Approbation of a Homologous Model of the Antitumor Vaccine Based on Mature Mouse Dendritic Cells to Study the Biodistribution of the Cell Product.

Homologous animal cell product was obtained in protocol developed for female BALB/c mice. Dendritic cell (DC) migration from the injection site into the draining lymph nodes was evaluated. The number of DC labeled with carboxyfluorescein succinimidyl ester (CFSE) in draining lymph nodes increased from 5.3% (16 h) to 13.3% (48 h) (p=0.028) with a maximum at 72 h (15.4%, p=0.003). The immunophenotype of CFSE-DC detected in murine lymph nodes corresponded to the immunophenotype of mature vaccine DCs: they expressed differentiation markers CD11c, CD80, CD83, and CD86 (p>0.05 vs initial DC).

Spatiotemporal coordination of antigen presentation and co-stimulatory signal for enhanced anti-tumor vaccination.

Controlled-release systems enhance anti-tumor effects by leveraging local antigen persistence for antigen-presenting cells (APCs) recruitment and T cell engagement. However, constant antigen presentation alone tends to induce dysfunction in tumor-specific CD8+ T cells, neglecting the synergistic effects of co-stimulatory signal. To address this, we developed a soft particle-stabilized emulsion (SPE) to deliver lipopeptides with controlled release profiles by adjusting their hydrophobic chain lengths: C6-SPE (fast release), C10-SPE (medium release), and C16-SPE (slow release). Following administration, C6-SPE release antigen rapidly, inducing early antigen presentation, whereas C16-SPE's slow-release delays antigen presentation. Both scenarios missed the critical window for coordinating with the expression of CD86, leading to either T cell apoptosis or suboptimal activation. In contrast, C10-SPE achieved a spatiotemporally synergetic effect of the MHC-I-peptide complex and co-stimulatory signal (CD86), leading to effective dendritic cell (DC) activation, enhanced T cell activation, and tumor regression in EG7-OVA bearing mice. Additionally, co-delivery of cytosine-phosphate-guanine (CpG) with SPE provided a sustained expression of the CD86 window for DC activation, improving the immune response and producing robust anti-tumor effects with C6-SPE comparable to C10-SPE. These findings highlight that synchronizing the spatiotemporal dynamics of antigen presentation and APC activation may confer an optimal strategy for enhanced vaccinations.

A novel costimulatory molecule gene-modified leukemia cell-derived exosome enhances the anti-leukemia efficacy of DC vaccine in mouse models.

OBJECTIVES: Leukemia cell-derived exosomes (LEXs), carrying leukemia cell-specific antigens, can serve as a source of antigen for dendritic cell (DC) vaccine loading. However, LEX-targeted DC-based vaccines have demonstrated limited antitumor immune effects in clinical trials, attributed to the low immunogenicity of LEXs and the scant levels of costimulatory molecules on DCs. The costimulatory molecules CD80 and CD86, which are crucial to DC function, play a significant role in enhancing immune efficacy. In this study, we explored the anti-leukemia immune response of costimulatory molecule gene-modified LEX-targeted DCs (LEX-8086) in vitro and in animal models. METHODS: DCs were incubated with LEX-8086 to produce LEX-8086-targeted DCs (DCsLEX-8086). ELISA, cytotoxicity assays and flow cytometry utilized to assess the antitumor efficacy of DCsLEX8086 in vitro. Flow cytometry was used to evaluate the immunomodulatory function of DCsLEX8086 in animal models. RESULTS: Our findings indicated that LEX-8086 enhanced the maturation and antigen-presenting ability of DCs. Immunization with DCsLEX8086 significantly activated CD8+ T cells and boosted the CTL response in vitro. More importantly, DCsLEX-8086 effectively suppressed tumor growth and exerted anti-leukemia effects in both prophylactic and therapeutic animal models. Furthermore, DCsLEX-8086 promoted the proportion of CD4+ T cells, CD8+ T cells and M1 macrophages in the tumor environments both prophylactically and therapeutically. Treatment with DCsLEX-8086 showed no significant difference in the levels of M2 macrophages but decreased the proportion of Tregs within the tumor bed during therapeutic experiments. CONCLUSION: The results suggested that DCsLEX-8086 induces a more effective anti-leukemia immunity compared to DCsLEX-null in vivo and in vitro. DCsLEX-8086 might achieve antitumor effects by elevating the numbers of CD4+ T cells, CD8+ T cells, and M1 macrophages in tumors. Our findings indicate that DCsLEX-8086 could be leveraged to develop a new, highly effective vaccine for anti-leukemia immunity.

