Combination of rapamycin and IL-2 increases de novo induction of human CD4(+)CD25(+)FOXP3(+) T cells

The immune system protects itself from autoreactivity by maintaining a balance between effector and Treg responses. Peripheral induction of Treg is one mechanism by which this balance may be maintained. Thus, it is important to understand factors that influence de novo generation of CD4⁺CD25⁺FOXP3⁺ Treg. Here, we focus on the effects of cytokines and the cell cycle inhibitor rapamycin. The cytokines IL-2 and IL-7, but not IL-4, increased initial activation induced FOXP3 expression, increased proliferation and sustained expression of FOXP3⁺ cells throughout the culture. Addition of rapamycin to cultures containing IL-2 further increased the frequency and absolute number of functional CD4⁺CD25⁺FOXP3⁺ Treg. This increase was not due to selective proliferation of FOXP3 cells, but was instead, the result of an increase in the frequency of FOXP3⁺ cells induced in G0 through delayed activation while the addition of IL-2 promoted survival and proliferation of the FOXP3⁺ population. Thus, combination of rapamycin and IL-2 may provide improved treatment options in transplantation and autoimmunity by promoting induction, survival, and expansion of functional iTreg from CD4⁺CD25⁻ cells.     

Identification of FOXP3-negative regulatory T-like (CD4(+)CD25(+)CD127(low)) cells in patients with immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome

Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is an autoimmune disorder caused by mutations in the FOXP3 gene, which plays a key role in the generation of CD4⁺CD25⁺regulatory T (Treg) cells. We selected CD127 as the surface marker of Treg cells to illustrate the development and function of Treg cells in IPEX syndrome. CD4⁺CD25⁺FOXP3⁺ T cells, the putative Treg cells, were almost completely absent in all patients. Importantly, a substantial number of CD4⁺CD25⁺CD127^low T cells were observed in 3 IPEX patients with hypomorphic mutations in the FOXP3 gene. We demonstrated that CD4⁺CD25⁺CD127^low T cells isolated from these 3 patients exhibited an appreciable suppressive activity on effector T cell proliferation, although less than that displayed by Treg cells from healthy controls. These results suggest that genetically altered FOXP3 can drive the generation of functionally immature Treg cells, but that intact FOXP3 is necessary for the complete function of Treg cells.     

Co‐expression of TNFR2 and CD25 identifies more of the functional CD4+FOXP3+ regulatory T cells in human peripheral blood

Previously, we found that co‐expression of CD25 and TNFR2 identified the most suppressive subset of mouse Treg. In this study, we report that human peripheral blood (PB) FOXP3⁺ cells present in CD25^high, CD25^low and even CD25^– subsets of CD4⁺ cells expressed high levels of TNFR2. Consequently, TNFR2‐expressing CD4⁺CD25 ⁺ Treg included all of the FOXP3⁺ cells present in the CD4⁺CD25^high subset as well as a substantial proportion of the FOXP3⁺ cells present in the CD4⁺CD25^low subset. Flow cytometric analysis of PB identified five‐fold more Treg, determined by FOXP3 expression, in the CD4⁺CD25⁺TNFR2⁺ subset than in the CD4⁺CD25^high subset. In addition, similar levels of FOXP3⁺ cells were identified in both the CD4⁺CD25⁺TNFR2⁺ and CD4⁺CD25⁺CD127^low/? subsets. Furthermore, the CD4⁺CD25⁺TNFR2⁺ subset expressed high levels of CTLA‐4, CD45RO, CCR4 and low levels of CD45RA and CD127, a phenotype characteristic of Treg. Upon TCR stimulation, human PB CD4 ⁺ CD25 ⁺ TNFR2 ⁺ cells were anergic and markedly inhibited the proliferation and cytokine production of co‐cultured T‐responder cells. In contrast, CD4 ⁺ CD25 ⁺ TNFR2 ^– and CD4 ⁺ CD25 ^– TNFR2 ⁺ T cells did not show inhibitory activity. As some non‐Treg express TNFR2, the combination of CD25 and TNFR2 must be used to identify a larger population of human Treg, a population that may prove to be of diagnostic and therapeutic benefit in cancer and autoimmune diseases.     

