Identification of distinct and age‐dependent p16High microglia subtypes

Cells expressing high levels of the cyclin‐dependent kinase (CDK)4/6 inhibitor p16 (p16^High) accumulate in aging tissues and promote multiple age‐related pathologies, including neurodegeneration. Here, we show that the number of p16^High cells is significantly increased in the central nervous system (CNS) of 2‐year‐old mice. Bulk RNAseq indicated that genes expressed by p16^High cells were associated with inflammation and phagocytosis. Single‐cell RNAseq of brain cells indicated p16^High cells were primarily microglia, and their accumulation was confirmed in brains of aged humans. Interestingly, we identified two distinct subpopulations of p16^High microglia in the mouse brain, with one being age‐associated and one present in young animals. Both p16^High clusters significantly differed from previously described disease‐associated microglia and expressed only a partial senescence signature. Taken together, our study provides evidence for the existence of two p16‐expressing microglia populations, one accumulating with age and another already present in youth that could positively and negatively contribute to brain homeostasis, function, and disease.     

Synergistic effect of Abraxane that combines human IL15 fused with an albumin‐binding domain on murine models of pancreatic ductal adenocarcinoma

Nab‐paclitaxel (Abraxane), which is a nanoparticle form of albumin‐bound paclitaxel, is one of the standard chemotherapies for pancreatic ductal adenocarcinoma (PDAC). This study determined the effect of Abraxane in combination with a fusion protein, hIL15‐ABD, on subcutaneous Panc02 and orthotopic KPC C57BL/6 murine PDAC models. Abraxane combined with hIL15‐ABD best suppressed tumour growth and produced a 40%–60% reduction in the tumour size for Panc02 and KPC, compared to the vehicle group. In the combination group, the active form of interferon‐γ (IFN‐γ)‐secreting CD8⁺ T cells and CD11b⁺CD86⁺ M1 macrophages in tumour infiltrating lymphocytes (TILs) were increased. In the tumour drainage lymph nodes (TDLNs) of the combination group, there was a 18% reduction in CD8⁺IFN‐γ⁺ T cells and a 0.47% reduction in CD4⁺CD25⁺FOXP3⁺ regulatory T cells, as opposed to 5.0% and 5.1% reductions, respectively, for the control group. Superior suppression of CD11b⁺GR‐1⁺ myeloid‐derived suppressor cells (MDSCs) and the induction of M1 macrophages in the spleen and bone marrow of mice were found in the combination group. Abraxane and hIL15‐ABD effectively suppressed NF‐κB‐mediated immune suppressive markers, including indoleamine 2,3‐dioxygenase (IDO), Foxp3 and VEGF. In conclusion, Abraxane combined with hIL15‐ABD stimulates the anticancer activity of effector cells, inhibits immunosuppressive cells within the tumour microenvironment (TME) of PDAC, and produces a greater inhibitory effect than individual monotherapies.     

Regulatory KIR+RA+ T cells accumulate with age and are highly activated during viral respiratory disease

Severe respiratory viral infectious diseases such as influenza and COVID‐19 especially affect the older population. This is partly ascribed to diminished CD8⁺ T‐cell responses a result of aging. The phenotypical diversity of the CD8⁺ T‐cell population has made it difficult to identify the impact of aging on CD8⁺ T‐cell subsets associated with diminished CD8⁺ T‐cell responses. Here we identify a novel human CD8⁺ T‐cell subset characterized by expression of Killer‐cell Immunoglobulin‐like Receptors (KIR⁺) and CD45RA (RA⁺). These KIR⁺RA⁺ T cells accumulated with age in the blood of healthy individuals (20–82 years of age, n = 50), expressed high levels of aging‐related markers of T‐cell regulation, and were functionally capable of suppressing proliferation of other CD8⁺ T cells. Moreover, KIR⁺RA⁺ T cells were a major T‐cell subset becoming activated in older adults suffering from an acute respiratory viral infection (n = 36), including coronavirus and influenza virus infection. In addition, older adults with influenza A infection showed that higher activation status of their KIR⁺RA⁺ T cells associated with longer duration of respiratory symptoms. Together, our data indicate that KIR⁺RA⁺ T cells are a unique human T‐cell subset with regulatory properties that may explain susceptibility to viral respiratory disease at old age.     