Obesity induces PD-1 on macrophages to suppress anti-tumour immunity.

Obesity is a leading risk factor for progression and metastasis of many cancers1,2, yet can in some cases enhance survival3-5 and responses to immune checkpoint blockade therapies, including anti-PD-1, which targets PD-1 (encoded by PDCD1), an inhibitory receptor expressed on immune cells6-8. Although obesity promotes chronic inflammation, the role of the immune system in the obesity-cancer connection and immunotherapy remains unclear. It has been shown that in addition to T cells, macrophages can express PD-19-12. Here we found that obesity selectively induced PD-1 expression on tumour-associated macrophages (TAMs). Type I inflammatory cytokines and molecules linked to obesity, including interferon-γ, tumour necrosis factor, leptin, insulin and palmitate, induced macrophage PD-1 expression in an mTORC1- and glycolysis-dependent manner. PD-1 then provided negative feedback to TAMs that suppressed glycolysis, phagocytosis and T cell stimulatory potential. Conversely, PD-1 blockade increased the level of macrophage glycolysis, which was essential for PD-1 inhibition to augment TAM expression of CD86 and major histocompatibility complex I and II molecules and ability to activate T cells. Myeloid-specific PD-1 deficiency slowed tumour growth, enhanced TAM glycolysis and antigen-presentation capability, and led to increased CD8+ T cell activity with a reduced level of markers of exhaustion. These findings show that obesity-associated metabolic signalling and inflammatory cues cause TAMs to induce PD-1 expression, which then drives a TAM-specific feedback mechanism that impairs tumour immune surveillance. This may contribute to increased cancer risk yet improved response to PD-1 immunotherapy in obesity.

Development of a Protocol for Obtaining a Homologous Cell Product of Animal Origin Intended for Preclinical Studies of Antitumor Vaccine CaTeVac.

When developing a program of preclinical studies of human cell-based drugs intended for adoptive immunotherapy of cancer patients, the biological effect should be substantiated by data describing their immunological action. Administration and study of human autologous dendritic cell vaccine to immunocompetent animals are not adequate in terms of immunological compatibility. It is possible to use immunocompromised, knockout, or transgenic animals or to obtain a homologous cellular product, namely, a preparation based on animal cells using a technology similar to obtaining the original preparation for clinical practice in humans. Within the framework of this study, we have developed a protocol for obtaining a homologous cell product based on animal dendritic cells (mice, rats) according to a similar technology for obtaining human vaccine dendritic cells, and demonstrated the comparability of morphological characteristics and expression of differentiation antigens of dendritic cells (CD11c, CD80, CD86, and CD83) of animals (mice) and humans.

Blockade of CD80/CD86-CD28 co-stimulation augments the inhibitory function of peptide antigen-specific regulatory T cells.

Mixed lymphocyte culture under the blockade of CD80/CD86-CD28 co-stimulation induces anergic (completely hyporesponsive) T cells with immune suppressive function (inducible suppressing T cells: iTS cells). Previously, iTS cell therapy has demonstrated outstanding benefits in clinical trials for organ transplantation. Here, we examined whether peptide antigen-specific iTS cells are inducible. DO 11.10 iTS cells were obtained from splenocytes of BALB/c DO 11.10 mice by stimulation with OVA peptide and antagonistic anti-CD80/CD86 mAbs. When DO 11.10 iTS or Foxp3- DO 11.10 iTS cells were stimulated with OVA, these cells produced IL-13, but not IL-4. DO 11.10 iTS cells decreased IL-4 and increased IL-13 production from OVA-stimulated naïve DO 11.10 splenocytes. When Foxp3+ DO 11.10 iTS cells were prepared, these cells significantly inhibited the production of IL-4 and IL-13 compared with freshly isolated Foxp3+ DO 11.10 T cells. Moreover, an increase in the population expressing OX40, ICOS, and 4-1BB suggested activation of Foxp3+ DO 11.10 iTS cells. Thus, blockade of CD80/CD86-CD28 co-stimulation during peptide antigen stimulation augments the inhibitory function of Foxp3+ regulatory T cells, and does not induce anergic Foxp3- conventional T cells. Peptide-specific Foxp3+ regulatory iTS cells could be useful for the treatment of allergic and autoimmune diseases without adverse effects.