In vivo depletion of CD4CD25 regulatory T cells is associated with improved antiviral responses in cats chronically infected with feline immunodeficiency virus

Regulatory T (Treg) cells are activated and suppress immune responses during infection, and are characterized as CD4⁺CD25^hiFOXP3⁺. Ex vivo studies demonstrate that Treg cells potentially suppress anti-HIV-1 T cell responses. Lentivirus-induced CD4⁺CD25^hi Treg cells were first described in feline immunodeficiency virus (FIV)-infected cats. In the present study we demonstrate that anti-feline CD25 monoclonal antibody (mAb) therapy depletes Treg cells in FIV-infected cats for 4 weeks and does not exacerbate viral replication or proinflammatory cytokine production. Significant FIV-specific immune responses are revealed in Treg cell-depleted cats. These anti-FIV effector cells exist prior to Treg cell depletion and are not expanded while Treg cells are depleted. Importantly, cats receiving the Treg cell-depleting mAb are able to produce a robust humoral response to new antigen. We propose that short-term in vivo Treg cell depletion during chronic HIV-1 infection could provide a window of opportunity for therapeutic vaccination in individuals with controlled viral replication.     

Staphylococcus aureus convert neonatal conventional CD4+ T cells into FOXP3+ CD25+ CD127low T cells via the PD‐1/PD‐L1 axis

The gut microbiota provides an important stimulus for the induction of regulatory T (Treg) cells in mice, whether this applies to newborn children is unknown. In Swedish children, Staphylococcus aureus has become a common early colonizer of the gut. Here, we sought to study the effects of bacterial stimulation on neonatal CD4⁺ T cells for the induction of CD25⁺ CD127^low Treg cells in vitro. The proportion of circulating CD25⁺ CD127^low Treg cells and their expression of FOXP3, Helios and CTLA‐4 was examined in newborns and adults. To evaluate if commensal gut bacteria could induce Treg cells, CellTrace violet‐stained non‐Treg cells from cord or peripheral blood from adults were co‐cultured with autologous CD25⁺ CD127^low Treg cells and remaining mononuclear cells and stimulated with S. aureus. Newborns had a significantly lower proportion of CD25⁺ CD127^low Treg cells than adults, but these cells were Helios⁺ and CTLA‐4⁺ to a higher extent than in adults. FOXP3⁺ CD25⁺ CD127^low T cells were induced mainly in neonatal CellTrace‐stained non‐Treg cells after stimulation with S. aureus. In cell cultures from adults, S. aureus induced CD25⁺ CD127^low T cells only if sorted naive CD45RA⁺ non‐Treg cells were used, but these cells expressed less FOXP3 than those induced from newborns. Sorted neonatal CD25⁺ CD127^low T cells from S. aureus‐stimulated cultures were still suppressive. Finally, blocking PD‐L1 during stimulation reduced the induction of FOXP3⁺ CD25⁺ CD127^low T cells. These results suggest that newborns have a higher proportion of circulating thymically derived Helios⁺ Treg cells than adults and that S. aureus possess an ability to convert neonatal conventional CD4⁺ T cells into FOXP3⁺ CD25⁺ CD127^low Treg cells via the PD‐1/PD‐L1 axis.     

Phenotypic analysis of human peripheral blood regulatory T cells (CD4+FOXP3+CD127lo/–) ex vivo and after in vitro restimulation with malaria antigens

Regulatory T cells (Treg) play crucial roles in regulating autoimmune responses and immunity to tumors and infectious diseases. However, numerous subpopulations of Treg are now being described and the utility of various Treg markers is being reassessed. Here we report the results of a detailed phenotypic comparison of two supposedly regulatory human T‐cell populations, namely CD4⁺FOXP3⁺ T cells and CD4⁺CD25^hi T cells. We find that CD4⁺FOXP3⁺ cells are extremely heterogeneous with respect to CD25 expression and that FOXP3⁺ and CD25^hi CD4⁺ T cells differ in their expression of chemokine receptors (CCR), CD95 and Bcl‐2, suggestive of distinct migration characteristics and susceptibility to apoptosis. Further, we propose that CD25 expression should be regarded as an activation marker rather than as a defining marker of Treg. Lastly, CD4⁺FOXP3⁺ T cells activated in vitro with malaria antigen expressed the highest levels of CCR4 and CD95, and the lowest levels of CCR7, indicating that they are most likely generated from effector memory cells during an immune response and rapidly succumb to apoptosis at the end of the response.     

