Malcolm H.A. Rustin

Human anergic/suppressive CD4+CD25+ T cells: a highly differentiated and apoptosis‐prone population

Anergic/suppressive CD4+CD25+ T cells exist in animal models but their presence has not yet been demonstrated in humans. We have identified and characterized a human CD4+CD25+ T cell subset, which constitutes 7–10 % of CD4+ T cells in peripheral blood and tonsil. These cells are a CD45RO+CD45RBlow highly differentiated primedT cell population that is anergic to stimulation. Depletion of this small subset from CD4+ T cells significantly enhances proliferation by threefold in the remaining CD4+CD25– T cells, while the addition of isolated CD4+CD25+ T cells to CD4+CD25– T cells significantly inhibits proliferative activity. Blocking experiments suggest that suppression is not mediated via IL‐4, IL‐10 or TGF‐β and is cell‐contact dependent. Isolated CD4+CD25+ T cells are susceptible to apoptosis that is associated with low Bcl‐2 expression, but this death can be prevented by IL‐2 or fibroblast‐secreted IFN‐β. However, the anergic/suppressive state of these cells is maintained after cytokine rescue. These human regulatory cells are therefore a naturally occurring, highly suppressive, apoptosis‐prone population which are at a late stage of differentiation. Further studies into their role in normal and pathological situations in humans are clearly essential.

Antigen-specific T cell suppression by human CD4+CD25+ regulatory T cells

Anergic/suppressive CD4+CD25+ T cells have been proposed to play an important role in the maintenance of peripheral tolerance. Here we demonstrate that in humans these cells suppress proliferation to self antigens, but also to dietary and foreign antigens. The suppressive CD4+CD25+ T cells display a broad usage of the T cell receptor Vβ repertoire,suggesting that they recognize a wide variety of antigens. They reside in the primed/memory CD4+CD45RO+CD45RBlow subset and have short telomeres, indicating that these cells have the phenotype of highly differentiated CD4+ T cells that have experienced repeated episodes of antigen‐specific stimulation in vivo. This suggests that anergic/suppressiveCD4+CD25+ T cells may be generated in the periphery as a consequence of repeated antigenic encounter. This is supported by the observation that highly differentiated CD4+T cells can be induced to become anergic/suppressive when stimulated by antigen presented by non‐professional antigen‐presenting cells. We suggest that besides being generated in the thymus, CD4+CD25+ regulatory T cells may also be generated in the periphery. This would provide a mechanism for the generation of regulatory cells that induce tolerance to a wide array of antigens that may not be encountered in the thymus.

Cytomegalovirus-Specific CD4+ T Cells in Healthy Carriers Are Continuously Driven to Replicative Exhaustion

Repeated antigenic encounter drives proliferation and differentiation of memory T cell pools. An important question is whether certain specific T cells may be driven eventually to exhaustion in elderly individuals since the human life expectancy is increasing. We found that CMV-specific CD4+ T cells were significantly expanded in healthy young and old carriers compared with purified protein derivative-, varicella zoster virus-, EBV-, and HSV-specific populations. These CMV-specific CD4+ T cells exhibited a late differentiated phenotype since they were largely CD27 and CD28 negative and had shorter telomeres. Interestingly, in elderly CMV-seropositive subjects, CD4+ T cells of different specificities were significantly more differentiated than the same cells in CMV-seronegative individuals. This suggested the involvement of bystander-secreted, differentiation-inducing factors during CMV infection. One candidate was IFN-α, which induced loss of costimulatory receptors and inhibited telomerase in activated CD4+ T cells and was secreted at high levels by CMV-stimulated plasmacytoid dendritic cells (PDC). The CMV-specific CD4+ T cells in elderly subjects had severely restricted replicative capacity. This is the first description of a human memory T cell population that is susceptible to being lost through end-stage differentiation due to the combined effects of lifelong virus reactivation in the presence of bystander differentiation-inducing factors.

Different Proliferative Potential and Migratory Characteristics of Human CD4+ Regulatory T Cells That Express either CD45RA or CD45RO

Although human naturally occurring regulatory T cells (Tregs) may express either CD45RA or CD45RO, we find in agreement with previous reports that the (∼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.

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

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.

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

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-α 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-α 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.

Scleromyxoedema-like changes in four renal dialysis patients.

Summary We describe four renal dialysis patients from our hospital who, over a 6‐month period, developed erythematous, thickened, indurated dermal plaques. The plaques were limited to the limbs and in three patients there were associated flexion contractures. The clinical features most resembled scleromyxoedema. All patients had previously received at least one renal transplant. Histopathology of the plaques showed features of scleromyxoedema in two patients, whereas the other two showed a different picture, more suggestive of a morphoea‐like process. There are important differences between our patients and classical scleromyxoedema. All four patients had normal immunoglobulins and no paraprotein was detected. Almost all cases of classical scleromyxoedema are associated with an IgGλ paraproteinaemia. We have not yet identified an underlying cause for this cluster of cases in our hospital. It is possible that the skin changes seen may have been precipitated by an environmental agent, such as in ‘toxic oil syndrome’ and vinyl chloride‐induced scleroderma. We discuss the differences between our patients and those with scleromyxoedema, localized or generalized morphoea and environmentally induced scleroderma. We feel that our patients show a constellation of features similar, but not identical, to scleromyxoedema. There has been only one previous report of similar patients. We believe this to be a new and distinct phenomenon.

The treatment of chilblains with nifedipine: the results of a pilot study, a double-blind placebo-controlled randomized study and a long-term open trial

A pilot study, a double‐blind placebo‐controlled randomised study and a long term open trial have indicated that nifedipine is effective in the treatment of perniosis. At a dose of 20 mg to 60 mg daily, nifedipine significantly reduced the time to clearance of existing lesions and prevented the development of new chilblains. Nifedipine also reduced the pain, soreness and irritation of the lesions. A comparison of the pre‐and post‐treatment skin biopsies showed resolution of the dermal oedema and diminution of the perivascular infiltrate. An increase in cutaneous blood flow following administration of nifedipine suggests that the vasodilator action of this drug may be important in its action.

Silica‐associated systemic sclerosis is clinically, serologically and immunologically indistinguishable from idiopathic systemic sclerosis

To determine whether the clinical, immunological and serological features of patients with silica‐associated systemic sclerosis are different from patients with the‘idiopathic’ form of systemic sclerosis (SS) we studied 22 underground coal miners who were exposed to silica dust (SD), 30 mine workers who later developed silicosis (S) and 17 mine workers exposed to silica dust who subsequently developed a systemic sclerosis‐like disease (SA‐SS).

The treatment of atopic dermatitis with adjunctive high-dose intravenous immunoglobulin: a report of three patients and review of the literature.

There are few reports of the use of high‐dose intravenous immunoglobulin (hdIVIg) in the treatment of atopic dermatitis (AD). We describe our experience using this therapy in three patients with severe AD, all of whom had steroid‐related side‐effects. These patients received either Alphaglobin® or Sandoglobulin® 2 g/kg monthly: all had improved skin scores, allowing reduction of their steroid dose. Total IgE fell in one of three patients. We discuss the side‐effects of hdIVIg and their management, and detail the differences between the available immunoglobulin products available in the U.K. There are several proposed mechanisms of action of this therapy which may be operative, and those most important in AD are discussed. In view of the time and expense involved in the treatment of patients with hdIVIg, careful patient assessment is vital. We describe dose reduction strategies and methods for cost containment. In addition, one of the patients has embarked on IVIg home therapy training. This will be the first time this has been attempted for a dermatological indication. Training of this type may be available through an immunotherapy service such as exists for patients with primary immunodeficiencies.