Evaluation of CD49f as a novel surface marker to identify functional adipose‐derived mesenchymal stem cell subset

ObjectivesCD49f is expressed on a variety of stem cells and has certain effects on their cytological functions, such as proliferation and differentiation potential. However, whether CD49f is expressed on the surface of adipose tissue‐derived mesenchymal stem cells (ADSCs) and its effect on ADSCs has not been clarified.Materials and methodsThe effects of in vitro culture passage and inflammatory factor treatment on CD49f expression and the adhesion ability of ADSCs from mice and rats were investigated. CD49f⁺ cells were selected from rat ADSCs (rADSCs) by magnetic‐activated cell sorting (MACS), and the cellular functions of CD49f⁺ ADSCs and unsorted ADSCs, including their clonogenic, proliferation, adipogenic and osteogenic differentiation, migration and anti‐apoptotic capacities, were compared.ResultsCD49f expression and the adhesion ability of ADSCs decreased with increasing in vitro culture passage number. TNF‐α and IFN‐γ treatment decreased CD49f expression but increased the adhesion ability of ADSCs. After CD49f was blocked with an anti‐CD49f antibody, the adhesion ability of ADSCs was decreased. No significant difference in clonogenic activity was observed between unsorted ADSCs and CD49f⁺ ADSCs. CD49f⁺ ADSCs had greater proliferation, adipogenic and osteogenic differentiation, migration and anti‐apoptotic capacities than unsorted ADSCs.ConclusionIn the current study, the expression of CD49f on ADSCs was identified for the first time. The expression of CD49f on ADSCs was influenced by in vitro culture passage number and inflammatory factor treatment. Compared with unsorted ADSCs, CD49f ⁺ ADSCs exhibited superior cellular functions, thus may have great application value in mesenchymal stem cell (MSC)‐based therapies.     

T cell–derived exosomes induced macrophage inflammatory protein‐1α/β drive the trafficking of CD8+ T cells in oral lichen planus

Oral lichen planus (OLP) is a T cell–mediated chronic inflammatory disease with uncertain aetiology. Exosomes are nanosized particles with biological capacities. Here, we aimed to study the effects of T cell–derived exosomes (T‐exos) on the pathogenesis of OLP and its mechanism. T‐exos were incubated with Jurkat cells for 48 hours, and 26 cytokines in the supernatant were measured by luminex assay. The expression of macrophage inflammatory protein (MIP)‐1α/β was detected using immunohistochemistry and ELISA; that of CCR1/3/5 on peripheral T cells was determined by flow cytometry. Transwell assay was performed to investigate the chemotactic effect of MIP‐1α/β, and cells in the lower chambers were examinated by flow cytometry. As a result, OLP T‐exos elevated the production of MIP‐1α/β, which were highly expressed in OLP tissues and plasma. CCR1/5 were markedly expressed on OLP peripheral T cells, and the majority of CCR1/5⁺ T cells were CD8⁺ T cells. Besides, MIP‐1α/β promoted the migration of OLP mononuclear cells, while inhibiting CCR1/5 significantly decreased the trafficking of mononuclear cells, especially that of CD8⁺ T cells. Conclusively, OLP T‐exos‐induced MIP‐1α/β may drive the trafficking of CD8⁺ T cells after binding with CCR1/5 in OLP, contributing to the development of OLP.     

Dynamics of NK,CD8 and Tfh cell mediated the production of cytokines and antiviral antibodies in Chinese patients with moderate COVID‐19