The Increase of IFN-γ Production through Aging Correlates with the Expanded CD8CD28CD57 Subpopulation

The use of flow cytometry to detect intracellular cytokines at the single cell level has the potential to quantify cytokine production together with the possibility of phenotypic identification of the cell population concerned. The unbalanced presence of intracellular cytokines produced by T cells has been recognized in some pathological conditions. To better address this issue, we studied the production of IFN-γ and IL-4 in CD4⁺ and CD8^+high T cells in healthy donors of a broad range of age (17–62 years). Given that an increase of IFN-γ and IL-4 with aging had been reported by some authors in healthy controls, we have performed a multivariate analysis to assess the intrinsic role of aging or of other external factors, such as chronic antigenic exposures (i.e., viruses), over the cytokine production of phenotypically characterized T cells. In this respect we show that, mainly in CD8^+high T cells, the production of IFN-γ is directly correlated with age. Besides, the cytokine production correlates with the CD8^+highCD28⁻CD57⁺ T-cell population, which we have recently reported elevated in aged individuals. Perhaps this T-cell subpopulation plays a regulatory role as a Tc1 response in aging individuals.     

Surface expression of CD39 identifies an enriched Treg‐cell subset in the rheumatic joint,which does not suppress IL‐17A secretion

Treg cells are important for the maintenance of self‐tolerance and are implicated in autoimmunity. Despite enrichment of Treg cells in joints of rheumatoid arthritis (RA) patients, local inflammation persists. As expression of the ATP‐hydrolyzing enzymes CD39 and CD73 and the resulting anti‐inflammatory adenosine production have been implicated as an important mechanism of suppression, we characterized FOXP3⁺ Treg cells in blood and synovial fluid samples of RA patients in the context of CD39 and CD73 expression. Synovial FOXP3⁺ Treg cells displayed high expression levels of rate‐limiting CD39, whereas CD73 was diminished. FOXP3⁺CD39⁺ Treg cells were also abundant in synovial tissue. Furthermore, FOXP3⁺CD39⁺ Treg cells did not secrete the proinflammatory cytokines IFN‐γ and TNF after in vitro stimulation in contrast to FOXP3⁺CD39^? T cells. FOXP3⁺CD39⁺ Treg cells could be isolated by CD39 and CD25 coexpression, displayed a demethylated Treg‐specific demethylated region and coculture assays confirmed that CD25⁺CD39⁺ T cells have suppressive capacity, while their CD39^? counterparts do not. Overall, our data show that FOXP3⁺CD39⁺ Treg cells are enriched at the site of inflammation, do not produce proinflammatory cytokines, and are good suppressors of many effector T‐cell functions including production of IFN‐γ, TNF, and IL‐17F but do not limit IL‐17A secretion.     

Homeostatic regulation of T effector to Treg ratios in an area of seasonal malaria transmission

An important aspect of clinical immunity to malaria is the ability to down‐regulate inflammatory responses, once parasitaemia is under control, in order to avoid immune‐mediated pathology. The role of classical (CD4⁺CD25⁺CD127^lo/?FOXP3⁺) Treg in this process, however, remains controversial. Thus, we have characterized the frequency, phenotype and function of Treg populations, over time, in healthy individuals in The Gambia. We observed that both the percentage and the absolute number of CD4⁺FOXP3⁺CD127^lo/? T cells were higher among individuals living in a rural village with highly seasonal malaria transmission than among individuals living in an urban area where malaria rarely occurs. These CD4⁺FOXP3⁺CD127^lo/? T cells exhibited an effector memory and apoptosis‐prone phenotype and suppressed cytokine production in response to malaria antigen. Cells from individuals exposed to malaria expressed significantly higher levels of mRNA for forkhead box P3 and T‐box 21 (T‐BET) at the end of the malaria transmission season than at the end of the non‐transmission season. Importantly, the ratio of T‐BET to forkhead box P3 was remarkably consistent between populations and over time, indicating that in healthy individuals, a transient increase in Th1 responses during the malaria transmission season is balanced by a commensurate Treg response, ensuring that immune homeostasis is maintained.     

Loss of FOXP3 expression in natural human CD4+CD25+ regulatory T cells upon repetitive in vitro stimulation

The adoptive transfer of CD4⁺CD25⁺ 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⁺ cells was shown to separate activated conventional T cells from natural Treg cell populations allowing the isolation of highly enriched FOXP3⁺ 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⁺CD25⁺CD127^low Treg, correlating with loss of FOXP3 expression and emergence of pro‐inflammatory cytokines. Further analysis identified CD45RA^?FOXP3⁺ memory‐type Treg as the main source of converting cells, whereas CD45RA⁺FOXP3⁺ 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.     

