Artificial antigen-presenting cells plus IL-15 and IL-21 efficiently induce melanoma-specific cytotoxic CD8^+CD28^+ T lymphocyte responses

Objective:To develop a novel artificial antigen-presenting system for efficiently inducing melanoma-specific CD8+CD28+ cytotoxic T lymphocyte（CTL） responses.Methods:Cell-sized Dynabeads? M-450 Epoxy beads coated with H-2Kb:Ig-TRP2(181111K and anti-CD28 antibody were used as artificial antigen-presenting cells（aAPCs） lo induce melanoma-specific CD8*CD28’ CTL responses with the help of IL-2I and IL-I5.Dimer staining,proliferation,ELISPOT,and cytotoxicity experiments were conducted to evaluate the frequency and activity of induced CTLs.Results:Dimer staining demonstrated that the new artificial antigen-presenting system efficiently induced melanoma TRP2-specific CD8CD28' CTLs.Proliferation and ELISPOT assays indicated that the induced CTLs rapidly proliferate and produce increased IFN- y under the slimulalion of H-2K:Ig-TRP2-aAPCs,TL-15,and IL-21.In addition,cytoloxicily experiments showed lhat induced CTLs have specific killing activity of target cells.Conclusions:The new artificial antigen-presenting system including aAPCs plus IL-21 and IL-15 can induce a large number of antigen-specific CD8+CD28+ CTLs against the melanoma.Our study provides evidence for a novel adoptive immunotherapy against tumors.

Over-expression of programmed death-ligand 1 and programmed death-1 on antigen-presenting cells as a predictor of organ dysfunction and mortality during early sepsis: a prospective cohort study

BACKGROUND:This study aimed to explore the changes of programmed death-ligand 1(PDL1)and programmed death-1(PD-1)expression on antigen-presenting cells(APCs)and evaluate their association with organ failure and mortality during early sepsis.METHODS:In total,40 healthy controls and 198 patients with sepsis were included in this study.Peripheral blood was collected within the first 24 h after the diagnosis of sepsis.The expression of PDL1 and PD-1 was determined on APCs,such as B cells,monocytes,and dendritic cells(DCs),by flow cytometry.Cytokines in plasma,such as interferon-γ(IFN-γ),tumor necrosis factor-α(TNF-α),interleukin-4(IL-4),IL-6,IL-10,and IL-17A were determined by Luminex assay.RESULTS:PD-1 expression decreased significantly on B cells,monocytes,myeloid DCs(mDCs),and plasmacytoid DCs(pDCs)as the severity of sepsis increased.PD-1 expression was also markedly decreased in non-survivors compared with survivors.In contrast,PD-L1 expression was markedly higher on mDCs,pDCs,and monocytes in patients with sepsis than in healthy controls and in non-survivors than in survivors.The PD-L1 expression on APCs(monocytes and DCs)was weakly related to organ dysfunction and infl ammation.The area under the receiver operating characteristic curve(AUC)of the PD-1 percentage of monocytes(monocyte PD-1%)+APACHE II model(0.823)and monocyte PD-1%+SOFA model(0.816)had higher prognostic value than other parameters alone.Monocyte PD-1%was an independent risk factor for 28-day mortality.CONCLUSION:The severity of sepsis was correlated with PD-L1 or PD-1 over-expression on APCs.PD-L1 in monocytes and DCs was weakly correlated with infl ammation and organ dysfunction during early sepsis.The combination of SOFA or APACHE II scores with monocyte PD-1%could improve the prediction ability for mortality.