Recent studies have demonstrated a marked decrease in peripheral lymphocyte levels in patients with coronavirus disease 2019 (COVID‐19) caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). Few studies have focused on the changes of NK, T‐ and B‐cell subsets, inflammatory cytokines and virus‐specific antibodies in patients with moderate COVID‐19. A total of 11 RT‐PCR‐confirmed convalescent patients with COVID‐19 and 11 patients with non‐SARS‐CoV‐2 pneumonia (control patients) were enrolled in this study. NK, CD8⁺ T, CD4⁺ T, Tfh‐like and B‐cell subsets were analysed using flow cytometry. Cytokines and SARS‐CoV‐2‐specific antibodies were analysed using an electrochemiluminescence immunoassay. NK cell counts were significantly higher in patients with COVID‐19 than in control patients (P = 0.017). Effector memory CD8⁺ T‐cell counts significantly increased in patients with COVID‐19 during a convalescent period of 1 week (P = 0.041). TIM‐3⁺ Tfh‐like cell and CD226⁺ Tfh‐like cell counts significantly increased (P = 0.027) and decreased (P = 0.022), respectively, during the same period. Moreover, ICOS⁺ Tfh‐like cell counts tended to decrease (P = 0.074). No abnormal increase in cytokine levels was observed. The high expression of NK cells is important in innate immune response against SARS‐CoV‐2. The increase in effector memory CD8⁺ T‐cell counts, the up‐regulation of inhibitory molecules and the down‐regulation of active molecules on CD4⁺ T cells and Tfh‐like cells in patients with COVID‐19 would benefit the maintenance of balanced cellular and humoural immune responses, may prevent the development of severe cases and contribute to the recovery of patients with COVID‐19.     

CD4+CD25?Nrp1+ T Cells Synergize with Rapamycin to Prevent Murine Cardiac Allorejection in Immunocompetent Recipients

Besides CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs), other immunosuppressive T cells also participated in the regulation of immune tolerance. Reportedly, neuropilin-1 (Nrp1) might be one of the molecules by which regulatory cells exert their suppressive effects. Indeed, CD4⁺CD25⁻Nrp1⁺ T cells exhibit potent suppressive function in autoimmune inflammatory responses. Here we investigated the specific role of CD4⁺CD25⁻Nrp1⁺ T cells in the setting of the transplant immune response. Through MLR assays, we found that CD4⁺CD25⁻Nrp1⁺ T cells suppressed the proliferation of naive CD4⁺CD25⁻ T cells activated by allogeneic antigen-stimulation. Adoptive transfer of CD4⁺CD25⁻Nrp1⁺ T cells synergized with rapamycin to induce long-term graft survival in fully MHC-mismatched murine heart transplantation, which was associated with decreased IFN-γ, IL-17 and increased IL-10, TGF-β, Foxp3 and Nrp1 expression in the grafts. Importantly, our data indicated that CD4⁺CD25⁻Nrp1⁺ T cell transfer augments the accumulation of Tregs in the recipient, and creates conditions that favored induction of hyporesponsiveness of the T effector cells. In conclusion, this translational study indicates the possible therapeutic potential of CD4⁺CD25⁻Nrp1⁺ T cells in preventing allorejection. CD4⁺Nrp1⁺ T cells might therefore be used in bulk as a population of immunosuppressive cells with more beneficial properties concerning ex vivo isolation as compared to Foxp3⁺ Tregs.     

Induction of γδT cells from HSC‐enriched BMCs co‐cultured with iPSC‐derived thymic epithelial cells

T cells bearing γδ antigen receptors have been investigated as potential treatments for several diseases, including malignant tumours. However, the clinical application of γδT cells has been hampered by their relatively low abundance in vivo and the technical difficulty of inducing their differentiation from hematopoietic stem cells (HSCs) in vitro. Here, we describe a novel method for generating mouse γδT cells by co‐culturing HSC‐enriched bone marrow cells (HSC‐eBMCs) with induced thymic epithelial cells (iTECs) derived from induced pluripotent stem cells (iPSCs). We used BMCs from CD45.1 congenic C57BL/6 mice to distinguish them from iPSCs, which expressed CD45.2. We showed that HSC‐eBMCs and iTECs cultured with IL‐2 + IL‐7 for up to 21 days induced CD45.1⁺ γδT cells that expressed a broad repertoire of Vγ and Vδ T‐cell receptors. Notably, the induced lymphocytes contained few or no αβT cells, NK1.1⁺ natural killer cells, or B220⁺ B cells. Adoptive transfer of the induced γδT cells to leukemia‐bearing mice significantly reduced tumour growth and prolonged mouse survival with no obvious side effects, such as tumorigenesis and autoimmune diseases. This new method suggests that it could also be used to produce human γδT cells for clinical applications.     

