Differential requirements for CD80/86-CD28 costimulation in primary and memory CD4 T cell responses to vaccinia virus

Vaccinia virus infection can confer immunity to smallpox by inducing potent T cell and antibody responses. While the CD8 T cell response to vaccinia virus has been well characterized, less is known about factors required for priming and memory for the CD4 T cells. Focusing on two recently described epitopes, we show that after intranasal infection, both I1L and L4R epitopes are co-dominant during the acute response, but the I1L epitope dominates during memory. CD4 T cell priming was intact in the absence of CD80/86, however secondary responses were reduced. This contrasts with our previous data showing CD80/86–CD28 interaction is required for optimal primary and memory CD8 T cell responses. The absence of CD80/86 also changed the immunodominance hierarchy during memory, with the I1L and L4R responses becoming co-dominant in knockout mice. These data highlight different costimulatory requirements for primary CD4 and CD8 T cell responses to vaccinia virus.     

Intratumoral immune heterogeneity of prostate cancer characterized by typing and hub genes

Discordant abundances of different immune cell subtypes is regarded to be an essential feature of tumour tissue. Direct studies in Prostate cancer (PC) of intratumoral immune heterogeneity characterized by immune cell subtype, are still lacking. Using the single sample gene set enrichment analysis (ssGSEA) algorithm, the abundance of 28 immune cells infiltration (ICI) were determined for PC. A NMF was performed to determine tumour-sample clustering based on the abundance of ICI and PFS information. Hub genes of clusters were identified via weighted gene co-expression network analysis (WGCNA). The multivariate dimensionality reduction analysis of hub genes expression matrix was carried out via principal component analysis (PCA) to obtain immune score (IS). We analysed the correlation between clustering, IS and clinical phenotype. We divided the 495 patients into clusterA (n = 193) and clusterB (n = 302) on the basis of ICI and PFS via NMF. The progression-free survival (PFS) were better for clusterA than for clusterB (p < 0.001). Each immune cell subtypes was more abundant in clusterA than in clusterB (p < 0.001). The expression levels of CTAL-4 and PD-L1 were lower in clusterB than in clusterA (p < 0.001 and p = 0.006). We obtained 103 hub genes via WGCNA. In the training and validation cohorts, the prognosis of high IS group was worse than that of the low IS group (p < 0.05). IS had good predictive effect on 5-year PFS. The expression of immune checkpoint genes was higher in the low IS group than in the high IS group (p < 0.01). Patients with low IS and receiving hormone therapy had better prognosis than other groups. The combination of IS and clinical characteristics including lymph node metastasis and gleason score can better differentiate patient outcomes than using it alone. IS was a practical algorithm to predict the prognosis of patients. Advanced PC patients with low IS may be more sensitive to hormone therapy. CXCL10, CXCL5, MMP1, CXCL12, CXCL11, CXCL2, STAT1, IL-6 and TLR2 were hub genes, which may drive the homing of immune cells in tumours and promote immune cell differentiation.     

MTOR signaling is essential for the development of thymic epithelial cells and the induction of central immune tolerance

Thymic epithelial cells (TECs) are critical for the establishment and maintenance of appropriate microenvironment for the positive and negative selection of thymocytes and the induction of central immune tolerance. Yet, little about the molecular regulatory network on TEC development and function is understood. Here, we demonstrate that MTOR (mechanistic target of rapamycin [serine/threonine kinase]) is essential for proper development and functional maturation of TECs. Pharmacological inhibition of MTOR activity by rapamycin (RPM) causes severe thymic atrophy and reduction of TECs. TEC-specific deletion of Mtor causes the severe reduction of mTECs, the blockage of thymocyte differentiation and output, the reduced generation of thymic regulatory T (Treg) cells and the impaired expression of tissue-restricted antigens (TRAs) including Fabp2, Ins1, Tff3 and Chrna1 molecules. Importantly, specific deletion of Mtor in TECs causes autoimmune diseases characterized by enhanced tissue immune cell infiltration and the presence of autoreactive antibodies. Mechanistically, Mtor deletion causes overdegradation of CTNNB1/Beta-Catenin due to excessive autophagy and the attenuation of WNT (wingless-type MMTV integration site family) signaling in TECs. Selective inhibition of autophagy significantly rescued the poor mTEC development caused by Mtor deficiency. Altogether, MTOR is essential for TEC development and maturation by regulating proliferation and WNT signaling activity through autophagy. The present study also implies that long-term usage of RPM might increase the risk of autoimmunity by impairing TEC maturation and function.     

