A characteristic feature of tumors is high production of lactic acid due to enhanced glycolysis. Here, we show a positive correlation between lactate serum levels and tumor burden in cancer patients and examine the influence of lactic acid on immune functions in vitro. Lactic acid suppressed the proliferation and cytokine production of human cytotoxic T lymphocytes (CTLs) up to 95% and led to a 50% decrease in cytotoxic activity. A 24-hour recovery period in lactic acid-free medium restored CTL function. CTLs infiltrating lactic acid-producing multicellular tumor spheroids showed a reduced cytokine production. Pretreatment of tumor spheroids with an inhibitor of lactic acid production prevented this effect. Activated T cells themselves use glycolysis and rely on the efficient secretion of lactic acid, as its intracellular accumulation disturbs their metabolism. Export by monocarboxylate transporter-1 (MCT-1) depends on a gradient between cytoplasmic and extracellular lactic acid concentrations and consequently, blockade of MCT-1 resulted in impaired CTL function. We conclude that high lactic acid concentrations in the tumor environment block lactic acid export in T cells, thereby disturbing their metabolism and function. These findings suggest that targeting this metabolic pathway in tumors is a promising strategy to enhance tumor immunogenicity.

Next-generation sequencing of the hypervariable V3 region of the 16s rRNA gene isolated from serial stool specimens collected from 31 patients receiving allogeneic stem cell transplantation (SCT) was performed to elucidate variations in the composition of the intestinal microbiome in the course of allogeneic SCT. Metagenomic analysis was complemented by strain-specific enterococcal PCR and indirect assessment of bacterial load by liquid chromatography-tandem mass spectrometry of urinary indoxyl sulfate. At the time of admission, patients showed a predominance of commensal bacteria. After transplantation, a relative shift toward enterococci was observed, which was more pronounced under antibiotic prophylaxis and treatment of neutropenic infections. The shift was particularly prominent in patients that developed subsequently or suffered from active gastrointestinal (GI) graft-versus-host disease (GVHD). The mean proportion of enterococci in post-transplant stool specimens was 21% in patients who did not develop GI GVHD as compared with 46% in those that subsequently developed GI GVHD and 74% at the time of active GVHD. Enterococcal PCR confirmed predominance of Enterococcus faecium or both E. faecium and Enterococcus faecalis in these specimens. As a consequence of the loss of bacterial diversity, mean urinary indoxyl sulfate levels dropped from 42.5 ± 11 µmol/L to 11.8 ± 2.8 µmol/L in all post-transplant samples and to 3.5 ± 3 µmol/L in samples from patients with active GVHD. Our study reveals major microbiome shifts in the course of allogeneic SCT that occur in the period of antibiotic treatment but are more prominent in association with GI GVHD. Our data indicate early microbiome shifts and a loss of diversity of the intestinal microbiome that may affect intestinal inflammation in the setting of allogeneic SCT.

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Thymus-derived CD4 ؉ CD25 ؉ regulatory T cells suppress autoreactive CD4 ؉ and CD8 ؉ T cells and thereby protect from autoimmunity. In animal models, adoptive transfer of CD4 ؉ CD25 ؉ regulatory T cells has been shown to prevent and even cure autoimmune diseases as well as pathogenic alloresponses after solid organ and stem-cell transplantations. We recently described methods for the efficient in vitro expansion of human regulatory T cells for clinical applications. We now demonstrate that only CCR7-and L-selectin (CD62L) - IntroductionSelf-tolerance within the T-cell compartment is primarily ensured by positive and negative selection during T-cell development in the thymus. Autoreactive T cells that escape central deletion are frequently controlled by peripheral tolerance mechanisms, including cell-mediated suppression by CD4 ϩ CD25 ϩ regulatory T (Treg) cells. [1][2][3] Thymic maturation and suppressive function of natural Treg cells depend on expression of the transcriptional repressor forkhead box P3 (FOXP3), as nonsense mutations in this gene result in loss of Treg-cell function and severe autoimmunity in mice and humans. [4][5][6][7][8] Apart from suppression of autoreactive T cells, FOXP3 ϩ CD4 ϩ CD25 ϩ Treg cells also dampen immune responses against infectious pathogens, 9 cancer, 10 and allogeneic organ 11 and stem-cell grafts. 12 Thus, depletion of Treg cells seems a promising strategy to augment immune responsiveness to tumors, chronic infections, and vaccination, while an enhancement of Treg-cell activity is envisaged for the prevention and treatment of T-cellinduced diseases. 13 In animal models, the adoptive transfer of CD4 ϩ CD25 ϩ Treg cells has been shown to protect from type 1 diabetes 14,15 or experimental autoimmune encephalomyelitis 16 and even revert ongoing disease in colitis 17,18 and arthritis. 19 Similarly, adoptively transferred Treg cells protected against rejection and graft-versus-host disease (GVHD) after allogeneic organ transplantation and bone marrow transplantation (BMT), respectively. 11,[20][21][22][23][24][25] In human peripheral blood, natural Treg cells mainly reside within the subpopulation of CD4 ϩ T cells with high CD25 expression levels (CD25 high ), 26 while cells with intermediate CD25 expression (CD25 int ) consist mainly of recently activated and memory T cells, with only 5% to 15% FOXP3 ϩ Treg cells (P.H. and M.E., unpublished results, June 2006). Due to lack of Treg cell-specific surface markers, isolation of CD4 ϩ CD25 high T cells is thus far considered the most promising strategy for the generation of pure Treg-cell products. 27 As they represent only 1% to 3% of peripheral-blood mononuclear cells (PBMCs), we and others recently described methods for the in vitro expansion of Treg cells for future clinical trials. [28][29][30] Cross-linking of stimulating CD3 and CD28 antibodies by beads or Fc receptor-bearing fibroblasts in combination with high-dose interleukin-2 (IL-2) resulted in a 3-to 4-log expansion within 3 to 4 weeks. Cultured cells maintained Treg-c...