Allogeneic induced human FOXP3+IFN‐γ+ T cells exhibit selective suppressive capacity

Human induced CD4⁺CD25⁺ T cells have been shown to express FOXP3, similar to naturally occurring Treg cells (nTreg). However, the suppressive capacity of these cells is still under debate. The current study was designed to investigate functional characteristics of CD25⁺FOXP3⁺ derived from CD25^? T cells. Stimulation of CD25^? PBMC with allogeneic PBMC resulted in production of CD4⁺CD25^high T cells. This process was more rapid and prominent when highly mature DC were used for stimulation. The resultant CD4⁺CD25^high population concurrently exhibited regulatory markers FOXP3, CTLA‐4, GITR, and inflammatory cytokines IL‐2 and IFN‐γ. These human‐induced FOXP3⁺IFN‐γ⁺ T cells were shown, for the first time, to markedly inhibit alloreactive T‐cell expansion, similar to nTreg. However, in contrast to nTreg, the induced CD4⁺CD25⁺FOXP3⁺ cells did not suppress proliferation against a third party donor stimulus or CMV. This suggested that the cell population possessed a more selective suppressive capacity targeted against the original stimulus only. The induced human CD4⁺CD25⁺FOXP3⁺ subset derived from CD25^? T cells, while expressing inflammatory cytokines, exhibits a suppressive cell contact‐dependent effect, restricted against T cells responding to the original stimulus. Such unique properties suggest that these cells are potentially ideal for the use as post‐transplant GVH disease prophylaxis.     

Functional islet‐specific Treg can be generated from CD4+CD25− T cells of healthy and type 1 diabetic subjects

CD4⁺CD25⁺FOXP3⁺ Treg cells require TCR engagement for suppressive function, thus ensuring that suppression occurs only in the presence of specific antigens; however, to date no studies have addressed the function of self‐antigen‐specific Treg in humans. These studies were designed to determine whether peripheral generation and function of islet antigen‐specific adaptive Treg are defective in human subjects with type 1 diabetes (T1D). Islet antigen‐specific adaptive Treg were induced in vitro by activation of CD4⁺FOXP3^? T cells with glutamic acid decarboxylase and islet‐specific glucose‐6‐phosphate catalytic subunit‐related protein peptides in the context of T1D‐associated HLA‐DRβ alleles. Antigen‐specific Treg were characterized using flow cytometry for FOXP3 and class II tetramer and assessed for the ability to inhibit proliferation. These adaptive Treg were then compared with influenza‐specific Treg from the same study population. The function of tetramer⁺ cells that expressed FOXP3 was similar for both influenza and islet antigens generated from control and T1D subjects. In fact, the potency of suppression correlated with FOXP3 expression, not antigen specificity. Thus, these data suggest that development of functional adaptive Treg can occur in response to islet antigens and activation of islet‐specific Treg may potentially be used as a targeted immunotherapy in T1D.     

1142292807Expression of CD4+CD25+FOXP3+ regulatory T cells in women with idiopathic recurrent spontaneous abortion and implantation failure: 3-color flow cytometric analysis

Introduction: A population of CD4⁺CD25⁺ regulatory T (Treg) cells is thought to regulate alloreactive T cells in many autoimmune diseases. Lack of Treg cells resulted in abortions in mice and transfer of them prevented miscarriage. FOXP3 is now considered the most specific marker for Treg cells. In this study, we investigated whether levels of peripheral blood Treg cells in women with recurrent spontaneous abortion (RSA) of unknown etiology or with repeated implantation failures (IF) are different from those of normal fertile women. Materials and Methods: Non‐pregnant women with a history of idiopathic RSA or repeated IF were enrolled in the study group (n = 15) and non‐pregnant fertile females served as controls (n = 7). A flow cytometry assay was used. Peripheral blood mononuclear cells (PBMCs) were isolated and stained with appropriate monoclonal antibodies that identify Treg cells: for surface markers, such as anti‐CD4 and anti‐CD25, and for intracellular marker, anti‐FOXP3. Results: The proportions of CD4⁺CD25⁺FOXP3⁺ Treg cells were significantly different between the patients and controls, 1.5% versus 2.4%, respectively (P < 0.05). In addition, an alteration in the expression of surface CD25 was noted after permeabilization of PBMC which allows antibodies to enter the cells and bind to FOXP3. The mean percentage of CD25+ cells before permeabilization, 9.9 + 4.8%, was decreased to 5.3+3.5% after permeabilization (P < 0.01). The ratios of CD4⁺CD25⁺/CD4⁺ between the surface and the intracellular staining also decreased by 46% (P < 0.001). Conclusion: CD4⁺CD25⁺FOXP3⁺ cells were significantly lower in the patients with idiopathic RSA or multiple IF than in the controls. Permeabilization for intracellular staining induces a decrease in expression of surface markers.     