Senolytics alleviate the degenerative disorders of temporomandibular joint in old age

Aging is one of the major risk factors for degenerative joint disorders, including those involving the temporomandibular joint (TMJ). TMJ degeneration occurs primarily in the population over 65, significantly increasing the risk of joint discomfort, restricted joint mobility, and reduced quality of life. Unfortunately, there is currently no effective mechanism‐based treatment available in the clinic to alleviate TMJ degeneration with aging. We now demonstrate that intermittent administration of senolytics, drugs which can selectively clear senescent cells, preserved mandibular condylar cartilage thickness, improved subchondral bone volume and turnover, and reduced Osteoarthritis Research Society International (OARSI) histopathological score in both 23‐ to 24‐month‐old male and female mice. Senolytics had little effect on 4 months old young mice, indicating age‐specific benefits. Our study provides proof‐of‐concept evidence that age‐related TMJ degeneration can be alleviated by pharmaceutical intervention targeting cellular senescence. Since the senolytics used in this study have been proven relatively safe in recent human studies, our findings may help justify future clinical trials addressing TMJ degeneration in old age.     

Decreased antiviral immune response within the central nervous system of aged mice is associated with increased lethality of West Nile virus encephalitis

West Nile virus (WNV) is an emerging pathogen that causes disease syndromes ranging from a mild flu‐like illness to encephalitis. While the incidence of WNV infection is fairly uniform across age groups, the risk of lethal encephalitis increases with advanced age. Prior studies have demonstrated age‐related, functional immune deficits that limit systemic antiviral immunity and increase mortality; however, the effect of age on antiviral immune responses specifically within the central nervous system (CNS) is unknown. Here, we show that aged mice exhibit increased peripheral organ and CNS tissue viral burden, the latter of which is associated with alterations in activation of both myeloid and lymphoid cells compared with similarly infected younger animals. Aged mice exhibit lower MHCII expression by microglia, and higher levels of PD1 and lower levels of IFNγ expression by WNV‐specific CD8⁺ T cells in the CNS and CD8⁺CD45⁺ cells. These data indicate that the aged CNS exhibits limited local reactivation of T cells during viral encephalitis, which may lead to reduced virologic control at this site.     

Ectopic cervical thymi and no thymic involution until midlife in naked mole rats

Immunosenescence is a hallmark of aging and manifests as increased susceptibility to infection, autoimmunity, and cancer in the elderly. One component of immunosenescence is thymic involution, age‐associated shrinkage of the thymus, observed in all vertebrates studied to date. The naked mole rat (Heterocephalus glaber) has become an attractive animal model in aging research due to its extreme longevity and resistance to disease. Here, we show that naked mole rats display no thymic involution up to 11 years of age. Furthermore, we found large ectopic cervical thymi in addition to the canonical thoracic thymus, both being identical in their cell composition. The developmental landscape in naked mole rat thymi revealed overt differences from the murine T‐cell compartment, most notably a decrease of CD4⁺/CD8⁺ double‐positive cells and lower abundance of cytotoxic effector T cells. Our observations suggest that naked mole rats display a delayed immunosenescence. Therapeutic interventions aimed at reversing thymic aging remain limited, underscoring the importance of understanding the cellular and molecular mechanisms behind a sustained immune function in the naked mole rat.     

Comparative kinetic analyses of gene profiles of naïve CD4+ and CD8+ T cells from young and old animals reveal novel age-related alterations