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IntroductionImmunosuppression is an intrinsic capacity of the immune system and is partially mediated by T cells. The best-defined T-cell population with immunosuppressive activity is CD4 ϩ and constitutively expresses the interleukin 2 receptor (IL-2R) ␣-chain (CD25). 1 These thymus-derived suppressor cells 2,3 contribute to the maintenance of self-tolerance and thereby give protection from a variety of autoimmune diseases. 1,4 They control the size of the peripheral T-cell pool, 5 modulate immune responses after infection 6 and against tumors, 7,8 and contribute to tolerance induction after solid organ transplantation. 9,10 These findings confirm the central role of T reg cells in the modulation of immune responses under physiologic as well as pathologic conditions.We and others recently showed that donor-type CD4 ϩ CD25 ϩ T reg cells themselves do not induce graft-versus-host disease (GVHD) after major histocompatibility complex (MHC)-mismatched bone marrow transplantation but suppress GVHD induced by nonregulatory donor T cells. [11][12][13][14] Importantly, cotransfer of CD4 ϩ CD25 ϩ T reg cells neither interferes with stem cell engraftment [15][16][17] nor abrogates the beneficial antitumor activity of donor T-cell infusions. 15,18,19 These murine studies encouraged clinical transplanters to further investigate the role of donor CD4 ϩ CD25 ϩ T reg cells for the improvement of stem cell transplantation (SCT).CD4 ϩ T cells with high expression levels of CD25 have been isolated from human blood, peripheral lymphoid organs, umbilical cord blood, and thymus. [20][21][22][23][24][25] Phenotypically, they are comparable to their murine counterparts; that is, they constitutively express CD25, glucocorticoid-induced tumor necrosis factor receptor family-related gene (GITR) and intracellular cytotoxic T lymphocyteassociated antigen-4 (CTLA-4). In addition, expression of the transcription factor forkhead box P3 (FoxP3), described recently as a key regulatory gene for the development of CD4 ϩ CD25 ϩ T cells in mice, 26 could now be demonstrated in human CD4 ϩ CD25 ϩ T reg cells. 27 Most importantly, however, functional characteristics of murine CD4 ϩ CD25 ϩ T reg cells could be confirmed for human CD4 ϩ CD25 ϩ/high T reg cells, such as hyporesponsiveness to T-cell receptor (TCR)-mediated stimulation and cytokine-independent, cell contact-dependent suppression of cocultured CD25 Ϫ T cells. 20,21,23,25 Thus, immunoregulation by CD4 ϩ CD25 ϩ T reg cells seems to be a conserved mechanism, and their adoptive transfer or therapeutic manipulation in vivo might therefore be an attractive strategy for the prevention or treatment of T cell-mediated diseases and, in particular, for the protection from GVHD after allogeneic SCT.The main obstacle for the clinical application of human CD4 ϩ CD25 ϩ T reg cells so far is their paucity in peripheral blood and, in consequence, the necessity to develop reliable expansion protocols. Although several studies demonstrated that exogenous IL-2 can abrogate the anergic state of human and muri...

