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.

Optimal conditions for adenoviral transduction of immature dendritic cells without affecting the tolerogenic activity of DC-based immunotherapy.

Dendritic cells (DCs) play a pivotal role in maintaining immune tolerance. Using recombinant adenovirus (rAd) to deliver vectors to immature dendritic cells (imDCs) is an important method for studying the tolerogenic function of DCs. We found that using RPMI medium and a higher MOI during transduction increased the expression of CD80, CD86, and MHC-II on the surface of imDCs. Our data reveal a significant increase in the secretion of the pro-inflammatory cytokine IL-6 in the group showing the most pronounced phenotypic changes. In the mouse heart transplant model, imDCs with unstable phenotype and function due to adenoviral transduction resulted in an increased proportion of Th1 and Th17 cells in recipients. However, these effects can be managed, and our proposed optimized transduction strategy significantly minimizes these adverse effects. Our study holds significant implications for the development and optimization of immunotherapy utilizing tolerogenic dendritic cells.

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.

The levels of serum soluble CD86 are correlated with the expression of CD86 variant 3 gene and are prognostic indicators in patients with myeloma.

We previously showed that cell-surface CD86 expressed on multiple myeloma (MM) cells contributed to not only tumor growth but also antitumor cytotoxic T-lymphocyte responses mediated by induction of IL-10-producing CD4+ T cells. The soluble form of CD86 (sCD86) was also detected in serum from patients with MM. Thus, to determine whether sCD86 levels are a useful prognostic factor, we investigated the association of serum sCD86 levels with disease progression and prognosis in 103 newly diagnosed patients with MM. Serum sCD86 was detected in 71% of the patients with MM but was only rarely detected in patients with monoclonal gammopathy of undetermined significance and healthy controls, and the level was significantly increased in patients with advanced-stage MM. When we examined differences in clinical characteristics according to the level of serum sCD86, those in the high (≥2.18 ng/mL, n = 38) group exhibited more aggressive clinical characteristics, with shorter overall survival times compared with those in the low (<2.18 ng/mL, n = 65) group. On the other hand, it was difficult to stratify the patients with MM into different risk groups based on the expression levels of cell-surface CD86. The levels of serum sCD86 were significantly correlated with the expression levels of the messenger RNA (mRNA) transcripts of CD86 variant 3, which lack exon 6, resulting in a truncated transmembrane region, and its variant transcripts were upregulated in the high group. Thus, our findings suggest that sCD86 can be easily measured in peripheral blood samples and is a useful prognostic marker in patients with MM.

In silico engineering a CD80 variant with increased affinity to CTLA-4 and decreased affinity to CD28 for optimized cancer immunotherapy.

CD80 or cluster of differentiation 80, also known as B7-1, is a member of the immunoglobulin super family, which binds to CTLA-4 and CD28 T cell receptors and induces inhibitory and inductive signals respectively. Although CTLA-4 and CD28 receptors belong to the same protein family, slight differences in their structures leads to CD80 having a higher binding affinity to CTLA-4 (-14.55 kcal/mol) compared with CD28(-12.51 kcal/mol). In this study, we constructed a variant of CD80 protein with increased binding affinity to CTLA-4 and decreased binding affinity to CD28. This variant has no signaling capability, and can act as a cap for these receptors to protect them from natural CD80 proteins existing in the body. The first step was the evolutionary and alanine scanning analysis of CD80 protein to determine conserved regions in this protein. Next, complex alanine scanning technique was employed to determine CD80 protein hotspots in CD80-CTLA-4 and CD80-CD28 protein complexes. This information was fed into a computational model developed in R for in silico mutagenesis and CD80 variant library construction. The 3D structures of variants were modeled using the Swiss model webserver. After modeling the 3D structures, HADDOCK server was employed to build all protein-protein complexes, which contain CTLA-4-CD80 variant complexes, Wild type CD80-CD28 complexes and CD28-CD80 variant complexes. Protein-protein binding free energy was determined using FoldX and the variant number 316 with mutations at 29, 31, 33 positions showed increased binding affinity to CTLA-4 (-21.43 kcal/mol) and decreased binding affinity to CD28 (- 9.54 kcal/mol). Finally, molecular dynamics (MD) simulations confirmed the stability of variant 316. In conclusion, we designed a new CD80 protein variant with potential immunotherapeutic applications.

Therapeutic gene editing of T cells to correct CTLA-4 insufficiency.

Heterozygous mutations in CTLA-4 result in an inborn error of immunity with an autoimmune and frequently severe clinical phenotype. Autologous T cell gene therapy may offer a cure without the immunological complications of allogeneic hematopoietic stem cell transplantation. Here, we designed a homology-directed repair (HDR) gene editing strategy that inserts the CTLA-4 cDNA into the first intron of the CTLA-4 genomic locus in primary human T cells. This resulted in regulated expression of CTLA-4 in CD4+ T cells, and functional studies demonstrated CD80 and CD86 transendocytosis. Gene editing of T cells isolated from three patients with CTLA-4 insufficiency also restored CTLA-4 protein expression and rescued transendocytosis of CD80 and CD86 in vitro. Last, gene-corrected T cells from CTLA-4-/- mice engrafted and prevented lymphoproliferation in an in vivo murine model of CTLA-4 insufficiency. These results demonstrate the feasibility of a therapeutic approach using T cell gene therapy for CTLA-4 insufficiency.