Dexamethasone upregulates FOXP3 expression without increasing regulatory activity

Recent studies have shown the capacity of corticoids to increase forkhead box p3 (FOXP3) expression, which suggests that these drugs may be able to generate regulatory T cells (Treg). Therefore, corticoids may possibly be employed in protocols to generate or expand Treg cells with the aim of being used in cell transfer therapy. However, given that in humans FOXP3 is not necessarily associated with regulatory function, it is of great importance to ascertain whether FOXP3-expressing cells generated with corticoids are “truly” Treg cells. To this end, we studied the effect of dexamethasone on both human activated lymphocytes and in vitro generated Treg cells as well as regulatory activity of CD4⁺CD25^high cells from SLE patients users and non users of prednisone. Results show that dexamethasone markedly enhances FOXP3 expression and generates CD25^high cells with phenotypic characteristics attributable to natural Treg cells. Unexpectedly, in spite of their hyporesponsiveness and enhanced FOXP3 expression, these cells did not exert suppressive activity. Moreover, although dexamethasone was able to enhance FOXP3 expression in in vitro generated Treg cells, once again this effect was not correlated with increased regulatory activity. These results were supported by the fact that CD4⁺CD25^high cells from steroid-treated SLE patients did not show a higher antiproliferative function than those from non-steroid-treated patients. We conclude that the increment on FOXP3 expression caused by dexamethasone is not connected with regulatory function, supporting the fact that FOXP3 expression in humans is not an exclusive attribute of Treg cells. Subsequently, the use of FOXP3 as a Treg cell marker must be done cautiously, especially in patients with systemic inflammatory diseases or those under corticoid treatment.     

Histone methylation mediates plasticity of human FOXP3+ regulatory T cells by modulating signature gene expressions

CD4⁺ FOXP3⁺ regulatory T (Treg) cells constitute a heterogeneous and plastic T-cell lineage that plays a pivotal role in maintaining immune homeostasis and immune tolerance. However, the fate of human Treg cells after loss of FOXP3 expression and the epigenetic mechanisms contributing to such a phenotype switch remain to be fully elucidated. In the current study, we demonstrate that human CD4⁺ CD25^high CD127^low/− Treg cells convert to two subpopulations with distinctive FOXP3⁺ and FOXP3⁻ phenotypes following in vitro culture with anti-CD3/CD28 and interleukin-2. Digital gene expression analysis showed that upon in vitro expansion, human Treg cells down-regulated Treg cell signature genes, such as FOXP3, CTLA4, ICOS, IKZF2 and LRRC32, but up-regulated a set of T helper lineage-associated genes, especially T helper type 2 (Th2)-associated, such as GATA3, GFI1 and IL13. Subsequent chromatin immunoprecipitation-sequencing of these subpopulations yielded genome-wide maps of their H3K4me3 and H3K27me3 profiles. Surprisingly, reprogramming of Treg cells was associated with differential histone modifications, as evidenced by decreased abundance of permissive H3K4me3 within the down-regulated Treg cell signature genes, such as FOXP3, CTLA4 and LRRC32 loci, and increased abundance of H3K4me3 within the Th2-associated genes, such as IL4 and IL5; however, the H3K27me3 modification profile was not significantly different between the two subpopulations. In conclusion, this study revealed that loss of FOXP3 expression from human Treg cells during in vitro expansion can induce reprogramming to a T helper cell phenotype with a gene expression signature dominated by Th2 lineage-associated genes, and that this cell type conversion may be mediated by histone methylation events.     

CXCL4 promoted the production of CD4+CD25+FOXP3+treg cells in mouse sepsis model through regulating STAT5/FOXP3 pathway

Abstract

Background: CXCL4 plays an essential role in the regulation of multiple immune diseases. However, the underlying role of CXCL4 is still not clear in sepsis. Aim: In the present study, we aimed to investigate the function of CXCL4 in sepsis.