It is well established that immune responses are diminished in the old. However, we still do not have a clear understanding of what dictates the dysfunction of old T cells at the molecular level. Although microarray analysis has been used to compare young and old T cells, identifying hundreds of genes that are differentially expressed among these populations, it has been difficult to utilize this information to pinpoint which biological pathways truly affect the function of aged T cells. To better define differences between young and old naïve CD4⁺ and CD8⁺ T cells, microarray analysis was performed pre‐ and post‐TCR stimulation for 4, 12, 24 and 72 h. Our data indicate that many genes are differentially expressed in the old compared to the young at all five time points. These genes encode proteins involved in multiple cellular functions such as cell growth, cell cycle, cell death, inflammatory response, cell trafficking, etc. Additionally, the information from this microarray analysis allowed us to underline both intrinsic deficiencies and defects in signaling only seen after activation, such as pathways involving T‐cell signaling, cytokine production, and Th2 differentiation in old T cells. With the knowledge gained, we can proceed to design strategies to restore the function of old T cells. Therefore, this microarray analysis approach is a powerful and sensitive tool that reveals the extensive changes seen between young and old CD4⁺ and CD8⁺ naïve T cells. Evaluation of these differences provides in‐depth insight into potential functional and phenotypical differences among these populations.     

Aging impairs human bone marrow function and cardiac repair following myocardial infarction in a humanized chimeric mouse

Ventricular remodeling following myocardial infarction (MI) is a major cause of heart failure, a condition prevalent in older individuals. Following MI, immune cells are mobilized to the myocardium from peripheral lymphoid organs and play an active role in orchestrating repair. While the effect of aging on mouse bone marrow (BM) has been studied, less is known about how aging affects human BM cells and their ability to regulate repair processes. In this study, we investigate the effect aging has on human BM cell responses post‐MI using a humanized chimeric mouse model. BM samples were collected from middle aged (mean age 56.4 ± 0.97) and old (mean age 72.7 ± 0.59) patients undergoing cardiac surgery, CD34^+/− cells were isolated, and NOD‐scid‐IL2rγ^null (NSG) mice were reconstituted. Three months following reconstitution, the animals were examined at baseline or subjected to coronary artery ligation (MI). Younger patient cells exhibited greater repopulation capacity in the BM, blood, and spleen as well as greater lymphoid cell production. Following MI, CD34⁺ cell age impacted donor and host cellular responses. Mice reconstituted with younger CD34⁺ cells exhibited greater human CD45⁺ recruitment to the heart compared to mice reconstituted with old cells. Increased cellular responses were primarily driven by T‐cell recruitment, and these changes corresponded with greater human IFNy levels and reduced mouse IL‐1β in the heart. Age‐dependent changes in BM function led to significantly lower survival, increased infarct expansion, impaired host cell responses, and reduced function by 4w post‐MI. In contrast, younger CD34⁺ cells helped to limit remodeling and preserve function post‐MI.     

Phosphodiesterase 4A confers resistance to PGE2‐mediated suppression in CD25+/CD54+ NK cells

Inadequate persistence of tumor‐infiltrating natural killer (NK) cells is associated with poor prognosis in cancer patients. The solid tumor microenvironment is characterized by the presence of immunosuppressive factors, including prostaglandin E2 (PGE2), that limit NK cell persistence. Here, we investigate if the modulation of the cytokine environment in lung cancer with IL‐2 or IL‐15 renders NK cells resistant to suppression by PGE2. Analyzing Cancer Genome Atlas (TCGA) data, we found that high NK cell gene signatures correlate with significantly improved overall survival in patients with high levels of the prostaglandin E synthase (PTGES). In vitro, IL‐15, in contrast to IL‐2, enriches for CD25⁺/CD54⁺ NK cells with superior mTOR activity and increased expression of the cAMP hydrolyzing enzyme phosphodiesterase 4A (PDE4A). Consequently, this distinct population of NK cells maintains their function in the presence of PGE2 and shows an increased ability to infiltrate lung adenocarcinoma tumors in vitro and in vivo. Thus, strategies to enrich CD25⁺/CD54⁺ NK cells for adoptive cell therapy should be considered.     

