Here, we provide a demonstration of the suction blister cutaneous recall model. The model allows a simple access to study human in vivo adaptive immune responses, for instance in the context of vaccine development.
Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model offers accessibility to rare populations of antigen-specific T-cells representative of the cellular memory response as well as the cytokine microenvironment in situ.
This report describes the practical procedure of a cutaneous recall, an SB induction, and a harvest of antigen-specific T-cells. To exemplify the method, the tuberculin skin test is used for antigenic recall in individuals who, prior to this study, underwent a Bacillus Calmette-Guérin vaccination against an infection with Mycobacterium tuberculosis. Finally, examples of multiplex and flow cytometric analyses of SB specimens are provided, illustrating high fractions of antigen-specific polyfunctional CD4+ T-cells available by this sampling method compared with cells isolated from the blood.
The method described here is safe and minimally invasive, provides a unique opportunity to study both innate and adaptive immune responses in vivo, and may be beneficial to a broad community of researchers working with cell-mediated immunity and human memory responses, in the context of vaccine development.
A skin SB is an artificially induced blister, which allows for the harvest of cells and fluid directly from the skin. The technique of raising SBs by vacuum is a well-known tool within the field of dermatology used for the study of skin immunology in health and disease1,2,3,4,5,6,7,8. This report demonstrates how the SB technique combined with cutaneous antigenic recall (SB cutaneous recall method) can provide direct insights into adaptive immune responses in vivo.
The principle behind the SB induction is simple: a light vacuum is applied to a small area of the skin. The negative pressure will eventually force the epidermis to separate from the dermis, creating a local blister filled with fluid and cells1,2,9. The blister fluid can be harvested by a fine needle aspiration and the content can be used for further study in vitro.
In recent years, there has been an increasing interest in the SB method for the study of in vivo immune responses other than diseases restricted to the skin10,11. The study of the adaptive immunity in humans is limited by the fact that the cells and cytokines of interest are sampled from peripheral blood because an invasive sampling of the lymph node or gastrointestinal mucosal tissue may be unacceptable and unethical. An example is the study of long-lived human memory T-cell responses after vaccination12. In such trials, the sampling of relevant T-cells can be a great challenge, because the relevant population of cells that mediate immunity resides in the lymphoid tissue, and only a very limited number of specific T-cells circulate in the peripheral blood.
The SB technique offers a unique opportunity to the study of memory T-cells and other specific cell populations. Following the cutaneous inoculation of antigen, T-cells specific for the antigen are recruited from their lymphoid hideaways to the skin and can easily be sampled from the SB. The methodology and research applications of this cutaneous recall model were described by Akbar et al. in 20132. A commonly used antigen for skin recall is the commercially available purified protein derivative (PPD) of mycobacteria used to perform a tuberculin skin test (TST)13, where PPD is injected into the dermal layer of the skin. In individuals with an existing immunological memory towards PPD [e.g., individuals with an Mycobacterium tuberculosis (Mtb) infection or a prior Bacillus Calmette-Guérin (BCG) vaccination], the antigen deposition results in a recall response with migration to the skin and an in vivo clonal expansion of PPD-specific CD4+ T-cells2,11,14,15. As a result, high fractions of PPD-specific polyfunctional CD4+ T-cells accumulate in the skin ready for SB sampling. T-cells collected by this method have proven to be robust and are sufficiently abundant to be characterized by a range of immunoassays and by a long-term in vitro culture15. Thus, the cutaneous recall model and the SB induction may prove a valuable method to study in vivo T-cell responses by ex vivo analyses, and increased knowledge of this approach may benefit researchers with interests in cellular immunology and vaccinology.
This report provides the first stepwise guide on how to induce human skin suction blisters in PPD-injected skin. The cutaneous antigen recall model is demonstrated using the TST in BCG-vaccinated volunteers. Finally, the relevant ex vivo analysis of the cells and cytokines isolated by SB is exemplified. Phenotypical and functional characteristics of PPD-specific T-cells obtained by the SB method are thoroughly described elsewhere2,10,11,16,17,18,19. This report aims to discuss the practical and immunological aspects of the methodology to ease the application of this technique by other research groups.
This manuscript describes a practical procedure for the study of human immune memory responses in vivo, using cutaneous antigen recall and cell harvest by suction blister induction. TST was used as an example of intradermal antigen deposition and BCG-vaccine recall. Finally, an example of SB specimen characterization by flow cytometric and multiplex cytokine analysis was provided, demonstrating that roughly a third of the SB cells were antigen-specific polyfunctional T-cells of the effector memory phenotype.