Enhancement of HLA class II-restricted CD4+ T cell recognition of human melanoma cells following treatment with bryostatin-1

The majority of melanoma cells express detectable levels of HLA class II proteins, and an increased threshold of cell surface class II is crucial for the stimulation of CD4+ T cells. Bryostatin-1, a protein kinase C (PKC) activator, has been considered as a potent chemotherapeutic agent in a variety of in vitro tumor models. Little is known about the role of bryostatin-1 in HLA class II Ag presentation and immune activation in malignant tumors, especially in melanoma. In this study, we show that bryostatin-1 treatment enhances CD4+ T cell recognition of melanoma cells in the context of HLA class II molecules. We also show that bryostatin-1 treatment of melanoma cells increases class II protein levels by upregulating the class II transactivator (CIITA) gene. Flow cytometry and confocal microscopic analyses revealed that bryostatin-1 treatment upregulated the expression of costimulatory molecules (CD80 and CD86) in melanoma cells, which could prolong the interaction of immune cells and tumors. Bryostatin-1 also induced cellular differentiation in melanoma cells, and reduced tumorigenic factors such as pro-cathepsins and matrix-metalloproteinase-9. These data suggest that bryostatin-1 could be used as a chemo-immunotherapeutic agent for reducing tumorigenic potential of melanoma cells while enhancing CD4+ T cell recognition to prevent tumor recurrence.     

Downregulation of IL-12 and a novel negative feedback system mediated by CD25+CD4+ T cells

CD25(+)CD4(+) regulatory T cells suppress immune responses and are believed to play roles in preventing autoimmune diseases. However, the mechanism(s) underlying the suppression and the regulation of their homeostasis remain to be elucidated. Here we show that these regulatory T cells downregulated CD25(-)CD4(+) T-cell-mediated production of IL-12 from antigen-presenting cells, which can act as a growth factor for CD25(-)CD4(+) T cells. We further found that CD25(+)CD4(+) T cells, despite their well-documented "anergic" nature, proliferate significantly in vitro only when CD25(-)CD4(+) T cells are present. Notably, this proliferation was strongly dependent on IL-2 and relatively independent of IL-12. Thus, CD25(+)CD4(+) T cells suppress CD25(-)CD4(+) T-cell responses, at least in part, by inhibiting IL-12 production while they themselves can undergo proliferation with the mediation of CD25(-)CD4(+) T cells in vitro. These results offer a novel negative feedback system involving a tripartite interaction among CD25(+)CD4(+) and CD25(-)CD4(+) T cells, and APCs that may contribute to the termination of immune responses.     

Wharton’s jelly‐derived stromal cells and their cell therapy applications in allogeneic haematopoietic stem cell transplantation

For decades, mesenchymal stromal cells (MSCs) have been of great interest in the fields of regenerative medicine, tissue engineering and immunomodulation. Their tremendous potential makes it desirable to cryopreserve and bank MSCs to increase their accessibility and availability. Postnatally derived MSCs seem to be of particular interest because they are harvested after delivery without ethical controversy, they have the capacity to expand at a higher rate than adult‐derived MSCs, in which expansion decreases with ageing, and they have demonstrated immunological and haematological supportive properties similar to those of adult‐derived MSCs. In this review, we focus on MSCs obtained from Wharton''s jelly (the mucous connective tissue of the umbilical cord between the amniotic epithelium and the umbilical vessels). Wharton''s jelly MSCs (WJ‐MSCs) are a good candidate for cellular therapy in haematology, with accumulating data supporting their potential to sustain haematopoietic stem cell engraftment and to modulate alloreactivity such as Graft Versus Host Disease (GVHD). We first present an overview of their in‐vitro properties and the results of preclinical murine models confirming the suitability of WJ‐MSCs for cellular therapy in haematology. Next, we focus on clinical trials and discuss tolerance, efficacy and infusion protocols reported in haematology for GVHD and engraftment.     

