The Characterization of Varicella Zoster Virus-Specific T Cells in Skin and Blood during Aging.

Vukmanovic-Stejic M, Sandhu D, Seidel JA, Patel N, Sobande TO, Agius E, Jackson SE, Fuentes-Duculan J, Suárez-Fariñas M, Mabbott NA, Lacy KE, Ogg G, Nestle FO, Krueger JG, Rustin MH, Akbar AN.

Journal of Investigative Dermatology 2015 Jul;135(7):1752-62.


Reactivation of the varicella zoster virus (VZV) increases during aging. Although the effects of VZV reactivation are observed in the skin (shingles), the number and functional capacity of cutaneous VZV-specific T cells have not been investigated. The numbers of circulating IFN-γ-secreting VZV-specific CD4(+) T cells are significantly decreased in old subjects. However, other measures of VZV-specific CD4(+) T cells, including proliferative capacity to VZV antigen stimulation and identification of VZV-specific CD4(+) T cells with an major histocompatibility complex class II tetramer (epitope of IE-63 protein), were similar in both age groups. The majority of T cells in the skin of both age groups expressed CD69, a characteristic of skin-resident T cells.
VZV-specific CD4(+) T cells were significantly increased in the skin compared with the blood in young and old subjects, and their function was similar in both age groups. In contrast, the number of Foxp3(+) regulatory T cells and expression of the inhibitory receptor programmed cell death -1 PD-1 on CD4(+) T cells were significantly increased in the skin of older humans.
Therefore, VZV-specific CD4(+) T cells in the skin of older individuals are functionally competent. However, their activity may be restricted by multiple inhibitory influences in situ.

Revisiting the role of B cells in skin immune surveillance.

Egbuniwe IU, Karagiannis SN, Nestle FO, Lacy KE.

Trends in Immunology 2015 Feb;36(2):102-11.


Whereas our understanding of the skin immune system has increased exponentially in recent years, the role of B cells in cutaneous immunity remains poorly defined. Recent studies have revealed the presence of B cells within lymphocytic infiltrates in chronic inflammatory skin diseases and cutaneous malignancies including melanoma, and have examined their functional significance in these settings. We review these findings and discuss them in the context of the current understanding of the role of B cells in normal skin physiology, as well as in both animal and human models of skin pathology. We integrate these findings into a model of cutaneous immunity wherein crosstalk between B cells and other skin-resident immune cells plays a central role in skin immune homeostasis.

IFN-α inhibits telomerase in human CD8⁺ T cells by both hTERT downregulation and induction of p38 MAPK signaling.

Lanna A, Coutavas E, Levati L, Seidel J, Rustin MH, Henson SM, Akbar AN, Franzese O.

Journal of Immunology 2013 Oct 1;191(7):3744-52.


The cytokine IFN-α is secreted during viral infections and has been shown to inhibit telomerase activity and accelerate T cell differentiation in vivo. However, the mechanism for this inhibition is not clear. In this study, we show that IFN-α inhibits both the transcription and translation of human telomerase reverse transcriptase (hTERT), the catalytic component of telomerase, in activated CD8(+) T cells. This was associated with increased activity of the repressor of hTERT transcription E2 transcription factor and decreased activation of NF-κB that promotes hTERT transcription. However IFN-α did not affect the translocation of hTERT from the cytoplasm to the nucleus. IFN-α also inhibits AKT kinase activation but increases p38 MAPK activity, and both of these events have been shown previously to inhibit telomerase activity. Addition of BIRB796, an inhibitor of p38 activity, to IFN-α-treated cells reversed, in part, the inhibition of telomerase by this cytokine. Therefore, IFN-α can inhibit the enzyme telomerase in CD8(+) T cells by transcriptional and posttranslational mechanisms. Furthermore, the addition of IFN-α to CD8(+)CD27(+)CD28(+) T cells accelerates the loss of both these costimulatory molecules. This suggests that persistent viral infections may contribute to the accumulation of highly differentiated/senescent CD8(+)CD27(-)CD28(- ) T cells during aging by promoting IFN-α secretion during repeated episodes of viral reactivation.

Investigation of the cutaneous response to recall antigen in humans in vivo.

Akbar AN, Reed JR, Lacy KE, Jackson SE, Vukmanovic-Stejic M, Rustin MH.

Clinical and Experimental Immunology 2013 Aug;173(2):163-72.