Antigen-presenting effects of effector memory Vγ9Vδ2 T cells in rheumatoid arthritis

Rheumatoid arthritis is an autoimmune disease that primarily affects the limbs, but the pathogenic mechanism remains unclear. 78 T cells, a T-cell subpopulation, are characterized by multiple biological functions and associated with a variety of diseases. This study investigated the antigen-presenting effects of γδ2 cells and their relationship with rheumatoid arthritis development. We found that Vγ9Vδ2 T cells （the predominant subtype of γδ T cells in peripheral blood） were activated by isopentenyl pyrophosphate to continuously proliferate and differentiate into effector memory cells. The effector memory Vγ9Vδ2 T cells exhibited phenotypic characteristics of specific antigen-presenting cells, including high HLA-DR and CD80/86 expression. These Vγ9Vδ2 T cells could present soluble antigens and synthetic peptides to CD4＋ T cells. Vγ9Vδ2 T cells with different phenotypes showed different cytokine secretion patterns. Effector memoryVγ9Vδ2 T cells simultaneously secreted not only interferon （IFN）-γbut also IL-17. The peripheral blood and joint synovial fluid from RA patients contained numerous heterogeneous γδ T cells that were predominantly effector memory Vγ9Vδ2 T cells with the ability to secrete inflammatory factors. We also found that γδ T cells had a similar antigen-presenting capability to B cells. These results suggest that during the development of rheumatoid arthritis, 78 T cells can aggravate immune dysfunction and produce abnormal immune damage by secreting cytokines and inducing inflammatory cells to participate in synergistic inflammatory responses. Furthermore, γδ T cells can behave similarly to B cells to present viral peptides and autoantigen peptides to CD4＋ T cells, thus sustaining CD4＋ T-cell activation.

Establishment and Characterization of a Cell Based Artificial Antigen-Presenting Cell for Expansion and Activation of CD8^+ T Cells Ex Vivo

Artificial antigen-presenting cells are expected to stimulate the expansion and acquisition of optimal therapeutic features of T cells before infusion. Here CD32 that binds to a crystallizable fragment of IgG monoclonal antibody was genetically expressed on human K562 leukemia cells to provide a ligand for T-cell receptor. CD86 and 4-1BBL, which are ligands of co-stimulating receptors of CD28 and 4-1BB, respectively, were also expressed on K562 cells. Then we accomplished the artificial antigen-presenting cells by coupling K32/CD86/4-1BBL cell with OKT3 monoclonal antibody against CD3, named K32/CD86/4-1BBL/OKT3 cells. These artificial modified cells had the abilities of inducing CD8^＋ T cell activation, promoting CD8^＋ T cell proliferation, division, and long-term growth, inhibiting CD8^＋ T cell apoptosis, and enhancing CD8^＋ T cell secretion of IFN-T and perforin. Furthermore, antigen-specific cytotoxic T lymphocytes could be retained in the culture stimulated with K32/CD86/4-1BBL/OKT3 cells at least within 28 days. This approach was robust, simple, reproducible and economical for expansion and activation of CD8^＋ T cells and may have important therapeutic implications for adoptive immunotherapy. Cellular ＆ Molecular Immunology.

Criculating fibrocytes： a potent cell population in antigen-presenting and wound healing

Fibrocytes are bone marrow-derived mesenchymal progenitors that co-express hematopoietic cell antigens and markers of monocytic lineage as well as fibroblast products. During wound healing, fibrocytes have been found to possess the ability of antigen-presentation to naive T cells in the inflammatory phase. Moreover, they can promote the endothelial cell proliferation, migration and angiogenesis by secreting several proteins. Fibrocytes can further differentiate into mature mesenchymocyte lineage, such as fibroblasts, myofibroblasts and adipocytes, and they may represent the systemic source of myofibroblasts that exert a contractile force required to close tissue wounds. A deep understanding of the mechanism involved in fibrocyte migration and differentiation may lead to the development of a novel theory of normal physiology and pathology.

STING negatively regulates allogeneic T-cell responses by constraining antigen-presenting cell function