Methods: Sepsis model was constructed on mouse. Flow cytometry was used to determine the ratio of CD4⁺CD25⁺FOXP3⁺Treg cells. ELISA assays were used to determine the levels of CXCL4, IL-6, IL-10, and TNF-α respectively. Western blot was used to examine protein contents.

Results: Our results suggested that the serum level of CXCL4 was upregulated in patients with sepsis and positively associated with the ratio of human CD4⁺CD25⁺FOXP3⁺Treg cells. To further examine the role of CXCL4 in sepsis, we constructed the mouse sepsis model. Our results indicated that the mouse antibody of CXCL4 treatment reduced the expression of urine creatinine and urea nitrogen in sepsis model. Moreover, the frequency of CD25⁺FOXP3⁺ mouse regulatory T cells (Tregs) cells was decreased in mouse CD4⁺ T cells in the presence of mouse CXCL4 antibody. Further, the mouse recombinant protein CXCL4 was used to culture normal mouse CD4⁺ T cells in vitro. Our finding indicated that the recombinant protein CXCL4 promoted the percentage of mouse CD25⁺FOXP3⁺Treg cells and enhanced the phosphorylation of STAT5 in mouse CD4⁺ T cells in a dose-dependent manner. However, these effects were significantly reversed by the STAT5 inhibitor (p?<?.001). Conclusion: our findings not only indicated the function and signalling pathway of CXCL4 in CD4⁺ T cells but also provided novel insight and target in sepsis treatment.     

VEGFR2 is selectively expressed by FOXP3high CD4+ Treg

CD25⁺ FOXP3⁺CD4⁺ T cells (Treg) have been considered to play an important role in immune tolerance against several tumor antigens. It has also been indicated that high‐level expression of FOXP3 (FOXP3^high) is sufficient to confer suppressive activity to normal non‐Treg. Here, we showed for the first time that vascular endothelial growth factor receptor 2 (VEGFR2) is selectively expressed by FOXP3^high but not FOXP3^low Treg. Such VEGFR2⁺ Treg exist in several tissues including PBMC and malignant effusion‐derived lymphocytes. In conclusion, VEGFR2 may be a novel target for controlling Treg with highly suppressive function.     

Lactobacillus crispatus strain SJ‐3C‐US induces human dendritic cells (DCs) maturation and confers an anti‐inflammatory phenotype to DCs

Lactobacillus crispatus is one of the most predominant species in the healthy vagina microbiota. Nevertheless, the interactions between this commensal bacterium and the immune system are largely unknown. Given the importance of the dendritic cells (DCs) in the regulation of the immunity, this study was performed to elucidate the influence of vaginal isolated L. crispatus SJ‐3C‐US from healthy Iranian women on DCs, either directly by exposure of DCs to ultraviolet‐inactivated (UVI) and heat‐killed (HK) L. crispatus SJ‐3C‐US or indirectly to its cell‐free supernatant (CFS), and the outcomes of immune response. In this work we showed that L. crispatus SJ‐3C‐US induced strong dose‐dependent activation of dendritic cells and production of high levels of IL‐10, whereas IL‐12p70 production was induced at low level in an inverse dose‐dependent manner. This stimulation skewed T cells polarization toward CD4⁺ CD25⁺ FOXP3⁺ Treg cells and production of IL‐10 in a dose‐dependent manner in mixed leukocyte reaction (MLR) test. The mode of bacterial inactivation did not affect the DCs activation pattern, upon encounter with L. crispatus SJ‐3C‐US. Moreover, while DCs stimulated with CFS showed moderate phenotypic maturation and IL‐10 production, it failed to skew T cells polarization toward CD4⁺ CD25⁺ FOXP3⁺ regulatory T cells (Treg) and production of IL‐10. This study showed that L. crispatus SJ‐3C‐US confers an anti‐inflammatory phenotype to DCs through up‐regulation of anti‐inflammatory/regulatory IL‐10 cytokine production and induction of CD4⁺ CD25⁺ FOXP3⁺ T cells at optimal dosage. Our findings suggest that L. crispatus SJ‐3C‐US could be a potent candidate as protective probiotic against human immune‐mediated pathologies, such as chronic inflammation, vaginitis or pelvic inflammatory disease (PID).