Whole‐body senescent cell clearance alleviates age‐related brain inflammation and cognitive impairment in mice

Cellular senescence is characterized by an irreversible cell cycle arrest and a pro‐inflammatory senescence‐associated secretory phenotype (SASP), which is a major contributor to aging and age‐related diseases. Clearance of senescent cells has been shown to improve brain function in mouse models of neurodegenerative diseases. However, it is still unknown whether senescent cell clearance alleviates cognitive dysfunction during the aging process. To investigate this, we first conducted single‐nuclei and single‐cell RNA‐seq in the hippocampus from young and aged mice. We observed an age‐dependent increase in p16^Ink4a senescent cells, which was more pronounced in microglia and oligodendrocyte progenitor cells and characterized by a SASP. We then aged INK‐ATTAC mice, in which p16^Ink4a‐positive senescent cells can be genetically eliminated upon treatment with the drug AP20187 and treated them either with AP20187 or with the senolytic cocktail Dasatinib and Quercetin. We observed that both strategies resulted in a decrease in p16^Ink4a exclusively in the microglial population, resulting in reduced microglial activation and reduced expression of SASP factors. Importantly, both approaches significantly improved cognitive function in aged mice. Our data provide proof‐of‐concept for senolytic interventions'' being a potential therapeutic avenue for alleviating age‐associated cognitive impairment.     

Reducing TGF‐β1 cooperated with StemRegenin 1 promoted the expansion ex vivo of cord blood CD34+ cells by inhibiting AhR signalling

ObjectiveAs an inhibitor of the AhR signalling pathway, StemRegenin 1 (SR1) not only promotes the expansion of CD34⁺ cells but also increases CD34⁻ cell numbers. These CD34⁻ cells influenced the ex vivo expansion of CD34⁺ cells. In this work, the effects of periodically removing CD34⁻ cells combined with SR1 addition on the ex vivo expansion and biological functions of HSCs were investigated.Materials and methodsCD34⁻ cells were removed periodically with SR1 addition to investigate cell subpopulations, cell expansion, biological functions, expanded cell division mode and supernatant TGF‐β1 contents.ResultsAfter 10‐day culture, the expansion of CD34⁺ cells in the CD34⁻ cell removal plus SR1 group was significantly higher than that in the control group and the SR1 group. Moreover, periodically removing CD34⁻ cells with SR1 addition improved the biological function of expanded CD34⁺ cells and significantly increased the percentage of self‐renewal symmetric division of CD34⁺ cells. In addition, the concentration of total TGF‐β1 and activated TGF‐β1 in the supernatant was significantly lower than those in the control group and the SR1 group. RT‐qPCR results showed that the periodic removal of CD34⁻ cells with cooperation from SR1 further reduced the expression of AhR‐related genes.ConclusionsPeriodic removal of CD34⁻ cells plus cooperation with SR1 improved the expansion of CD34⁺ cells, maintained better biological function of expanded CD34⁺ cells and reduced the TGF‐β1 contents by downregulating AhR signalling.

SR1 increased self‐renewal symmetric division of cord blood CD34⁺ cells. Removal of CD34⁻ cells cooperated with SR1 increased ex vivo expansion of cord blood CD34⁺ cells. Removal of CD34⁻ cells cooperated with SR1 further downregulated AhR signalling     

Thioredoxin Priming Prolongs Lung Allograft Survival by Promoting Immune Tolerance

Tolerance to allograft antigen is the major challenge and final goal of transplant medicine. Our previous study demonstrated that thioredoxin-1 (Trx) priming of donor lung significantly protected allogeneic lung graft. To determine whether Trx priming of donor lung inhibits allograft rejection, extends allograft survival and induces immune tolerance, orthotopic left lung transplantation was performed from Lewis to Sprague-Dawley rats without immunosuppression. Donor lungs were primed with Trx at 4°C for 4 hr prior to transplantation. After up to 37 days post-transplantation, allograft lung morphology, recipient T cell and humoral alloantigen-specific immune responses were examined. We found that Trx-primed lungs exhibited much reduced acute rejection and associated lung injuries resulting in loss of graft functional area at 5-37 days post-transplant in contrast to the control groups. CD4⁺ T cells from the recipients with Trx-primed grafts responded to the stimulation of dendritic cells (DCs) of donor origin, in contrast to DCs from the third party, with significantly reduced proliferation. Consistent with above findings, we observed that CD4⁺Foxp3⁺ regulatory T cells in spleen cells from the recipients with Trx-primed grafts were significantly increased compared to controls, and CD4⁺ T cells from the recipients with Trx-primed grafts produced much higher levels of immunosuppressive cytokine, IL-10 when stimulated with allogeneic donor DCs. In addition, humoral immune tolerance was also induced as there was no significant increase levels of serum antibodies against donor antigens in Trx-lung recipients when re-challenged with allogeneic donor antigens. Our results demonstrate that one-time Trx-priming of donor lung grafts prior to transplantation significantly prolongs the survival of the grafts through inducing or promoting cellular and humoral alloantigen-specific immune tolerance, which might be associated with the induction of immunosuppressive regulatory T cells.     