K33 only mutant ubiquitin augments bone marrow-derived dendritic cell-mediated CTL priming via PI3K-Akt pathway.

To explore the effect of K33 only mutant ubiquitin (K33O) on bone marrow-derived dendritic cells' (BMDCs') maturity, antigen uptake capability, surface molecule expressions and BMDC-mediated CTL priming, and further investigate the role of PI3K-Akt engaged in K33O-increased BMDC maturation, antigen uptake and presentation, surface molecule expressions and BMDC-based CTL priming. BMDCs were conferred K33O and other ubiquitin mutants (K33R, K48R, K63R-mutant ubiquitin) incubation or LY294002 and wortmannin pretreatment. PI3K-Akt phosphorylation, antigen uptake, antigenic presentation and CD86/MHC class I expression in BMDC were determined by western blot or flow cytometry. BMDC-based CTL proliferation and priming were determined by in vitro mixed lymphocyte reaction (MLR), ex vivo enzyme-linked immunospot assay (Elispot) and flow cytometry with intracellular staining, respectively. The treatment with K33O effectively augmented PI3K-Akt phosphorylation, BMDCs' antigen uptake, antigenic presentation, CD86/MHC class I and CD11c expressions. MLR, Elispot and flow cytometry revealed that K33O treatment obviously enhanced CTL proliferation, CTL priming and perforin/granzyme B expression. The pretreatment with PI3K-Akt inhibitors efficiently abrogated K33O's effects on BMDC. The replenishment of K33 only mutant ubiquitin augments BMDC-mediated CTL priming in bone marrow-derived dendritic cells via PI3K-Akt signalling.

Ingestion of miso regulates immunological robustness in mice.

In Japan, there is a long history of consumption of miso, a fermented soybean paste, which possesses beneficial effects on human health. However, the mechanism behind these effects is not fully understood. To clarify the effects of miso on immune cells, we evaluated its immunomodulatory activity in mice. Miso did not alter the percentage of B and T cells in the spleen; however, it increased CD69+ B cells, germinal center B cells and regulatory T cells. Anti-DNA immunoglobulin M antibodies, which prevent autoimmune disease, were increased following ingestion of miso. Transcriptome analysis of mouse spleen cells cultured with miso and its raw material revealed that the expression of genes, including interleukin (IL)-10, IL-22 and CD86, was upregulated. Furthermore, intravital imaging of the small intestinal epithelium using a calcium biosensor mouse line indicated that miso induced Ca2+ signaling in a manner similar to that of probiotics. Thus, ingestion of miso strengthened the immune response and tolerance in mice. These results appear to account, at least in part, to the salubrious effects of miso.

Elevated number of IL-21+ TFH and CD86+CD38+ B cells in blood of renal transplant recipients with AMR under conventional immuno-suppression.

The objective of this study is to detect the number of different subsets of TFH and B cells in renal transplant recipients (RTR) with antibody-mediated acute rejection (AMR), acute rejection (AR), chronic rejection (CR), or transplant stable (TS). The present study was a prospective study. The numbers of ICOS +, PD-1+ and IL-21+ TFH, CD86+, CD38+, CD27+, and IgD- B cells in 21 patients with end-stage renal disease (ESRD) and post-transplant times were measured by flow cytometry. The level of serum IL-21 was detected by ELISA. The numbers of circulating CD4+CXCR5+, CD4+CXCR5+ICOS+, CD4+CXCR5+PD-1+, CD4+CXCR5+IL-21+ TFH, CD19+CD86+, and CD19 +CD86+CD38+ B cells as well as the level of serum IL-21 in the AMR, AR, and CR groups at post-transplantation were significantly higher than those at pre-transplantation. In contrast, the number of circulating CD19+CD27+IgD B cells was significantly increased in the TS groups in respect to the other groups. Moreover, the numbers of circulating CD4+CXCR5+IL-21+ TFH cells, CD19+CD86+CD38+ B cells as well as the level of serum IL-21 were positive related to the level of serum Cr while showing negative correlated with the values of eGFR in the AMR groups at post-transplantation for 4 and 12 weeks. Circulating TFH cells may be a biomarker in RTR with AMR, which can promote the differentiation of B cells into plasma cells by activating B cells, thereby promoting disease progression.

