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

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.

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 ( 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.

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.

Nitric oxide generation in children with malaria and the NOS2G-954C promoter polymorphism.

Previous epidemiological studies have demonstrated a protective association between the NOS2G-954C (NOS2(Lambaréné)) polymorphism in inducible nitric oxide synthase and severe malaria. The polymorphism is commoner in children with uncomplicated compared with severe malaria. We now show that the likely mechanism for such protection is increased flux of nitrogen from arginine to nitric oxide (NO) during episodes of malaria. Forty-seven boys with uncomplicated malaria received an infusion of (15)N-arginine to measure directly whole body in vivo NO production. The NOS2G-954C genotype previously associated with reduced risk of severe malaria in Gabon was also assessed. Evaluable data were obtained from 40 boys, of whom 6 were NOS2G-954C heterozygotes. Heterozygotes had higher urinary (15)N nitrate enrichments, 2.3 ± 0.6 vs. 1.4 ± 0.5 atoms percent excess (P = 0.001) and higher ratios of (15)N between urine nitrate and plasma arginine (87 ± 11 vs. 57 ± 18%, P = 0.001) consistent with accelerated NO production. We also derived total NO production rates, combining data with total urine production rate and nitrate concentration; these showed no difference by genotype (0.62 ± 0.36, n = 6 vs. 0.83 ± 0.50 µmol/kg·h, n = 16; P = 0.36), but data were confounded by very high variability in measurements of urine output and nitrate concentrations. This study supports the idea that NOS2 genotype protects against severe malaria by increasing NO production during episodes of uncomplicated malaria.

Human Neutrophil Elastase Proteolytic Activity in Ulcerative Colitis Favors the Loss of Function of Therapeutic Monoclonal Antibodies.

PURPOSE: Proteases play an essential role in the pathophysiology of inflammatory bowel disease (IBD), contributing to the intestinal mucosal lesions through the degradation of the extracellular matrix and alteration of the barrier function. Ulcerative colitis (UC) is characterized by an extensive infiltrate of neutrophils into the mucosa and hence, increased proteolytic activity. Human neutrophil elastase (HNE) is a serine protease that has been reported to be increased in UC patients' intestinal mucosa. Based on our previous studies, we hypothesized that HNE might induce proteolytic degradation and loss of function of therapeutic monoclonal antibodies in IBD patients. PATIENTS AND METHODS: Elastase expression and elastinolytic activity were determined in mucosal explants from ulcerative colitis patients (n=6) and cultured ex vivo in the presence or absence of recombinant elafin. Enzymatic digestions of therapeutic monoclonal antibodies were performed using recombinant HNE and elafin. The integrity of the therapeutic antibodies was evaluated by immunoblotting and protein G binding assay, whereas their TNF-neutralizing activity was assessed with a reporter cell line. RESULTS: We found that HNE and its elastinolytic activity were increased in the gut mucosa of UC patients. We also demonstrated that HNE cleaved biological drugs, impairing the TNF-α neutralizing capacity of anti-TNF monoclonal antibodies. This proteolytic degradation was inhibited by the addition of the specific inhibitor, elafin. CONCLUSION: Our results suggest that the high level of proteolytic degradation by mucosal neutrophil elastase, along with a potential imbalance with elafin, contributes to the loss of function of biologic agents, which are currently used in patients with IBD. These findings might explain the non-responsiveness of UC patients to therapeutic monoclonal antibodies and suggest the potential beneficial concomitant use of elafin in this treatment.

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

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.