Induction of xenogeneic islet transplantation tolerance by simultaneously blocking CD28-B7 and OX40-OX40L co-stimulatory pathways

It has been demonstrated that prolonged graft survival can be achieved through inhibiting the activation of T cells, and addition of soluble CTLA4Ig and OX40Ig proteins to mixed lymphocyte reactions can effectively inhibit T cell proliferation. To explore the potential of this type of treatment in xenotransplantation, we infected streptozotocin-induced diabetic BalB/c mice (H-2d) (200 mg/kg, IV) with 5×10⁸ pfu AdCTLA4Ig-IRES-OX40Ig on day 1 before islets transplantation through the tail vein. The results showed that this treatment prolonged the islet xenografts survival significantly. The reaction to exogenous glucose stimulation was normal and the cytokine secretion of the type Th1 cells was inhibited. The AdCTLA4Ig-IRES-OX40Ig-mediated treatment effectively induced the T cells into anergy and the Th1/Th2 cells into deviation. These results strongly supported the therapeutic potential of blockade of costimulation by Ad-CTLA4Ig-IRES-OX40Ig genes transfer in inducing the organ transplantation tolerance.     

Induction of xenogeneic islet transplantation tolerance by simultaneously blocking CD28-B7 and OX40-OX40L co-stimulatory pathways

Type 1 diabetes mellitus is an autoimmune disease caused by T cell-mediated destruction of pancreatic beta islets. With its incidence continuous rising in the pediatric age group in recent years, it is becoming a serious threat to the human health. Up to now, there has been no effective therapy for diabetes mellitus, and islet transplantation still remains a promising ap-proach to the treatment of Type 1 diabetes. However, two serious problems hinder the successful islets transplantation, tha…     

The promise of stem cell markers in the diagnosis and therapy of epithelial dysplasia and oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is the most common type of head and neck cancer. Epithelial dysplasia is often initiated in the cells and cell nuclei adjacent to the epithelial cell membrane. Reduced cell–cell adhesions enable cancer cells to detach from the tumor and disseminate to other organs. The mutations in epithelial dysplasia markers such as E‐cadherin and epithelial cell adhesion molecules (CD326) can lead to proliferation, growth and survival of the tumor cells and persistence of numerous malignancies that play a key role in epithelial dysplasia of OSCC. Accordingly, these genes can be considered prognostic markers or potential therapeutic targets for the tailored management of patients with OSCC. The gene expression profile of OSCC stem cells indicates a differential pattern that facilitates establishing a cell signature. Owing to the highly tumorigenic behavior of cancer stem cells and the role of these cells in tumor differentiation, treatment resistance, relapse, and metastasis, we reviewed the role of stem cell markers in epithelial dysplasia and OSCC.     

An immunophenotyping of renal clear cell carcinoma with characteristics and a potential therapeutic target for patients insensitive to immune checkpoint blockade

Renal clear cell carcinoma (RCC) patients who do not achieve optimal control of progression with immune checkpoint blockade (ICB) should be further studied. Unsupervised consensus clustering was used to group 525 RCC patients based on two typical ICB pathways, CTLA-4 and pogrammed death 1 (PD-1)/programmed death-ligand 1 (PD-L1), as well as two new discovered regulators, CMTM6 and CMTM4. Three immune molecular subtypes (IMMSs) with different clinical and immunological characteristics were identified (type I, II, and III), among which there were more stage I and low-grade tumors in type I RCC than in type II and III. The proportion of males was highest in type II RCC. Overall survival of type II and III was similar (5.2 and 6 years) and statistically shorter than that of type I (7.6 years) before and after adjusting for age and gender. When conducting stratified analysis, our IMMSs were able to identify high-risk patients among middle-aged patients, males, and stage IV patients. Among the differentially expressed genes, approximately 84% were highly expressed in type II and III RCC. Genes related to ICB (CTLA-4, CD274, and PDCD1LG2) and cytotoxic lymphocytes (CD8A, GZMA, and PRF1) were all highly expressed in type II and III RCC. These results documented that patients with type II and III cancer may be more sensitive to anti-CTLA-4 therapy, anti-PD-1/PD-L1 therapy, and a combination of immunotherapies. High expression of CMTM4 in type I RCC (69%) and a statistically significant interaction of CD274 and CMTM6 indicated that CMTM4/6 might be new therapy targets for type I, who are resistant to ICB.     