Stimulator of interferon genes(STING)-mediated innate immune activation plays a key role in tumor-and self-DNA-elicited antitumor immunity and autoimmunity.However,STING can also suppress tumor immunity and autoimmunity.STING signaling In host nonhematopoietic cells was reported to either protect against or promote graft-versus-host disease(GVHD),a major complication of allogeneic hematopoietic cell transplantation(allo-HCT).Host hematopoietic antigen-presenting cells(APCs)play key roles in donor T-cell priming during GVHD initiation.However,how STING regulates host hematopoietic APCs after allo-HCT remains unknown.We utilized murine models of allo-HCT to assess the role of STING in hematopoietic APCs.STING-deficient recipients developed more severe GVHD after major histocompatibility complex-mismatched allo-HCT.Using bone marrow chimeras,we found that STING deficiency in host hematopoietic cells was primarily responsible for exacerbating the disease.Furthermore,STING on host CD11c+cells played a dominant role in suppressing allogeneic T-cell responses.Mechanistically,STING deficiency resulted in increased survival,activation,and function of APCs,including macrophages and dendritic cells.Consistently,constitutive activation of STING attenuated the survival,activation,and function of APCs isolated from STING V154M knock-in mice.STING-deficient APCs augmented donor T-cell expansion,chemokine receptor expression,and migration into intestinal tissues,resulting in accelerated/exacerbated GVHD.Using pharmacologic approaches,we demonstrated that systemic administration of a STING agonist(bis-(3'-5')-cyclic dimeric guanosine monophosphate)to recipient mice before transplantation significantly reduced GVHD mortality.In conclusion,we revealed a novel role of STING in APC activity that dictates T-cell allogeneic responses and validated STING as a potential therapeutic target for controlling GVHD after allo-HCT.

Diverse immune cell profles in ASFV-associated lymphopenia

Pathogenic African swine fever virus(ASFV)remains a lethal causative agent in the domestic pig industry,which poses a burden on the swine market and causes substantial socioeconomic losses worldwide.Currently,there are no commercially efcacious vaccines or specifc treatments available for ASF prevention and control.Unfortunately,little is known about the swine immune response upon ASFV infection.Here,we investigated the host immune response discrepancy induced by the feld moderately virulent strain ASFV HB-2208 among healthy,diseased and asymptomatic pigs.In the peripheral blood of diseased swine,lymphopenia is caused by the massive loss of bystander lymphocytes,such asγδT cells,B cells and CD4^(+)T cells.Conversely,ASFV has a strong tropism for the mononuclear phagocyte system(MPS)and partial dendritic cells(DCs),whose antigen-presenting ability is impeded by the downregulation of CD80 and MHC I.However,no signifcant diference in the number of CD8α^(high) T cells was detected,whereas the frequencies of NK cells,NKT cells,and regulatory T cells(Tregs)were signifcantly increased.Additionally,an in vitro model was established with a coculture of primary pulmonary alveolar macrophages(PAMs)and peripheral blood mononuclear cells(PBMCs),which signifcantly reducedγδT cells,B cells and CD4^(+)T cells and increased Tregs.The diferentiated immune response might aid in enhancing the understanding of ASFV pathogenesis in suids and provide insights into the mechanism of ASFV-induced lymphopenia for further studies.

Targeting epidermal Langerhans cells by epidermal powder immunization

Immune reactions to foreign or self-antigens lead to protective immunity and, sometimes, immune disorders such as allergies and autoimmune diseases. Antigen presenting cells (APC) including epidermal Langerhans cells (LCs) play an important role in the course and outcome of the immune reactions. Epidermal powder immunization (EPI) is a technology that offers a tool to manipulate the LCs and the potential to harness the immune reactions towards prevention and treatment of infectious diseases and immune disorders.

Inducible expression of endomorphins in murine dendritic cells

Bone marrow precursor cells were extracted from C57BL/6J mice aged 7-8 weeks, and dendritic cells were purified using anti-CD1 lc （a specific marker for dendritic cells） antibody-coated magnetic beads. Immunofluorescence staining revealed that the expression levels of endomorphin-1 and endomorphin-2 were upregulated in dendritic cells activated by lipopolysaccharide. An enzyme Jmmunoassay showed that lipopolysaccharide and other Toll-like receptor ligands promoted the secretion of endomorphin-1 and endomorphin-2 from activated dendritic cells. [3H]-thymidine incorporation demonstrated that endomorphin-1 and endomorphin-2 both inhibited the proliferation of T lymphocyte induced by activated dendritic cells. Furthermore, this immunosuppressive effect was blocked by CTOP, a specific antagonist of IJ-opioid receptors. Our experimental findings indicate that activated dendritic cells can induce the expression and secretion of endomorphins, and that endomorphins suppress T lymphocyte proliferation through activation of iJ-opioid receptors.