The adoptive transfer of CD4 1 CD25 1 natural regulatory T cells (Treg) is a promising strategy for the treatment of autoimmune diseases and the prevention of alloresponses after transplantation. Clinical trials exploring this strategy require efficient in vitro expansion of this rare cell population. Protocols developed thus far rely on high-grade purification of Treg prior to culture initiation, a process still hampered by the lack of Treg cell-specific surface markers. Depletion of CD127 1 cells was shown to separate activated conventional T cells from natural Treg cell populations allowing the isolation of highly enriched FOXP3 1 cells with all functional and molecular characteristics of natural Treg. Here, we demonstrate that upon in vitro expansion, CpG methylation in a conserved region within the FOXP3 gene locus increased in CD4 1 CD25 1 CD127 low Treg, correlating with loss of FOXP3 expression and emergence of pro-inflammatory cytokines. Further analysis identified CD45RA À FOXP3 1 memory-type Treg as the main source of converting cells, whereas CD45RA 1 FOXP3 1 Treg from the same donors showed no conversion within 3 wk of in vitro expansion. Thus, Treg cell lineage differentiation does not seem to represent a final fate decision, as natural Treg can lose their cell-type-specific characteristics after repetitive TCR stimulation.Key words: Cellular therapy . Immune regulation . Treg Supporting Information available online Introduction CD4 1 CD25 1 Treg are pivotal for the maintenance of peripheral self-tolerance and imbalances in this T-cell compartment have been shown to contribute to various autoimmune diseases [1]. In murine disease models, adoptively transferred Treg prevent, and in some cases, even cure autoimmunity [2,3]. In addition, they protect from graft rejection after allogeneic organ transplantation [4] as well as from graft-versus-host disease after MHCmismatched stem cell transplantation [5][6][7][8]. Recently, a limited number of Phase I clinical trials exploring the adoptive transfer of Treg have been initiated and several additional trials in various clinical settings are in preparation [9,10]. Prerequisites for the initiation of such trials are (i) the availability of efficient in vitro expansion protocols for this rare cell population and (ii) the ability to unequivocally identify Treg to avoid contamination of 1088Treg cultures with potentially harmful conventional effector T cells (Tconv). While efficient cell culture protocols have recently been established by us and others for the polyclonal as well as antigen-specific Treg cell expansion [11][12][13], the search for an exclusive surface marker for Treg is still ongoing.Contrary to earlier reports, human CD4 1 CD25 high Treg in adult peripheral blood have recently been shown to comprise not only (self-)antigen-experienced, CD45RA À central and effector memory cells, but also a subpopulation of CD45RA 1 naïve recent thymic emigrants [14,15]. We previously demonstrated that these CD45RA 1 CD4 1 CD25 high T cells (RA 1 Treg) homogen...

High concentrations of lactic acid (LA) are found under various pathophysiological conditions and are accompanied by an acidification of the environment. To study the impact of LA on TNF secretion, human LPS-stimulated monocytes were cultured with or without LA or the corresponding pH control. TNF secretion was significantly suppressed by low concentrations of LA (< or = 10 mM), whereas only strong acidification had a similar effect. This result was confirmed in a coculture model of human monocytes with multicellular tumor spheroids. Blocking synthesis of tumor-derived lactate by oxamic acid, an inhibitor of lactate dehydrogenase, reversed the suppression of TNF secretion in this coculture model. We then investigated possible mechanisms underlying the suppression. Uptake of [3-(13)C]lactate by monocytes was shown by hyphenated mass spectrometry. As lactate might interfere with glycolysis, the glycolytic flux of monocytes was determined. We added [1,2-(13)C(2)]glucose to the culture medium and measured glucose uptake and conversion into [2,3-(13)C(2)]lactate. Activation of monocytes increased the glycolytic flux and the secretion of lactate, whereas oxygen consumption was decreased. Addition of unlabeled LA resulted in a highly significant decrease in [2,3-(13)C(2)]lactate secretion, whereas a mere corresponding decrease in pH exerted a less pronounced effect. Both treatments increased intracellular [2,3-(13)C(2)]lactate levels. Blocking of glycolysis by 2-deoxyglucose strongly inhibited TNF secretion, whereas suppression of oxidative phosphorylation by rotenone had little effect. These results support the hypothesis that TNF secretion by human monocytes depends on glycolysis and suggest that LA and acidification may be involved in the suppression of TNF secretion in the tumor environment.