Apoptosis of CD4<Superscript>+</Superscript>CD25<Superscript>high</Superscript> T cells in response to Sirolimus requires activation of T cell receptor and is modulated by IL-2

Targeted molecular therapies inhibit proliferation and survival of cancer cells but may also affect immune cells. We have evaluated the effects of Sirolimus and Sorafenib on proliferation and survival of lymphoid cell subsets. Both drugs were cytotoxic to CD4⁺CD25^high T cells, and were growth inhibitory for CD4⁺ and CD8⁺ T cells. Cytotoxicity depended on CD3/CD28 stimulation and was detectable within 12 h, with 80–90% of CD4⁺CD25^high cells killed by 72 h. Cell death was due to apoptosis, based on Annexin V and 7AAD staining. Addition of IL-2 prevented the apoptotic response to Sirolimus, potentially accounting for reports that Sirolimus can enhance proliferation of CD4⁺CD25^high cells. These results predict that Sirolimus or Sorafenib would reduce CD4⁺CD25^high cells if administered prior to antigenic stimulation in an immunotherapy protocol. However, administration of IL-2 protects CD4⁺CD25^high T cells from cytotoxic effects of Sirolimus, a response that may be considered in design of therapeutic protocols.     

Tailoring host immune responses to Listeria by manipulation of virulence genes -- the interface between innate and acquired immunity

Although attenuated strains of microbial pathogens have triggered vaccine development from its origin, the role of virulence factors in determining host immunity has remained largely unexplored. Using the murine listeriosis model, we investigated whether the induction and expansion of protective and inflammatory T cell responses may be modified by selective manipulation of virulence genes. We intentionally deleted specific genes of Listeria monocytogenes, including those encoding the positive regulatory factor (prfA), hemolysin (hly), the actin nucleator (actA), and phospholipase B (plcB). The resulting strains showed decisive differences in their immunogenic properties. In particular, we identified a double-deletion mutant that retained Listeria's profound ability to induce protective CD8(+) T cells, but that is strongly attenuated and exhibits a significantly reduced ability to induce CD4(+) T cell-mediated inflammation. We conclude that this mutant, L. monocytogenes DeltaactADeltaplcB, is at present the most promising mutant for a bacterial vaccine vector and is able to safely induce potent CD8(+) T cell-mediated immunity.     

Preferential effects of leptin on CD4 T cells in central and peripheral immune system are critically linked to the expression of leptin receptor

Leptin can enhance thymopoiesis and modulate the T-cell immune response. However, it remains controversial whether these effects correlate with the expression of leptin receptor, ObR. We herein addressed this issue by using in vivo animal models and in vitro culture systems. Leptin treatment in both ob/ob mice and normal young mice induced increases of CD4 SP thymocytes in thymus and CD4 T cells in the periphery. Interestingly, expression of the long form ObR was significantly restricted to DN, DP and CD4 SP, but not CD8 SP thymocytes. Moreover, in the reaggregated DP thymocyte cultures with leptin plus TSCs, leptin profoundly induced differentiation of CD4 SP but not CD8 SP thymocytes, suggesting that the effects of leptin on thymocyte differentiation might be closely related to the expression of leptin receptor in developing thymocytes. Surprisingly, ObR expression was markedly higher in peripheral CD4 T cells than that in CD8 T cells. Furthermore, leptin treatment with or without IL-2 and PHA had preferential effects on cell proliferation of CD4 T cells compared to that of CD8 T cells. Collectively, these data provide evidence that the effects of leptin on differentiation and proliferation of CD4 T cells might be closely related to the expression of leptin receptor.     