The critical steps of this protocol include the intradermal injection technique, the suction blister induction, and the blister puncture. Firstly, a correct intradermal deposition requires trained personnel. An incorrect deposition can lead to suboptimal results. PPD is generally a well-known and safe cutaneous antigen, but its composition may vary between manufacturers, limiting comparability13,20. This report provides instructions for a single intradermal deposition of 2 TU, and optionally two parallel depositions (2 x 2 TU), which may require additional ethical approval. However, other studies have used 10 TU, which increase the likelihood of a strong skin reaction10,21. During the SB induction step, small stepwise increases in the negative pressure will reduce the risk of hemorrhage and blister rupture. The chances of contamination with red blood cells or leucocytes from the bloodstream are generally low2. The aseptic puncture technique prevents microbial contamination and avoiding contact between the puncture needle and the dermal blister floor reduces impurities of debris or resident skin cells. However, some researchers prefer to harvest SB fluid by applying a rolling pressure to the punctured blister10. It may be necessary to puncture the septa within the blister. The SB technique itself is minimally invasive; collapsed SBs heal without scarring and infections are very rare.2 However, some degree of hypo-pigmentation may occur and SB induction should probably be avoided in people with a history of colloid scarring.
Technical limitations include low total cell yields and consequently limited options for long-term storage. Relationships between the leucocyte yield, clinical TST responses, the blister size, and erythrocyte contamination has been thoroughly described elsewhere2,10. In the BCG-recall experiments presented here (Figure 1), the median total yield from each blister was 50,000, implying a small-scale experimental set-up using these cells, especially if SBCs are studied alone15. However, the specific T-cell population in an SB sample mostly exceeds what is found in PBMC samples in both relative and absolute cell counts. In the example presented in Figure 2, the number of triple-positive PPD-specific T-cells in a sample of 100,000 SB cells were more than 2x greater than the number found in 1 x 106 PBMCs from the same donor (data not shown). A visual scoring of the TST reaction is the most common clinical method for the evaluation of M. tuberculosis memory. Of note, the underlying adaptive immunological reaction does not peak at the same time as the clinical skin reaction2,21. Not all BCG-vaccinated or Mtb-exposed individuals will develop strong TST reactions and strategies for classification, and a handling of samples from TST non-responders, as well as a preexisting mycobacterial sensitization in the study population, need to be considered before initiating a recall trial using this method. Also, in both SB sampling and visual scoring, there is a potential for a theoretical bias in individuals with reduced skin responses due to global or skin-related impaired immunity as seen, for instance, in HIV-infection and certain age groups2,13,18,20. In addition, an immunological boosting of the TST reaction with repeated testing is well known20,22. However, the SB cell phenotypes remain rather constant when TSTs are repeated10. This observation supports the role of SB recall in longitudinal studies of cellular immune responses.
For T-cell immunologists, SB recall allows for the harvest of high fractions of antigen-specific cells. However, the timing of SB for a sampling from a PPD-deposition is critical as both the cellular composition and the cytokine microenvironment change over time. In this protocol, blisters were induced 7-day post-challenge and the isolated cells were primarily CD4+ effector memory T-cells including high fractions of cells with a co-expression of IFN-γ, TNF-α, and IL-2. These observations are in line with previous studies describing how T-cell activation, proliferation, and differentiation occurs in PPD-primed skin2,15,21. Kinetic SB studies in PPD-sensitized individuals suggest that the very early skin response is unspecific; however, already within the first days following the PPD-challenge, the response becomes dominated by CD4+ T-cells (both central memory and effector memory phenotypes have been described) and, after 3 days, the response is dominated by high fractions of PPD-specific cytokine producing CD4+ T-cells2,8,10,15,21. This adaptive cytokine response remains detectable for more than 2 weeks2,8,10,23. Day 7 post-TST appears to be the most optimal time point for the collection of cytokine producing memory CD4+ T-cells2. However, other time points may, of course, be relevant depending on the antigen and response of interest. Of note, in one study, the adaptive PPD response has been reported to peak a little earlier with a decrease in the pro-inflammatory cytokine secretion already at 5 days post-TST10.
SB fluid contains high levels of both pro-inflammatory cytokines and other proteins shown to be representative of the skin microenvironment15,24. Kinetics studies have shown that TNF-α and IFN-γ levels in SB fluid peak after 3 days while IL-2 levels peak after 7 days2,10, probably reflecting the dominance of adaptive responses at this later stage. Because SB cells have been activated in vivo, they exhibit a high spontaneous cytokine release as well as a potential for a specific release upon restimulation (Figure 3 and 4).
This report focuses on CD4+ T-cell immunity. As demonstrated in Figure 2, the majority of the isolated T-cells were indeed CD4+, while there were almost no CD8+ T-cells in the suction blister fluid. This is in line with other SB PPD-recall studies reporting low proportions and an inferior migratory capacity of CD8+ T-cells compared to CD4+ T cells2,8,10,23. A further characterization of the CD8+ contribution would be preferable; however, this is beyond the scope of this report.