Cell-type specific regulation of MARCH1 E3 ubiquitin ligase by the anti-inflammatory cytokine IL-10

Membrane-associated RING-CH-1 (MARCH1) is an E3 ubiquitin ligase which targets MHC-II, CD86 and various other molecules for degradation. It is one of the most efficient post-translational regulators of antigen presentation. MARCH1 is expressed in resting immature dendritic cells and B cells but can be induced in other cell types. While activation of most immune cells results in a reduction in MARCH1 gene expression, its anti-inflammatory properties are highlighted by its induction in response to IL-10. Here, we review what is known about the regulation of MARCH1 gene expression in response to IL-10 by various immune cells. We speculate on the role of MARCH1 ininfection, its differential expression pattern and the promise that this E3 ubiquitin ligase holds to influence immune responses and mitigate immune pathologies.

Co-Inhibitors of Second Signal of Lymphocyte Response in Human Renal Transplants: PD-L2, GITR, and ILT-2/3/5 Positive Cells from Aspiration Biopsies Associate with Acute Rejection-Freedom

Following organ transplantation, the outcome of the encounter between an APC and a T lymphocyte is strongly dependent on the presence of costimulatory and co-inhibitory molecules, the former associated with allograft rejection and the latter with allograft acceptance. We evaluated the expression of PD-L2, GITR, ILT-2/3/5, and ILT-4 on graft-infiltrating cells procured by Fnab from human KTx under different immunosuppressive regimens. Methods: Fnab biopsies were performed on days 7 or 14 - 30 in stable KTx and on the day of acute rejection diagnosis. Cytopreparations were studied by the enzymatic avidin biotin complex staining. Results: Acute rejection group showed a significant down-regulated expression of PD-L2, GITR, and ILT-2/3/5 as compared to stable group, while for ILT-4 we did not find significant difference. Anti-IL2αR and rapamicyn treatment trend to down-regulate ILT-4 expression, although meaningless. A significantly positive correlation was observed between PD-L2 and GITR expression in Fnab. The PPV for acute rejection diagnosis for both PD-L2 and GITR was clearly above 0.8. Conclusions: Our findings point to an early entrance of cells expressing PD-L2, GITR and ILT-2/3/5 inside human KTx who are going to remain rejection-free. Both PD-L2 and GITR shared a high ability to rule-in and rule-out acute rejection.

STING-activating dendritic cell-targeted nanovaccines that evoke potent antigen cross-presentation for cancer immunotherapy

Recently,nanovaccine-based immunotherapy has been robustly investigated due to its potential in governing the immune response and generating long-term protective immunity.However,the presentation of a tumor peptide-major histocompatibility complex to T lymphocytes is still a challenge that needs to be addressed for eliciting potent antitumor immunity.Type 1 conventional dendritic cell(cDC1)subset is of particular interest due to its pivotal contribution in the cross-presentation of exogenous antigens to CD8+T cells.Here,the DC-derived nanovaccine(denoted as Si9GM)selectively targets cDC1s with marginal loss of premature antigen release for effective stimulator of interferon genes(STING)-mediated antigen cross-presentation.Bone marrow dendritic cell(BMDC)-derived membranes,conjugated to cDC1-specific antibody(αCLEC9A)and binding to tumor peptide(OVA257-264),are coated onto dendrimer-like polyethylenimine(PEI)-grafted silica nanoparticles.Distinct molecular weight-cargos(αCLEC9A-OVA257-264 conjugates and 2′3′-cGAMP STING agonists)are loaded in hierarchical center-radial pores that enables lysosome escape for potent antigen-cross presentation and activates interferon type I,respectively.Impressively,Si9GM vaccination leads to the upregulation of cytotoxic T cells,a reduction in tumor regulatory T cells(Tregs),M1/M2 macrophage polarization,and immune response that synergizes with αPD-1 immune checkpoint blockade.This nanovaccine fulfills a dual role for both direct T cell activation as an artificial antigen-presenting cell and DC subset maturation,indicating its utility in clinical therapy and precision medicine.