NKG2D Enhances Double-Negative T Cell Regulation of B Cells.

Numerous studies reported a small subpopulation of TCRαß+CD4-CD8- (double-negative) T cells that exert regulatory functions in the peripheral lymphocyte population. However, the origin of these double-negative T (DNT) cells is controversial. Some researchers reported that DNT cells originated from the thymus, and others argued that these cells are derived from peripheral immune induction. We report a possible mechanism for the induction of nonregulatory CD4+ T cells to become regulatory double-negative T (iDNT) cells in vitro. We found that immature bone marrow dendritic cells (CD86+MHC-II- DCs), rather than mature DCs (CD86+MHC-II+), induced high levels of iDNT cells. The addition of an anti-MHC-II antibody to the CD86+MHC-II+ DC group significantly increased induction. These iDNT cells promoted B cell apoptosis and inhibited B cell proliferation and plasma cell formation. A subgroup of iDNT cells expressed NKG2D. Compared to NKG2D- iDNT cells, NKG2D+ iDNT cells released more granzyme B to enhance B cell regulation. This enhancement may function via NKG2D ligands expressed on B cells following lipopolysaccharide stimulation. These results demonstrate that MHC-II impedes induction, and iDNT cells may be MHC independent. NKG2D expression on iDNT cells enhanced the regulatory function of these cells. Our findings elucidate one possible mechanism of the induction of peripheral immune tolerance and provide a potential treatment for chronic allograft rejection in the future.

A flow cytometry approach reveals heterogeneity in conventional subsets of murine renal mononuclear phagocytes.

Mononuclear phagocytes (MNPs) participate in inflammation and repair after kidney injury, reflecting their complex nature. Dissection into refined functional subunits has been challenging and would benefit understanding of renal pathologies. Flow cytometric approaches are limited to classifications of either different MNP subsets or functional state. We sought to combine these two dimensions in one protocol that considers functional heterogeneity in each MNP subset. We identified five distinct renal MNP subsets based on a previously described strategy. In vitro polarization of bone marrow-derived macrophages (BMDM) into M1- and M2-like cells suggested functional distinction of CD86 + MHCII + CD206- and CD206 + cells. Combination of both distinction methods identified CD86 + MHCII + CD206- and CD206 + cells in all five MNP subsets, revealing their heterologous nature. Our approach revealed that MNP composition and their functional segmentation varied between different mouse models of kidney injury and, moreover, was dynamically regulated in a time-dependent manner. CD206 + cells from three analyzed MNP subsets had a higher ex vivo phagocytic capacity than CD86 + MHCII + CD206- counterparts, indicating functional uniqueness of each subset. In conclusion, our novel flow cytometric approach refines insights into renal MNP heterogeneity and therefore could benefit mechanistic understanding of renal pathology.

CD86+ Antigen-Presenting B Cells Are Increased in Cancer, Localize in Tertiary Lymphoid Structures, and Induce Specific T-cell Responses.

The role of B cells in antitumor immunity and their impact on emerging immunotherapies is increasingly gaining attention. B-cell effector functions include not only secretion of antibodies, but also presentation of antigens to T cells. A physiologic B-cell subset with immunostimulatory properties was described in humans, defined by a high expression of CD86 and downregulation of CD21. We used multicolor flow cytometry and IHC to elucidate abundance and spatial distribution of these antigen-presenting B cells (BAPC) in blood (peripheral blood mononuclear cells, PBMC) and tumor samples of 237 patients with cancer. Antigen-specific T-cell responses to cancer testis antigens were determined using tetramer staining and sorted BAPCs in FluoroSpot assays for selected patients. We found that BAPCs were increased in the tumor microenvironment of 9 of 10 analyzed cancer types with site-specific variation. BAPCs were not increased in renal cell carcinoma, whereas we found a systemic increase with elevated fractions in tumor-infiltrating lymphocytes (TIL) and PBMCs of patients with colorectal cancer and gastroesophageal adenocarcinoma. BAPCs were localized in lymphoid follicles of tertiary lymphoid structures (TLS) and were enriched in tumors with increased numbers of TLSs. BAPCs isolated from tumor-draining lymph nodes of patients with cancer showed increased percentages of tumor antigen-specific B cells and induced responses of autologous T cells in vitro. Our results highlight the relevance of BAPCs as professional antigen-presenting cells in tumor immunity and provide a mechanistic rationale for the observed correlation of B-cell abundance and response to immune checkpoint inhibition.

