For a negative control, skin APCs were not loaded with Melan-A

protein or peptide.

Skin APCs and naïve CD8+

T cells were isolated from plastic surgery patients’ surplus,

healthy skin samples and peripheral blood stem cells respectively, by fluorescence activated

cell sorting (FACS), see Figure 2.

After 9 days, the induction of Melan-A-specific cytotoxic T lymphocytes was determined by

flow cytometry analysis of tetramer binding, see Figure 3.

Figure 2. Diagram of fluorescence activated cell sorting (FACS).

Figure 3. Diagram showing A) cross-priming of a naive CD8+ T cells against Melan-A by an

antigen presenting cell (APC), B) the structure of an MHC class I/Melan-A peptide tetramer,

and C) tetramer binding to a Melan-A-specific cytotoxic T lymphocyte (CTL).

Results and Discussion

I investigated the ability of five different skin APCs (dermal CD14+ cells, CD1c

+ DCs,

CD141hi

DCs, macrophages and epidermal Langerhans cells) to cross-prime naive CD8+ T

cells against Melan-A in vitro.

The results from this study show that epidermal Langerhans cells (LCs) are capable of cross-

priming naïve CD8+ T cells against the melanoma-associated antigen, Melan-A, in vitro see

Figure 4. Although it is difficult to prove unequivocally LCs are a cross-priming DC subset,

these findings complement evidence showing LCs to be cross-presenting DCs14-16

. There is

ample evidence to support the hypothesis that LCs are efficient at cross-priming naïve CD8+

T cells16-19

.

The cross-priming ability of dermal CD14+ cells, CD1c

+ DCs, CD141

hi DCs and

macrophages in vitro was unconvincing due to the relatively high detection of tetramer+ cells

in the negative control, see Figure 4. However, without further investigation, the potential

cross-priming ability of these dermal APCs cannot be disregarded.

Figure 4. Percentage of CD3+CD8

+ T cells induced to tetramer

+/Melan-A-specific cytotoxic T

lymphocytes after a 9 day co-culture of naive CD8+ T cells with unloaded, Melan-A short

peptide loaded or Melan-A protein loaded skin APCs. Composite data for two experiments is

shown with the mean.

Conclusions

Dendritic cell (DC) mediated cross-priming of endogenous cancer-associated antigens is

essential for the induction of de novo cancer antigen-specific cytotoxic T lymphocyte

responses. DC subsets with functional specialisations have been identified in human skin,

including migratory cross-presenting DCs20

but cross-priming DCs have not been isolated,

despite their potential importance in immunity and generating an anti-tumour cytotoxic

response.

This study shows epidermal LCs are capable of effectively cross-priming naïve CD8+ T cells

against Melan-A in vitro. However, the number of donors is not adequate to reach statistical

significance and merit LCs with the definitive status of a cross-priming DC subset; further

investigation is required in order to consolidate these results. Ultimately, these findings will

facilitate the development of novel anti-melanoma immunotherapies, through direct targeting

of cross-priming DC subsets.

Acknowledgements

I would like to thank the plastic surgery patients for participating in this study, Tara Shrestha

for coordinating the skin samples, Emma Matthews at the Stem Cell Transplantation

Research Group for the provision of peripheral blood stem cells, Andrew Fuller and David

Macdonald at the Flow Cytometry core facility for their assistance with FACS, Elizabeth

Poyner for her advice, Merry Gunawan for her help and assistance, Muzlifah Haniffa for her

guidance and supervision, and Professor Matthew Collin and everyone else at the Human

Dendritic Cell Lab for their support and encouragement.

This work was supported by grants from the British Association of Dermatologists, the

British Skin Foundation and the Wellcome Trust.

Human Dendritic Cell Lab

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