A two-pronged strategy utilizing exosomes extracted from antigenpresenting cells to combat hepatitis B

Chronic hepatitis B(CHB)is consistently challenging to conquer with numerous complications and fatalities.Despite the effectiveness of antiviral therapies in suppressing hepatitis B virus(HBV)replication,there is an urgent need for novel and more effective treatment modalities.Current strategies predominantly emphasize immune activation but still face challenges in sufficiently eliciting T cell responses.Taking into account the targeted delivery of nanoparticles to the liver and spleen via intravenous injection,we have proposed a dual-pronged therapeutic strategy based on antigen-presenting cells(APCs)-derived exosomes.Specifically,exosomes targeted to the spleen can activate specific immune responses,while those targeted to the liver can modulate or reverse the liver’s immunosuppressive microenvironment.After immunization,exosome formulations exhibit the remarkable ability to effectively activate APCs,thereby triggering the proliferation of CD8^(+)T cells.Simultaneously,they also play an immunoregulatory role by converting M2 macrophages into M1 macrophages.This two-pronged therapeutic strategy precisely addresses the issues of T cell dysfunction and immune suppression,both characteristic features of CHB patients.When combined with Aluminum(Alum)-adjuvanted vaccine,these exosome formulations not only demonstrate a high level of cellular immune response but also secrete specific antibodies comparable to those induced by Alum adjuvant.This combined approach effectively enhances both cellular and humoral immunity,offering a promising avenue for the development of therapeutic hepatitis B vaccines based on exosome formulations.

An imbalance between innate and adaptive immune cells at the maternal-fetal interface occurs prior to endotoxin-induced preterm birth

Preterm birth （PTB） is the leading cause of neonatal morbidity and mortality worldwide. A transition from an anti-inflammatory state to a pro-inflammatory state in the mother and at the maternal-fetal interface has been implicated in the pathophysiology of microbial-induced preterm labor. However, it is unclear which immune cells mediate this transition. We hypothesized that an imbalance between innate and adaptive immune cells at the maternal-fetal interface will occur prior to microbial-induced preterm labor. Using an established murine model of endotoxin-induced PTB, our results demonstrate that prior to delivery there is a reduction of CD4 ＋ regulatory T cells （Tregs） in the uterine tissues. This reduction is neither linked to a diminished number of Tregs in the spleen, nor to an impaired production of ILIO, CCL17, or CCL22 by the uterine tissues. Endotoxin administration to pregnant mice does not alter effector CD4＋ T cells at the maternal-fetal interface. However, it causes an imbalance between Tregs （CD4＋ and CD8＋）, effector CD8＋ T cells, and Th17 cells in the spleen. In addition, endotoxin administration to pregnant mice leads to an excessive production of CCL2, CCL3, CCL17, and CCL22 by the uterine tissues as well as abundant neutrophils. This imbalance in the uterine microenvironment is accompanied by scarce APC-like cells such as macrophages and MHC II ＋ neutrophils. Collectively, these results demonstrate that endotoxin administration to pregnant mice causes an imbalance between innate and adaptive immune cells at the maternal-fetal interface.

Exploring the role of mononuclear phagocytes in the epididymis

The onslaught of foreign antigens carried by spermatozoa into the epididymis, an organ that has not demonstrated immune privilege, a decade or more after the establishment of central immune tolerance presents a unique biological challenge. Historically, the physical confinement of spermatozoa to the epididymal tubule enforced by a tightly interwoven wall of epithelial cells was considered sufficient enough to prevent cross talk between gametes and the immune system and, ultimately, autoimmune destruction. The discovery of an intricate arrangement of mononuclear phagocytes （MPs） comprising dendritic cells and macrophages in the murine epididymis suggests that we may have underestimated the existence of a sophisticated mucosal immune system in the posttesticular environment. This review consolidates our current knowledge of the physiology of MPs in the steady state epididymis and speculates on possible interactions between auto-antigenic spermatozoa, pathogens and the immune system by drawing on what is known about the immune system in the intestinal mucosa. Ultimately, further investigation will provide valuable information regarding the origins of pathologies arising as a result of autoimmune or inflammatory responses in the epididymis, including epididymitis and infertility.