Interferon Gamma Induces the Increase of Cell-Surface Markers (CD80/86, CD83 and MHC-II) in Splenocytes From Atlantic Salmon.

Type II interferon gamma (IFNγ) is a pleiotropic cytokine capable of modulating the innate and adaptive immune responses which has been widely characterized in several teleost families. In fish, IFNγ stimulates the expression of cytokines and chemokines associated with the pro-inflammatory response and enhances the production of nitrogen and oxygen reactive species in phagocytic cells. This work studied the effect of IFNγ on the expression of cell-surface markers on splenocytes of Atlantic salmon (Salmo salar). In vitro results showed that subpopulations of mononuclear splenocytes cultured for 15 days were capable of increasing gene expression and protein availability of cell-surface markers such as CD80/86, CD83 and MHC II, after being stimulated with recombinant IFNγ. These results were observed for subpopulations with characteristics associated with monocytes (51%), and features that could be related to lymphocytes (46.3%). In addition, a decrease in the expression of zbtb46 was detected in IFNγ-stimulated splenocytes. Finally, the expression of IFNγ and cell-surface markers was assessed in Atlantic salmon under field conditions. In vivo results showed that the expression of ifnγ increased simultaneously with the up-regulation of cd80/86, cd83 and mhcii during a natural outbreak of Piscirickettsia salmonis. Overall, the results obtained in this study allow us to propose IFNγ as a candidate molecule to stimulate the phenotypic progression of a small population of immune cells, which will increase antigen presenting cells markers. Thereby, modulatory strategies using IFNγ may generate a robust and coordinated immune response in fish against pathogens that affect aquaculture.

Effect of gingival mesenchymal stem cell-derived exosomes on inflammatory macrophages in a high-lipid microenvironment.

OBJECTIVE: The aim of this study was to examine the effect of gingival mesenchymal stem cells derived exosomes (GMSC-Exos) on lipopolysaccharide/interferon-gamma (LPS/INF-γ)-induced inflammatory macrophages in a high-lipid microenvironment. MATERIALS AND METHODS: Exosomes were obtained by culturing gingival mesenchymal stem cells (GMSCs) in alpha-MEM with exosome-free fetal bovine serum for 48 h. The control group was produced in vitro by inducing human acute monocytic leukemia cells (THP-1 cells) into naïve macrophages (M0). Inflammatory macrophages (M1) were made by activating M0 macrophages with LPS/IFN-γ. These M1 macrophages were treated with oxidized low-density lipoprotein (ox-LDL) to create the high-lipid group, of which some macrophages were further treated with GMSC-Exos for 24 h to form the GMSC-Exos group. Supernatants were collected, and total RNA were extracted for downstream analysis. The expression of surface markers in macrophages were analyzed by flow cytometry. The lipid accumulation level was assessed by oil red O staining. RESULTS: Exosomes were successfully isolated from GMSC medium. The GMSC-Exos group showed lower Tumor Necrosis Factor-α (TNF-α), Interleukin-6 (IL-6), Interleukin-1ß (IL-1ß), and cluster of differentiation 86 (CD86) expression levels than the high-lipid group, and the highest levels of Interleukin-10 (IL-10) among all groups. The GMSC-Exos group showed significant reductions in TNF-α levels than the high-lipid group, and significant escalations in IL-10 levels than the other two groups. Oil red o Staining showed that lipid accumulation in macrophages was inhibited in the GMSC-Exos group. CONCLUSIONS: GMSC-Exos reduce the release level and expression of inflammatory factors, inhibit lipid accumulation, and promote the polarization of pro-inflammatory macrophages into anti-inflammatory phenotype in a high-lipid microenvironment.

CD86 Is a Selective CD28 Ligand Supporting FoxP3+ Regulatory T Cell Homeostasis in the Presence of High Levels of CTLA-4.