Differences in CD80 and CD86 transendocytosis reveal CD86 as a key target for CTLA-4 immune regulation.

CD28 and CTLA-4 (CD152) play essential roles in regulating T cell immunity, balancing the activation and inhibition of T cell responses, respectively. Although both receptors share the same ligands, CD80 and CD86, the specific requirement for two distinct ligands remains obscure. In the present study, we demonstrate that, although CTLA-4 targets both CD80 and CD86 for destruction via transendocytosis, this process results in separate fates for CTLA-4 itself. In the presence of CD80, CTLA-4 remained ligand bound, and was ubiquitylated and trafficked via late endosomes and lysosomes. In contrast, in the presence of CD86, CTLA-4 detached in a pH-dependent manner and recycled back to the cell surface to permit further transendocytosis. Furthermore, we identified clinically relevant mutations that cause autoimmune disease, which selectively disrupted CD86 transendocytosis, by affecting either CTLA-4 recycling or CD86 binding. These observations provide a rationale for two distinct ligands and show that defects in CTLA-4-mediated transendocytosis of CD86 are associated with autoimmunity.

GPR174 signals via Gαs to control a CD86-containing gene expression program in B cells.

GPR174 is abundantly expressed in B and T lymphocytes and has a role in restraining T cell responses, but the function of GPR174 in B cells is less clear. Here we report that upon in vitro culture B cells undergo a spontaneous GPR174-dependent activation process that is associated with marked changes in gene expression, including up-regulation of Cd86, Nr4a1, Ccr7, and phosphodiesterases. B cells lacking Gαs show a block in induction of the GPR174-dependent program. Spontaneous up-regulation of CD86 in cultured B cells is dependent on protein kinase A. Both GPR174- and Gαs-deficient B cells show enhanced survival in culture. In vivo, GPR174 contributes to NUR77 expression in follicular B cells and is needed for establishing a marginal zone compartment of normal size. Treatment of mice with lysophosphatidylserine (lysoPS), a GPR174 ligand, is sufficient to promote CD86 up-regulation by follicular B cells. These findings demonstrate that GPR174 can signal via Gαs to modulate B cell gene expression and show this can occur in vivo in response to lysoPS. Additionally, the findings illuminate a pathway that might be targeted to improve systems for the in vitro study of B cell responses.

ß-Glucans from Trametes versicolor (L.) Lloyd Is Effective for Prevention of Influenza Virus Infection.

Coriolus versicolor (C. versicolor) is a higher fungi or mushroom which is now known by its accepted scientific names as Trametes versicolor (L.) Lloyd. Many studies have shown that ß-glucans from C. versicolor have various physiological activities, including activating macrophages to protect against Salmonella infection. However, whether ß-glucans have antiviral effects has not been reported. Hence, the objective of this study was to confirm whether ß-glucans could boost the immune response to combat influenza virus in mouse and chick models. The results show that ß-glucans induced the expression of Dectin-1, costimulatory molecules (CD80/86) and cytokines IL-6, IL-1ß, IFN-ß and IL-10 in murine bone marrow dendritic cells (BMDCs). In addition, orally administered ß-glucans reduced weight loss, mortality and viral titers in the lungs of mice infected with influenza virus and attenuated pathological lung damage caused by the virus in the mice. Orally administered ß-glucans improved survival and reduced lung viral titers in chickens infected with H9N2 avian influenza virus. These results suggest that ß-glucans have a significant antiviral effect. Therefore, ß-glucans could become a potential immunomodulator against influenza virus.

Gene-environment interactions increase the risk of pediatric-onset multiple sclerosis associated with ozone pollution.

