Regulatory T-cells (Tregs) act at the interface of host and pathogen interactions in human infectious diseases. Tregs are induced by a wide range of pathogens, but distinct effects of Tregs have been demonstrated for different pathogens and in different stages of infection. Moreover, Tregs that are induced by a specific pathogen may non-specifically suppress immunity against other microbes and parasites. Thus, Treg effects need to be assessed not only in homologous but also in heterologous infections and vaccinations. Though Tregs protect the human host against excessive inflammation, they probably also increase the risk of pathogen persistence and chronic disease, and the possibility of disease reactivation later in life. Mycobacterium leprae and Mycobacterium tuberculosis, causing leprosy and tuberculosis, respectively, are among the most ancient microbes known to mankind, and are master manipulators of the immune system toward tolerance and pathogen persistence. The majority of mycobacterial infections occur in settings co-endemic for viral, parasitic, and (other) bacterial coinfections. In this paper, we discuss recent insights in the activation and activity of Tregs in human infectious diseases, with emphasis on early, late, and non-specific effects in disease, coinfections, and vaccination. We highlight mycobacterial infections as important models of modulation of host responses and vaccine-induced immunity by Tregs.

T uberculosis (TB), caused by Mycobacterium tuberculosis, is the second greatest infectious cause of death worldwide after HIV, accounting for 1.3 million deaths in 2012 (1). The only available vaccine, Mycobacterium bovis bacillus Calmette-Guérin (BCG), protects infants from disseminated forms of TB but has insufficient and inconsistent efficacy in protecting adults from pulmonary TB (1, 2). A vaccine preventing active pulmonary TB, the contagious form of the disease, would greatly impact the epidemic (3), and a better understanding of vaccine-induced mechanisms of protection is essential in developing new surrogate endpoints (4).Both CD4 ϩ Th1 (gamma interferon-positive [IFN-␥ ϩ ]) cells and CD8 ϩ T cells are critical for protection against TB (5). Specifically, CD4ϩ IFN-␥ ϩ interleukin 2-positive (IL-2 ϩ ) tumor necrosis factor ␣-positive (TNF-␣ ϩ ) polyfunctional T cells have been proposed as a correlate of vaccine-induced protective immunity in murine infection models (6). In infants, BCG vaccination induced specific cytokine expression in CD4 ϩ and CD8 ϩ T cells (7-9), including IFN-␥ ϩ IL-2 ϩ TNF-␣ ϩ polyfunctional CD4 ϩ T cells (10). However, there was no relation between the presence of such cells and the development of TB during follow-up (11).In adults, BCG vaccination induced CD4 ϩ IFN-␥ ϩ responses (12-14) as well as IFN-␥-and TNF-␣-secreting CD8 ϩ T cells with cytotoxic activity (15). However, data on the induction of polyfunctional T cells by BCG vaccination in adults have been conflicting (16,17). In one report, the induction of polyfunctional CD4 ϩ T cells was similar in magnitude in BCG-vaccinated infants and adults; however, when induction was analyzed as the proportion of polyfunctional versus single-cytokine-producing T cells, the proportion of polyfunctional CD4 ϩ T cells was larger in children than in adults (16). Further, studies on latent (controlled) versus active TB in adults yielded variable results on changes in monoand triple-cytokine-producing T cell subsets (18,19), such that it was suggested that polyfunctional T cells are also present in active TB disease and that these cells are not a surrogate marker of protection against TB in humans (19,20).Another explanation for the inconsistent protection induced by BCG against TB in adults is the induction of regulatory T cells (Tregs) by mycobacteria, which can dampen proinflammatory responses (21). In that context, we reported that live BCG triggers the specific activation of CD8 ϩ (but not CD4 ϩ ) Tregs from peripheral blood mononuclear cells (PBMCs) of mycobacterial purified protein derivative (PPD)-responsive adults (22), while others found that BCG vaccination induced CD4 ϩ Tregs in newborns (23) and adults (24). Here, in a small, well-defined cohort of pre-