CD80 and CD86 are expressed on antigen presenting cells and are required to engage their shared receptor, CD28, for the costimulation of CD4 T cells. It is unclear why two stimulatory ligands with overlapping roles have evolved. CD80 and CD86 also bind the regulatory molecule CTLA-4. We explored the role of CD80 and CD86 in the homeostasis and proliferation of CD4+FoxP3+ regulatory T cells (Treg), which constitutively express high levels of CTLA-4 yet are critically dependent upon CD28 signals. We observed that CD86 was the dominant ligand for Treg proliferation, survival, and maintenance of a regulatory phenotype, with higher expression of CTLA-4, ICOS, and OX40. We also explored whether CD80-CD28 interactions were specifically compromised by CTLA-4 and found that antibody blockade, clinical deficiency of CTLA-4 and CRISPR-Cas9 deletion of CTLA-4 all improved Treg survival following CD80 stimulation. Taken together, our data suggest that CD86 is the dominant costimulatory ligand for Treg homeostasis, despite its lower affinity for CD28, because CD80-CD28 interactions are selectively impaired by the high levels of CTLA-4. These data suggest a cell intrinsic role for CTLA-4 in regulating CD28 costimulation by direct competition for CD80, and indicate that that CD80 and CD86 have discrete roles in CD28 costimulation of CD4 T cells in the presence of high levels of CTLA-4.

B cells reappear less mature and more activated after their anti-CD20-mediated depletion in multiple sclerosis.

B cell depletion via anti-CD20 antibodies is a highly effective treatment for multiple sclerosis (MS). However, little is known about the maturation/activation stage of the returning B cell population after treatment cessation and the wider effects on other immune cells. In the present study, 15 relapsing-remitting MS patients receiving 1,000 mg of rituximab were included. B, T, and myeloid cells were analyzed before anti-CD20 administration and in different time intervals thereafter over a period of 24 mo. In comparison to the phenotype before anti-CD20 treatment, the reappearing B cell pool revealed a less mature and more activated phenotype: 1) reappearing B cells were significantly enriched in transitional (before: 10.1 ± 1.9%, after: 58.8 ± 5.2%) and mature naive phenotypes (before: 45.5 ± 3.1%, after: 25.1 ± 3.5%); 2) the frequency of memory B cells was reduced (before: 36.7 ± 3.1%, after: 8.9 ± 1.7%); and 3) reappearing B cells showed an enhanced expression of activation markers CD25 (before: 2.1 ± 0.4%, after: 9.3 ± 2.1%) and CD69 (before: 5.9 ± 1.0%, after: 21.4 ± 3.0%), and expressed significantly higher levels of costimulatory CD40 and CD86. T cells showed 1) a persistent increase in naive (CD4+: before: 11.8 ± 1.3%, after: 18.4 ± 3.4%; CD8+: before: 12.5 ± 1.4%, after: 16.5 ± 2.3%) and 2) a decrease in terminally differentiated subsets (CD4+: before: 47.3 ± 3.2%, after: 34.4 ± 3.7%; CD8+: before: 53.7 ± 2.1%, after: 49.1 ± 2.7%).

Bacteroides fragilis alleviates the symptoms of lupus nephritis via regulating CD1d and CD86 expressions in B cells.

Emerging evidences indicated that the dysbiosis of microbiota was related to the onset of systemic lupus erythematosus (SLE). Bacteroides fragilis (B. fragilis) ATCC 25285, a human commensal, was discovered to improve inflammatory diseases. However, whether B. fragilis (ATCC 25285) has the beneficial effects on the treatment of lupus nephritis has still remained elusive. In the present study, oral treatment with B. fragilis (ATCC 25285) ameliorated the activity of MRL/lpr mice, including decreased levels of autoantibodies and symptoms of lupus nephritis. Furthermore, we demonstrated that treatment with B. fragilis (ATCC 25285) could promote CD1d expression in B cells by Est-1 pathway, while inhibit CD86 expression via SHP-2 signaling pathway to repair the immune response of B cells in MRL/lpr mice. In addition, our findings revealed a possible role of treatment with B. fragilis (ATCC 25285) in relieving intestinal inflammation in MRL/lpr mice. Meanwhile, it was uncovered that B. fragilis (ATCC 25285) restored the Th17/Treg balance in MRL/lpr mice that was consistent with the role of B. fragilis in other autoimmune diseases. Overall, the current study may highlight the potential application of B. fragilis (ATCC 25285) to treat manifestations of SLE in high-risk individuals.