T-cells are considered essential for the immune control of Mtb; however, it has been difficult to identify a reliable correlate of the protection reflected in the adaptive immune response from the blood25,26. This roadblock severely hinders the development of new TB vaccines, as there currently is no alternative to large and very costly efficacy trials27. TB vaccine developers determine vaccine immunogenicity by assessing small and transient changes in the vaccine-specific T-cell populations in the blood28,29. However, it is questionable whether the small fraction of circulating antigen-specific T-cells found in the blood is relevant (i.e., capable of migrating to the site of an infection and representative of the T-cell-rich microenvironment controlling Mtb)27,30,31.
Based on previous studies and the data presented herein, the SB cutaneous recall model represents an untapped potential for the study of vaccine-specific T-cells. Not only does the model enable a recall of a vaccine response generated decades ago, it also allows the evaluation of the true memory potential in a tissue-specific context15,18,19,21. Novel, specific skin tests, which include antigens also comprised of candidate subunit TB vaccines, suggest new opportunities for vaccine evaluation using this model28,32. Furthermore, transcriptomic analyses suggest that a cell-mediated immune response generated in PPD-challenged skin is similar to the response found in the Mtb-infected lung18.
While skin punch biopsies also allow for a cell harvest from the skin and provide spatial information, compared with SB sampling, the method is more invasive and requires enzymatic or mechanic processing to prepare single-cell suspensions33. Measurements of cytokines and cell markers are comparable between the two methods2,10.
The suction blister method has already been applied in many areas of medical research besides dermatology, either alone or in combination with a systemic or local skin challenge. Examples include studies of sepsis, Epstein-Barr virus-associated lymphoproliferative disease, diabetic neuropathy, glucocorticoid intake effects, and human trials testing therapeutic antibodies or models for T-cell-targeted therapies10,34,35,36,37,38.
From a therapeutic point of view, the SB cutaneous recall method offers unique advantages to study the central T-cell memory potential and-from both a biological and technical point of view-the skin seems to provide a relevant sampling compartment2,15,18,19,21. In particular, compared to traditional, passive sampling of circulating PBMCs, the SB cutaneous recall method allows for the study of T-cells that have proven the ability to migrate from the lymph node in response to their relevant antigen, and complete the local expansion and differentiation in a tissue-specific in vivo context15,18,19,21.
In conclusion, the model demonstrated here could be relevant for researchers within the field of human adaptive immunity and T-cell targeting agents (i.e., in infectious disease vaccinology or cancer research). The TST model applied in this protocol is, of course, of special relevance in the field of TB vaccinology. However, the basic concept of this model is highly applicable in other fields of research.
The authors have nothing to disclose.
We would like to thank Mads Radmer Jensen for his practical and academic advice; Lau Lindqvist and Kaare Svejstrup for providing technical expertise; Helene Bæk Juel, Jonathan Filskov, Signe T. Schmidt, and Solveig W. Harpøth for their technical assistance; the staff at the Gentofte TB outpatient clinic for their training in PPD administration; and all the volunteers for participating in the study. This project is funded by the European Commission H2020 program [grant number TBVAC2020 643381], and we would like to thank consortium partners for the fruitful discussions.
Gloves for laboratory use | Imtex, Denmark | 1013 | |
Desinfection spray | Apotek, Denmark | 82% alcohol, with glycerin, for human skin | |
Alcohol swaps | Mediq, Denmark | 1131892 | |
PPD RT 23 "SSI" (2 T.U./0.1ml) | AJ Vaccines | ||
Myjector syringe with fixed needle | Terumo | A236 | 1 ml/29G/12mm |
Ruler | not relevant | for skin reaction evaluation | |
Ballpoint pen | not relevant | for skin reaction evaluation | |
Portable Lung Suction Unit | Laerdal Medical, Denmark | 78000011 | NOTE. Modified for ranges below -40kPa and supplied with pressure gauge (ref. 1031107) by the Technical Dept. , Hvidovre Hospital |
Negative Pressure Chamber Assembly, unheated with connecting tubing | Electronic Diversities, Finksburg, MD, USA | ||
Orifice Plate, 47mm, 10mm Hole | Electronic Diversities, Finksburg, MD, USA | ||
Negative Pressure Instrument Seal Kit | Electronic Diversities, Finksburg, MD, USA | ||
Negative pressure Chamber Strap | Electronic Diversities, Finksburg, MD, USA | 12 inches | |
Micropore Surgical tape | 3M | 1533-1 | 2.5cm x 9.1m |
Cap from 15ml plastic tube | Sarstedt, Germany | 62,547,254 | |
Tubigrip bandage | Mölnlycke Health Care, Sweden | 1443 | |
Cosmopor steril adhesive dressing | Hartmann | 9008337 | 7.2 x 5 cm |
2ml Syringe Concentric Luer Slip | Becton Dickinson | 300185 | |
Microlance 23G/30mm needle | Becton Dickinson | 300700 | |
Safe-Lock microcentrifuge tubes (autoclaved) | Eppendorf | 0030120086 | Autoclaved |
Minispin plus centrifuge | Eppendorf | 5453000011 | |
Gibco AIM V Medium | Life Technologies | 12055-091 |