Advances in immunopathogenesis of adult immune thrombocytopenia

Primary immune thrombocytopenia(ITP)is an autoimmune disorder characterized by immune-mediated accelerated platelet destruction and/or suppressed platelet production.Although the development of autoantibodies against platelet glycoproteins remains central in the pathophysiology of ITP,several abnormalities involving the cellular mechanisms of immune modulation have been identified,and the pathways behind the immune-mediated destruction of platelets have opened new avenues for the design of specific immunotherapies in an attempt to reduce the platelet destruction.This review is primarily focused on the recent literature with respect to immunopathological mechanisms in patients with ITP.

B cell dysfunction in chronic hepatitis B virus infection

Chronic hepatitis B(CHB)remains a global health problem.The persistence of hepatitis B surface antigen(HBsAg)in the blood for longer than 6 months after the initial infection is a sign of CHB.The therapeutic goal for the functional cure of CHB is the generation of antibodies against HBsAg.However,the adaptive immune response of patients with CHB cannot generate an efficient antiviral response.Many previous studies have evaluated T cell function and T cell therapy specifically designed to counter hepatitis B virus(HBV)infection.As one of the major components of adaptive immunity,B cells also display dysfunctions in anti-HBsAg antibody(HBsAb)production and antigen presentation.Patients with CHB have amplification of CD19^(+)CD10^(-)CD27^(-)CD21^(-)atypical memory B cell subsets and CD19^(+)CD24^(hi)CD38^(hi) regulatory B cells.Currently,no reviews have summarized specific B cell responses during CHB infection.Thus,in this study,we summarized B cell dysfunction during CHB progression and the potential mechanisms behind these dysfunctions to further our understanding of the mechanisms of adaptive immune response of B cells in the process of CHB development and help provide new methods and ideas for the treatment of CHB.

Single-cell RNA sequencing reveals the process of CA19-9 production and dynamics of the immune microenvironment between CA19-9 (+) and CA19-9 (−) PDAC

Background:Pancreatic ductal adenocarcinoma(PDAC)is one of the main types of malignant tumor of the digestive system,and patient prognosis is affected by difficulties in early diagnosis,poor treatment response,and a high postoperative recurrence rate.Carbohydrate antigen 19-9(CA19-9)has been widely used as a biomarker for the diagnosis and postoperative follow-up of PDAC patients.Nevertheless,the production mechanism and potential role of CA19-9 in PDAC progression have not yet been elucidated.Methods:We performed single-cell RNA sequencing on six samples pathologically diagnosed as PDAC(three CA19-9-positive and three CA19-9-negative PDAC samples)and two paracarcinoma samples.We also downloaded and integrated PDAC samples(each from three CA19-9-positive and CA19-9-negative patients)from an online database.The dynamics of the proportion and potential function of each cell type were verified through immunofluorescence.Moreover,we built an in vitro coculture cellular model to confirm the potential function of CA19-9.Results:Three subtypes of cancer cells with a high ability to produce CA19-9 were identified by the markers TOP2A,AQP5,and MUC5AC.CA19-9 production bypass was discovered on antigen-presenting cancer-associated fibroblasts(apCAFs).Importantly,the proportion of immature ficolin-1 positive(FCN1+)macrophages was high in the CA19-9-negative group,and the proportion of mature M2-like macrophages was high in the CA19-9-positive group.High proportions of these two macrophage subtypes were associated with an unfavourable clinical prognosis.Further experiments indicated that CA19-9 could facilitate the transformation of M0 macrophages into M2 macrophages in the tumor microenvironment.Conclusions:Our study described CA19-9 production at single-cell resolution and the dynamics of the immune atlas in CA19-9-positive and CA19-9-negative PDAC.CA19-9 could promote M2 polarization of macrophage in the pancreatic tumor microenvironment.