Keywords: BCG r CD39 r CD8 + regulatory T (Treg) cell r Tuberculosis Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionMycobacterium tuberculosis was responsible for 1.4 million deaths in 2010 and is estimated to have infected one-third of the world population [1]. There is extensive evidence suggesting that M. tuberculosis strongly modulates the immune response, both innate and adaptive, to infection, with an important role for regulatory T (Treg) cells [2]. [20]. In damaged tissues, ATP is released in high concentrations, and functions as chemoattractant, generating a broad spectrum of pro-inflammatory responses [21]. ATP can also trigger mycobacterial killing in infected macrophages [22][23][24], can stimulate phagosome-lysosome fusion through P2X7 receptor activation [25], and can drive Th-17 cell differentiation in the murine lamina propria [26]. In a study focusing on the novel M. tuberculosis vaccine MVA85A, a drop in extracellular ATP consumption by PBMCs from subjects 2 weeks after vaccination corresponded with a decrease in CD4 + CD39 + Treg cells and a concomitant increase in the co-production of IL-17 and IFN-γ by CD4 + T cells [27]. Further hydrolysis of adenosine monophosphate by ecto-5 -nucleotidase (CD73) generates extracellular adenosine [20], which modulates inflammatory tissue damage, among others by inhibiting T-cell activation and multiple T-cell effector functions through A2A receptor-mediated signaling [28]. BCG, the only currently available vaccine for TB, fails to protect adults adequately and consistently from pulmonary TB [29], and part of this deficiency may be explained by induction of Treg cells by the BCG vaccine [7,30,31] (Fig. 3A). Flow cytometric analysis of sorted T-cell lines demonstrated enrichment for LAG-3, CD25, Foxp3, and CCL4 in the CD8 + CD39 + compared with the CD8 + CD39 − T cells (Fig. 3B).Blocking CD39 results in partial reversal of suppression by M. bovis BCG-stimulated CD8 + CD39 + T cellsas well as of other Treg-cell markers, including CD25, Foxp3, and CCL4 (Supporting Information Fig. 2) following further Eur. J. Immunol. 2013Immunol. . 43: 1925Immunol. -1932 (Fig. 4). Suppression by the CD8 + CD39 + T cells was also (partially) reversed by the anti-CD39 blocking monoclonal antibody BY40/OREG-103 [36, 37] (0-35% reversal of suppression; in four experiments; p = 0.005; Wilcoxon signed-ranks test) (Fig. 5); further supporting the direct functional involvement of CD39 in suppression mediated by CD8 + CD39 + Treg cells.To exclude that suppressive activity by CD8 + CD39 + T-cell lines was due to lysis rather than active suppression of the CD4 + Th1 responder clone, the Th1 responder clone and an equal number of cells of an irrelevant T-cell clone were labeled with low and high doses CFSE, respectively, and were added in equal numbers to the coculture assay, identical to previously described [13]. After 16 h, before division of the Th1 responder clone occurs, the percentages of responder ...

Mycobacterium bovis bacillus Calmette-Guérin (M. bovis BCG), the only currently available vaccine against tuberculosis, has been reported to induce regulatory T cells in humans. The activity of regulatory T cells may not only dampen immunogenicity and protective efficacy of tuberculosis-vaccines, but also hamper diagnosis of infection of tuberculosis, when using immune (e.g. IFNγ-release) assays. Still, in settings of infectious diseases and vaccination, most studies have focused on CD4+ regulatory T cells, and not CD8+ regulatory T-cells. Here, we present a comparative analysis of the suppressive phenotype and function of CD4+ versus CD8+ T cells after in vitro live BCG activation of human cells. Moreover, as BCG is administered as a (partly) live vaccine, we also compared the ability of live versus heatkilled BCG in activating CD4+ and CD8+ regulatory T cell responses. BCG-activated CD8+ T cells consistently expressed higher levels of regulatory T cell markers, and after live BCG activation, density and (co-)expression of markers were significantly higher, compared to CD4+ T cells. Furthermore, selection on CD25-expression after live BCG activation enriched for CD8+ T cells, and selection on co-expression of markers further increased CD8+ enrichment. Ultimately, only T cells activated by live BCG were functionally suppressive and this suppressive activity resided predominantly in the CD8+ T cell compartment. These data highlight the important contribution of live BCG-activated CD8+ Treg cells to immune regulation and emphasize their possible negative impact on immunity and protection against tuberculosis, following BCG vaccination.

Omicron BA.1 variant can readily infect people with vaccine-induced or naturally acquired SARS-CoV-2 immunity facilitated by escape from neutralizing antibodies. In contrast, T-cell reactivity against the Omicron BA.1 variant seems relatively well preserved. Here, we studied the preexisting T cells elicited by either vaccination with the mRNA-based BNT162b2 vaccine or by natural infection with ancestral SARS-CoV-2 for their cross-reactive potential to 20 selected CD4+ T-cell epitopes of spike-protein-harboring Omicron BA.1 mutations. Although the overall memory CD4+ T-cell responses primed by the ancestral spike protein was still preserved generally, we show here that there is also a clear loss of memory CD4+ T-cell cross-reactivity to immunodominant epitopes across the spike protein due to Omicron BA.1 mutations. Complete or partial loss of preexisting T-cell responsiveness was observed against 60% of 20 nonconserved CD4+ T-cell epitopes predicted to be presented by a broad set of common HLA class II alleles. Monitoring such mutations in circulating strains helps predict which virus variants may escape previously induced cellular immunity and could be of concern.