Cancer stem cells: implications for the progression and treatment of metastatic disease

Metastasis is the major cause of death for cancer patients with solid tumours, due mainly to the ineffectiveness of current therapies once metastases begin to form. Further insight into the biology of metastasis is therefore essential in order to gain a greater understanding of this process and ultimately to develop better cancer therapies. Metastasis is an inefficient process, such that very few cells that leave a tumour successfully form macrometastases in distant sites. This suggests that only a small subset of cells can successfully navigate the metastatic cascade and eventually re-initiate tumour growth to form life-threatening metastases. Recently, there has been growing support for the cancer stem cell (CSC) hypothesis which stipulates that primary tumours are initiated and maintained by a small subpopulation of cancer cells that possess "stem-like" characteristics. Classical properties of normal stem cells are strikingly reminiscent of the observed experimental and clinical behaviour of metastatic cancer cells, including an unlimited capacity for self renewal; the requirement for a specific 'niche' or microenvironment to grow; use of the stromal cell-derived factor 1 (SDF-1)/chemokine receptor 4 (CXCR4) axis for migration; enhanced resistance to apoptosis and an increased capacity for drug resistance. Therefore, in addition to playing a role in primary tumour formation, we believe that CSCs are also key players in the metastatic process. We will review the current evidence supporting this idea and discuss the potential implications of the CSC hypothesis with regards to experimental investigation and treatment of metastatic disease.     

Glial precursor cell transplantation therapy for neurotrauma and multiple sclerosis

Traumatic injury to the brain or spinal cord and multiple sclerosis (MS) share a common pathophysiology with regard to axonal demyelination. Despite advances in central nervous system (CNS) repair in experimental animal models, adequate functional recovery has yet to be achieved in patients in response to any of the current strategies. Functional recovery is dependent, in large part, upon remyelination of spared or regenerating axons. The mammalian CNS maintains an endogenous reservoir of glial precursor cells (GPCs), capable of generating new oligodendrocytes and astrocytes. These GPCs are upregulated following traumatic or demyelinating lesions, followed by their differentiation into oligodendrocytes. However, this innate response does not adequately promote remyelination. As a result, researchers have been focusing their efforts on harvesting, culturing, characterizing, and transplanting GPCs into injured regions of the adult mammalian CNS in a variety of animal models of CNS trauma or demyelinating disease. The technical and logistic considerations for transplanting GPCs are extensive and crucial for optimizing and maintaining cell survival before and after transplantation, promoting myelination, and tracking the fate of transplanted cells. This is especially true in trials of GPC transplantation in combination with other strategies such as neutralization of inhibitors to axonal regeneration or remyelination. Overall, such studies improve our understanding and approach to developing clinically relevant therapies for axonal remyelination following traumatic brain injury (TBI) or spinal cord injury (SCI) and demyelinating diseases such as MS.     

A subpopulation of bone marrow cells depleted by a novel antibody, anti-Liv8, is useful for cell therapy to repair damaged liver

We previously reported a new in vivo model named as "GFP/CCl(4) model" for monitoring the transdifferentiation of green fluorescent protein (GFP) positive bone marrow cell (BMC) into albumin-positive hepatocyte under the specific "niche" made by CCl(4) induced persistent liver damage, but the subpopulation which BMCs transdifferentiate into hepatocytes remains unknown. Here we developed a new monoclonal antibody, anti-Liv8, using mouse E 11.5 fetal liver as an antigen. Anti-Liv8 recognized both hematopoietic progenitor cells in fetal liver at E 11.5 and CD45-positive hematopoietic cells in adult bone marrow. We separated Liv8-positive and Liv8-negative cells and then transplanted these cells into a continuous liver damaged model. At 4 weeks after BMC transplantation, more efficient repopulation and transdifferentiation of BMC into hepatocytes were seen with Liv8-negative cells. These findings suggest that the subpopulation of Liv8-negative cells includes useful cells to perform cell therapy on repair damaged liver.