The balance of regulatory and stimulatory B cell subsets in breast cancer draining lymph nodes correlates with tumor prognostic factors.

AIMS: B cells can promote or inhibit immune responses against breast cancer. We investigated changes in the frequency of B cells with stimulatory or regulatory capacity in breast tumor draining lymph nodes during cancer progression. MAIN METHODS: We isolated mononuclear cells from fresh axillary lymph nodes (LNs) of 44 patients with breast cancer and stained lymphocytes with antibodies against CD19, CD80, CD86, CD39 and CD73. To assess programmed death-1 (PD-1) and PD-ligand 1 (PD-L1) expression, lymphocytes were briefly stimulated, stained for CD19, PD-1 and PD-L1, and examined with flow cytometry. KEY FINDINGS: The frequency of CD80+ B cells was higher in nonmetastatic lymph nodes, while the percentage of CD86+ B cells showed a positive relationship with higher tumor grade and higher numbers of involved LNs. A small proportion of unstimulated B cells expressed PD-1 or PD-L1 but these molecules were rapidly upregulated on B cells following activation. The frequency of stimulated PD-L1+ B cells showed an inverse association with estrogen and progesterone receptor expression and a nonsignificant positive association with tumor grade. In addition, the percentage of unstimulated PD-1+ B cells was higher in patients with higher-grade tumors. CD73 expression on B cells was associated with lower numbers of involved LNs, and the frequency of CD39+ B cells was higher in patients with larger tumors. SIGNIFICANCE: CD86+, CD39+, PD-1+ and PD-L1+ B cells showed associations with poor prognostic factors, therefore their potential role in the suppression of the immune responses against breast cancer should be evaluated in greater detail.

Activation of Human Vδ2+ γδ T Cells by Staphylococcus aureus Promotes Enhanced Anti-Staphylococcal Adaptive Immunity.

Murine studies have shown the potential for γδ T cells to mediate immunity to Staphylococcus aureus in multiple tissue settings by the secretion of diverse cytokines. However, the role played by γδ T cells in human immune responses to S. aureus is almost entirely unknown. In this study, we establish the capacity of human Vδ2+ γδ T cells for rapid activation in response to S. aureus In coculture with S. aureus-infected monocyte-derived dendritic cells (DCs), Vδ2+ cells derived from peripheral blood rapidly upregulate CD69 and secrete high levels of IFN-γ. DCs mediate this response through direct contact and IL-12 secretion. In turn, IFN-γ released by Vδ2+ cells upregulates IL-12 secretion by DCs in a positive feedback loop. Furthermore, coculture with γδ T cells results in heightened expression of the costimulatory molecule CD86 and the lymph node homing molecule CCR7 on S. aureus-infected DCs. In cocultures of CD4+ T cells with S. aureus-infected DCs, the addition of γδ T cells results in heightened CD4+ T cell activation. Our findings identify γδ T cells as potential key players in the early host response to S. aureus during bloodstream infection, promoting enhanced responses by both innate and adaptive immune cell populations, and support their consideration in the development of host-directed anti-S. aureus treatments.

Tet2 and Tet3 in B cells are required to repress CD86 and prevent autoimmunity.

A contribution of epigenetic modifications to B cell tolerance has been proposed but not directly tested. Here we report that deficiency of ten-eleven translocation (Tet) DNA demethylase family members Tet2 and Tet3 in B cells led to hyperactivation of B and T cells, autoantibody production and lupus-like disease in mice. Mechanistically, in the absence of Tet2 and Tet3, downregulation of CD86, which normally occurs following chronic exposure of self-reactive B cells to self-antigen, did not take place. The importance of dysregulated CD86 expression in Tet2- and Tet3-deficient B cells was further demonstrated by the restriction, albeit not complete, on aberrant T and B cell activation following anti-CD86 blockade. Tet2- and Tet3-deficient B cells had decreased accumulation of histone deacetylase 1 (HDAC1) and HDAC2 at the Cd86 locus. Thus, our findings suggest that Tet2- and Tet3-mediated chromatin modification participates in repression of CD86 on chronically stimulated self-reactive B cells, which contributes, at least in part, to preventing autoimmunity.