In this paper we provide a detailed description of an experimental method for investigating the induction and resolution of recall immune response to antigen in humans in vivo. This involves the injection of tuberculin purified protein derivative (PPD) into the skin, followed by inducing suction blisters at the site of injection, from which leucocytes and cytokines that are involved in the response can be isolated and characterized. Using this technique we found that although the majority of CD4(+) T cells in the skin that are present early in the response express cutaneous lymphocyte antigen (CLA), the expression of this marker is reduced significantly in later phases. This may enable these cells to leave the skin during immune resolution. Furthermore, interleukin (IL)-2 production can be detected both in CD4(+) T cells and also in the blister fluid at the peak of the response at day 7, indicating that mediators found in the blister fluid are representative of the cytokine microenvironment in vivo. Finally, we found that older humans have defective ability to respond to cutaneous PPD challenge, but this does not reflect a global immune deficit as they have similar numbers of circulating functional PPD-specific CD4(+) T cells as young subjects. The use of the blister technology enables further characterization of the skin specific defect in older humans and also general mechanisms that govern immune regulation in vivo.

Varicella zoster-specific CD4+Foxp3+ T cells accumulate after cutaneous antigen challenge in humans.

Vukmanovic-Stejic M, Sandhu D, Sobande TO, Agius E, Lacy KE, Riddell N, Montez S, Dintwe OB, Scriba TJ, Breuer J, Nikolich-Zugich J, Ogg G, Rustin MH, Akbar AN.

Journal of Immunology 2013 Feb 1;190(3):977-86.


We investigated the relationship between varicella zoster virus (VZV)-specific memory CD4(+) T cells and CD4(+)Foxp3(+) regulatory T cells (Tregs) that accumulate after intradermal challenge with a VZV skin test Ag. VZV-specific CD4(+) T cells were identified with a MHC class II tetramer or by intracellular staining for either IFN-γ or IL-2 after Ag rechallenge in vitro. VZV-specific T cells, mainly of a central memory (CD45RA(-)CD27(+)) phenotype, accumulate at the site of skin challenge compared with the blood of the same individuals. This resulted in part from local proliferation because >50% of tetramer defined Ag-specific CD4(+) T cells in the skin expressed the cell cycle marker Ki67. CD4(+)Foxp3(+) T cells had the characteristic phenotype of Tregs, namely CD25(hi)CD127(lo)CD39(hi) in both unchallenged and VZV challenged skin and did not secrete IFN-γ or IL-2 after antigenic restimulation.
The CD4(+)Foxp3(+) T cells from unchallenged skin had suppressive activity, because their removal led to an increase in cytokine secretion after activation. After VZV Ag injection, Foxp3(+)CD25(hi)CD127(lo)CD39(hi) T cells were also found within the VZV tetramer population. Their suppressive activity could not be directly assessed by CD25 depletion because activated T cells in the skin were also CD25(+). Nevertheless, there was an inverse correlation between decreased VZV skin responses and proportion of CD4(+)Foxp3(+) T cells present, indicating indirectly their inhibitory activity in vivo. These results suggest a linkage between the expansion of Ag-specific CD4(+) T cells and CD4(+) Tregs that may provide controlled responsiveness during Ag-specific stimulation in tissues.

Immune responses in the skin in old age.

Vukmanovic-Stejic M, Rustin MH, Nikolich-Zugich J, Akbar AN.

Current Opinion in Immunology 2011 Aug;23(4):525-31.


A marked increase in the susceptibility to cutaneous infections and malignancies has been observed in older humans indicating that cutaneous immunity becomes defective with age. In this review we will focus on recent developments in the understanding of age-related changes in immune function of the skin with a particular emphasis on how alterations in the interaction between cells involved in innate and adaptive immunity leads to decreased cutaneous antigen-specific T cell immunosurveillance.

Different proliferative potential and migratory characteristics of human CD4+ regulatory T cells that express either CD45RA or CD45RO.

Booth NJ, McQuaid AJ, Sobande T, Kissane S, Agius E, Jackson SE, Salmon M, Falciani F, Yong K, Rustin MH, Akbar AN, Vukmanovic-Stejic M.

Journal of Immunology 2010 Apr 15;184(8):4317-26.


Although human naturally occurring regulatory T cells (Tregs) may express either CD45RA or CD45RO, we find in agreement with previous reports that the ( approximately 80%) majority of natural Tregs in adults are CD45RO(+). The proportion of CD45RA(+) Tregs decreases, whereas CD45RO(+) Tregs increase significantly with age. Nevertheless, a small proportion of CD45RA(+) Tregs are found even in old (>80 y) adults and a proportion of these express CD31, a marker for recent thymic emigrants. We found that CD45RO(+) Tregs were highly proliferative compared with their CD45RA(+) counterparts. This was due in part to the conversion of CD45RA Tregs to CD45RO expression after activation. Another difference between these two Treg populations was their preferential migration to different tissues in vivo. Whereas CD45RA(+) Tregs were preferentially located in the bone marrow, associated with increased CXCR4 expression, CD45RO(+) Tregs were preferentially located in the skin, and this was associated with their increased expression of CLA and CCR4.
Our studies therefore show that proliferation features strongly in maintenance of the adult Treg pool in humans and that the thymus may make a minor contribution to the maintenance of the peripheral pool of these cells, even in older adults. Furthermore, the different tissue compartmentalization of these cells suggests that different Treg niches exist in vivo, which may have important roles for their maturation and function.