The Effect of Cellular Stress on T and B Cell Memory Pathways in Immunized and Unimmunized BALB/c Mice

Immunological memory is a fundamental function of vaccination. The antigenic breakdown products of the vaccine may not persist, and undefined tonic stimulation has been proposed to maintain the specific memory. We have suggested that cellular stress agents to which the immune cells are constantly exposed may be responsible for tonic stimulation. Here we have studied four stress agents: sodium arsenite, an oxidative agent; Gramicidin, eliciting K⁺ efflux and calcium influx; dithiocarbamate, a metal ionophore; and aluminum hydroxide (alum), an immunological adjuvant. The aims of this study are to extend these investigations to T and B cell responses of unimmunized and ovalbumin (OVA)-immunized BALB/c mice, and furthermore, to ascertain whether stress is involved in optimal expression of memory B cells, as demonstrated in CD4⁺ T cells. Examination of the homeostatic pathway defined by IL-15/IL-15R (IL-15 receptor) interaction and the inflammasome pathway defined by the IL-1-IL-1R interaction between dendritic cells (DC) and CD4⁺ T cells suggests that both pathways are involved in the development of optimal expression of CD4⁺CD45RO⁺ memory T cells in unimmunized and OVA-immunized BALB/c mice. Furthermore, significant direct correlation was found between CD4⁺CD44⁺ memory T cells and both IL-15 of the homeostatic and IL-1β of the inflammasome pathways. However, CD19⁺CD27⁺ memory B cells in vivo seem to utilize only the IL-15/IL-15R homeostatic pathway, although the proliferative responses are enhanced by the stress agents. Altogether, stress agents may up-regulate unimmunized and OVA-immunized CD4⁺CD44⁺ memory T cells by the homeostatic and inflammasome pathways. However, the CD19⁺CD27⁺ memory B cells utilize only the homeostatic pathway.     

The Immunosuppressant Protosappanin A Promotes Dendritic Cell-Mediated Expansion of Alloantigen-Specific Tregs and Prolongs Allograft Survival in Rats

Protosappanin A (PrA), an immunosuppressive ingredient of the medicinal herb Caesalpinia sappan L, prolongs heart allograft survival in rats, possibly by impairing the function of antigen-presenting cells (APCs). We examined the effects of PrA on the maturation and function of dendritic cells (DCs), a potent class of APCs, and the downstream cell–cell and intracellular signaling pathways mediating the immunosuppressive activity of PrA. PrA inhibited LPS-stimulated maturation of Wistar rat DCs in vitro as reflected by reduced expression of costimulatory molecules (CD80 and CD86) and reduced expression of TLR4 and NF-κB, two critical signaling components for antigen recognition. PrA also enhanced the release of IL-10 and decreased the release of IL-12 from DCs, but had no effect on the production of TGF-ß. In mixed cultures, Wistar DCs pretreated with PrA impaired the proliferation of Sprague Dawley (SD) rat T cells while promoting the expansion of SD rat CD4⁺CD25⁺ regulatory T cells (Tregs). Both oral PrA treatment and infusion of PrA-pretreated Wistar DCs prolonged cardiac allograft survival (Wistar donor, SD recipient) and expanded recipient CD4⁺CD25⁺Foxp3⁺ Tregs. Donor spleen cells, but not spleen cells from a third rat strain (DA), supported the expansion of recipient CD4⁺CD25⁺Foxp3⁺ Tregs and suppressed recipient T cell proliferation. We conclude that PrA triggers a tolerogenic state in DCs that allows for the induction of alloantigen-specific Tregs and the suppression of allograft rejection in vivo.