Regulatory T-cells (Treg) have been the focus of immunologic research due to their role in establishing tolerance for harmless antigens versus allowing immune responses against foes. Increased Treg frequencies measured by mRNA expression or protein synthesis of the Treg marker FOXP3 were found in various cancers, indicating that dysregulation of Treg levels contributes to tumor establishment. Furthermore, they constitute a key target of immunomodulatory therapies in cancer as well as transplantation settings. One core obstacle for understanding the role of Treg, thus far, is the inability of FOXP3 mRNA or protein detection methods to differentiate between Treg and activated T cells. These difficulties are aggravated by the technical demands of sample logistics and processing. Based on Treg-specific DNA demethylation within the FOXP3 locus, we present a novel method for monitoring Treg in human peripheral blood and solid tissues. We found that Treg numbers are significantly increased in the peripheral blood of patients with interleukin 2-treated melanoma and in formalin-fixed tissue from patients with lung and colon carcinomas. Conversely, we show that immunosuppressive therapy including therapeutic antibodies leads to a significant reduction of Treg from the peripheral blood of transplantation patients. In addition, Treg numbers are predictively elevated in the peripheral blood of patients with various solid tumors. Although our data generally correspond to data obtained with gene expression and protein-based methods, the results are less fluctuating and more specific to Treg. The assay presented here measures Treg robustly in blood and solid tissues regardless of conservation levels, promising fast screening of Treg in various clinical settings. [Cancer Res 2009;69(2):599-608]

Novel mRNA-based vaccines have been proven powerful tools to combat the global pandemic caused by SARS-CoV2 with BNT162b2 (trade name: Comirnaty) efficiently protecting individuals from COVID-19 across a broad age range. Still, it remains largely unknown how renal insufficiency and immunosuppressive medication affect development of vaccine induced immunity. We therefore comprehensively analyzed humoral and cellular responses in kidney transplant recipients after the standard second vaccination dose. As opposed to all healthy vaccinees and the majority of hemodialysis patients, only 4/39 and 1/39 transplanted individuals showed IgA and IgG seroconversion at day 8±1 after booster immunization with minor changes until day 23±5, respectively. Although most transplanted patients mounted spike-specific T helper cell responses, frequencies were significantly reduced compared to controls and dialysis patients, accompanied by a broad impairment in effector cytokine production, memory differentiation and activation-related signatures. Spike-specific CD8 + T cell responses were less abundant than their CD4 + counterparts in healthy controls and hemodialysis patients and almost undetectable in transplant patients. Promotion of anti-HLA antibodies or acute rejection was not detected after vaccination. In summary, our data strongly suggest revised vaccination approaches in immunosuppressed patients, including individual immune monitoring for protection of this vulnerable group at risk to develop severe COVID-19.

Regulatory T cells (T regs) IntroductionRegulatory T cells (T regs ) actively mediate self-tolerance and thus control autoimmunity. 1,2 T regs also limit antitumor T-cell responses and deleterious allogeneic T-cell responses that cause graft-versushost disease (GVHD) 3,4 and solid organ allograft rejection, 5 making them valuable therapeutic targets. We previously found that donor and host allogeneic responses are compromised in SH2 domaincontaining inositol 5-phosphatase (SHIP)-deficient hosts, which exhibit significantly reduced acute rejection of MHC-mismatched bone marrow grafts and GVHD. 6,7 Thus, an immunosuppressive environment prevails in SHIP-deficient hosts. We consistently observe a profound expansion of myeloid suppressor cells (MySC) in SHIP-deficient mice. [6][7][8][9] Because host and donor T regs limit GVHD 4,10 and Mac1 ϩ Gr1 ϩ cells, similar to SHIP Ϫ/Ϫ MySC, expand T regs in tumor and GVHD models 11,12 ; we considered that the T reg compartment in SHIP-deficient hosts may also be expanded. In addition, SHIP deficiency could intrinsically effect T reg homeostasis and function. SHIP can oppose PI3K signaling pathways triggered by engagement of costimulatory and cytokine receptors critical for the suppressive function, survival, and expansion of T regs , such as CD25 (interleukin-2 receptor ␣), IL-7R, and OX40. [13][14][15] Because T regs were initially characterized as CD4 ϩ T cells coexpressing CD25, most T reg studies focus on this phenotype, which is shared with activated CD4 ϩ T cells. 16 To distinguish between T regs and activated T cells, molecular markers correlated with or obligate for T reg function have been identified, such as the transcription factor FoxP3, as well as surface markers CD103, glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) and OX40, among others. FoxP3 functions as the master regulator in the development and suppressive ability of T regs . 17 CD103 expression among CD4 ϩ CD25 ϩ T regs distinguishes an effector/memory-like subset that displays an inflammationseeking phenotype and exhibits greater suppressive capacity. 18,19 In addition, CD103, which binds E-cadherin, mediates the retention of CD103 ϩ lymphocyte in epithelial compartments, 19 which are major sites of GVHD. Thus, CD103 ϩ T regs might have a prominent role in the prevention against GVHD. GITR and OX40, members of the tumor necrosis factor receptor superfamily of receptors, are costimulatory molecules known to play key roles in promoting the homeostasis, expansion, and suppressive capability of T regs . 20,21 Furthermore, CD4 ϩ CD25 ϩ OX40 ϩ T regs represent a mature population that does not require preactivation or stimulation to suppress antigen-specific T-cell responses. 22 Analysis of CD103, GITR, and FoxP3 expression has allowed the identification of a T reg population among "naive" CD4 ϩ CD25 Ϫ T cells. Specifically, CD4 ϩ CD25 Ϫ CD103 ϩ T cells display regulatory activity in both an in vitro proliferation assay and in vivo disease models, such as colitis and antigen-ind...