Decreased TNF-alpha synthesis by macrophages restricts cutaneous immunosurveillance by memory CD4+ T cells during aging.

Agius E, Lacy KE, Vukmanovic-Stejic M, Jagger AL, Papageorgiou AP, Hall S, Reed JR, Curnow SJ, Fuentes-Duculan J, Buckley CD, Salmon M, Taams LS, Krueger J, Greenwood J, Klein N, Rustin MH, Akbar AN.

Journal of Experimental Medicine 2009 Aug 31;206(9):1929-40.


Immunity declines during aging, however the mechanisms involved in this decline are not known. In this study, we show that cutaneous delayed type hypersensitivity (DTH) responses to recall antigens are significantly decreased in older individuals. However, this is not related to CC chemokine receptor 4, cutaneous lymphocyte-associated antigen, or CD11a expression by CD4(+) T cells or their physical capacity for migration. Instead, there is defective activation of dermal blood vessels in older subject that results from decreased TNF-alpha secretion by macrophages. This prevents memory T cell entry into the skin after antigen challenge. However, isolated cutaneous macrophages from these subjects can be induced to secrete TNF-alpha after stimulation with Toll-like receptor (TLR) 1/2 or TLR 4 ligands in vitro, indicating that the defect is reversible. The decreased conditioning of tissue microenvironments by macrophage-derived cytokines may therefore lead to defective immunosurveillance by memory T cells. This may be a predisposing factor for the development of malignancy and infection in the skin during aging.

The kinetics of CD4+Foxp3+ T cell accumulation during a human cutaneous antigen-specific memory response in vivo.

Vukmanovic-Stejic M, Agius E, Booth N, Dunne PJ, Lacy KE, Reed JR, Sobande TO, Kissane S, Salmon M, Rustin MH, Akbar AN.

Journal of Clinical Investigation 2008 Nov;118(11):3639-50.


Naturally occurring CD4(+)CD25(hi)Foxp3(+) Tregs (nTregs) are highly proliferative in blood. However, the kinetics of their accumulation and proliferation during a localized antigen-specific T cell response is currently unknown. To explore this, we used a human experimental system whereby tuberculin purified protein derivative (PPD) was injected into the skin and the local T cell response analyzed over time. The numbers of both CD4(+)Foxp3(-) (memory) and CD4(+)Foxp3(+) (putative nTreg) T cells increased in parallel, with the 2 populations proliferating at the same relative rate. In contrast to CD4(+)Foxp3(-) T cell populations, skin CD4(+)Foxp3(+) T cells expressed typical Treg markers (i.e., they were CD25(hi), CD127(lo), CD27(+), and CD39(+)) and did not synthesize IL-2 or IFN-gamma after restimulation in vitro, indicating that they were not recently activated effector cells.
To determine whether CD4(+)Foxp3(+) T cells in skin could be induced from memory CD4(+) T cells, we expanded skin-derived memory CD4(+) T cells in vitro and anergized them. These cells expressed high levels of CD25 and Foxp3 and suppressed the proliferation of skin-derived responder T cells to PPD challenge. Our data therefore demonstrate that memory and CD4(+) Treg populations are regulated in tandem during a secondary antigenic response. Furthermore, it is possible to isolate effector CD4(+) T cell populations from inflamed tissues and manipulate them to generate Tregs with the potential to suppress inflammatory responses.

Mantoux Test as a model for a secondary immune response in humans.

Vukmanovic-Stejic M, Reed JR, Lacy KE, Rustin MH, Akbar AN.

Immunology Letters 2006 Nov 15;107(2):93-101.


The Mantoux Test (MT) is a classical delayed-type hypersensitivity (DTH) response to the intradermal injection of tuberculin purified protein derivative (PPD). It represents a cutaneous T cell mediated memory recall immune response. The test is typically used to determine immunity to tuberculosis in humans and positive reactions develop in individuals previously exposed to Mycobacterium tuberculosis, and those immunised with the Bacillus of Calmette and Guérin (BCG) vaccine. In view of its relative accessibility human skin represents a convenient tissue for the investigation of human immune responses. Using the MT, we have been able to determine that significant cellular proliferation and clonal expansion occur at the site of antigen deposition in the skin. Furthermore, cells undergoing proliferation in the skin also undergo accelerated differentiation.
Taken together with other studies, in humans and in mice, these observations shed new light on the importance of the microenvironment at the site of the immune response for the proliferation and differentiation of memory T cells.