BACKGROUND: We previously reported a relationship between air pollutants and increased risk of pediatric-onset multiple sclerosis (POMS). Ozone is an air pollutant that may play a role in multiple sclerosis (MS) pathoetiology. CD86 is the only non-HLA gene associated with POMS for which expression on antigen-presenting cells (APCs) is changed in response to ozone exposure. OBJECTIVES: To examine the association between county-level ozone and POMS, and the interactions between ozone pollution, CD86, and HLA-DRB1*15, the strongest genetic variant associated with POMS. METHODS: Cases and controls were enrolled in the Environmental and Genetic Risk Factors for Pediatric MS study of the US Network of Pediatric MS Centers. County-level-modeled ozone data were acquired from the CDC's Environmental Tracking Network. Participants were assigned ozone values based on county of residence. Values were categorized into tertiles based on healthy controls. The association between ozone tertiles and having MS was assessed by logistic regression. Interactions between tertiles of ozone level and the GG genotype of the rs928264 (G/A) single nucleotide polymorphism (SNP) within CD86, and the presence of DRB1*15:01 (DRB1*15) on odds of POMS were evaluated. Models were adjusted for age, sex, genetic ancestry, and mother's education. Additive interaction was estimated using relative excess risk due to interaction (RERI) and attributable proportions (APs) of disease were calculated. RESULTS: A total of 334 POMS cases and 565 controls contributed to the analyses. County-level ozone was associated with increased odds of POMS (odds ratio 2.47, 95% confidence interval (CI): 1.69-3.59 and 1.95, 95% CI: 1.32-2.88 for the upper two tertiles, respectively, compared with the lowest tertile). There was a significant additive interaction between high ozone tertiles and presence of DRB1*15, with a RERI of 2.21 (95% CI: 0.83-3.59) and an AP of 0.56 (95% CI: 0.33-0.79). Additive interaction between high ozone tertiles and the CD86 GG genotype was present, with a RERI of 1.60 (95% CI: 0.14-3.06) and an AP of 0.37 (95% CI: 0.001-0.75) compared to the lowest ozone tertile. AP results indicated that approximately half of the POMS risk in subjects can be attributed to the possible interaction between higher county-level ozone carrying either DRB1*15 or the CD86 GG genotype. CONCLUSIONS: In addition to the association between high county-level ozone and POMS, we report evidence for additive interactions between higher county-level ozone and DRB1*15 and the CD86 GG genotype. Identifying gene-environment interactions may provide mechanistic insight of biological processes at play in MS susceptibility. Our work suggests a possible role of APCs for county-level ozone-induced POMS risk.

Multilocus evaluation of genetic predictors of multiple sclerosis.

BACKGROUND: Genome-wide association studies identified numerous susceptibility loci for multiple sclerosis in populations of European ancestry, but the associations are not always reproducible in other populations due to admixture and different linkage disequilibrium patterns obscuring true association signals. OBJECTIVE: Our aim was to identify genetic predictors of multiple sclerosis in three ethnically homogenous populations from the Volga-Ural region of Russian Federation. METHODS: In the largest to date study of multiple sclerosis in Russian population, involving 2048 participants from the Republic of Bashkortostan, Russian Federation (641 patients with multiple sclerosis and 1407 unaffected individuals), we performed replication analysis of previously identified genome-wide signals for multiple sclerosis. Associations were tested using logistic regression analysis under additive genetic model adjusted for sex. Meta-analysis of the study results in three populations was performed under fixed effects and random effects models. RESULTS: We demonstrate the association with multiple sclerosis of the five variants (INAVA rs7522462, EOMES rs11129295, C6orf10 rs3129934, CD86 rs9282641, and GPR65 rs2119704). The strongest association (OR = 2.16, CI:1.85-2.74, P = 2.53x10-13) was detected for rs3129934 polymorphism in the major histocompatibility region. Multilocus analysis has revealed 322 and 27 allelic patterns associated with multiple sclerosis in women and men, respectively. In women, the highest risk of MS was conferred by C6orf10 rs3129934*T/T + STAT3 rs744166*T combination (OR = 11.87), in men - by C6orf10 rs3129934*T + EOMES rs11129295*C + RPS6KB1 rs180515*C combination (OR = 3.25). CONCLUSION: We confirm five associations with multiple sclerosis previously reported in genome-wide scans in Europeans in three ethnic groups from the Volga-Ural region of Russia.

Development of Exhausted Memory Monocytes and Underlying Mechanisms.

Pathogenic inflammation and immuno-suppression are cardinal features of exhausted monocytes increasingly recognized in septic patients and murine models of sepsis. However, underlying mechanisms responsible for the generation of exhausted monocytes have not been addressed. In this report, we examined the generation of exhausted primary murine monocytes through prolonged and repetitive challenges with high dose bacterial endotoxin lipopolysaccharide (LPS). We demonstrated that repetitive LPS challenges skew monocytes into the classically exhausted Ly6Chi population, and deplete the homeostatic non-classical Ly6Clo population, reminiscent of monocyte exhaustion in septic patients. scRNAseq analyses confirmed the expansion of Ly6Chi monocyte cluster, with elevation of pathogenic inflammatory genes previously observed in human septic patients. Furthermore, we identified CD38 as an inflammatory mediator of exhausted monocytes, associated with a drastic depletion of cellular NAD+; elevation of ROS; and compromise of mitochondria respiration, representative of septic monocytes. Mechanistically, we revealed that STAT1 is robustly elevated and sustained in LPS-exhausted monocytes, dependent upon the TRAM adaptor of the TLR4 pathway. TRAM deficient monocytes are largely resistant to LPS-mediated exhaustion, and retain the non-classical homeostatic features. Together, our current study addresses an important yet less-examined area of monocyte exhaustion, by providing phenotypic and mechanistic insights regarding the generation of exhausted monocytes.

Micro/nano topography of selective laser melting titanium inhibits osteoclastogenesis via mediation of macrophage polarization.

Selective laser melting (SLM) titanium (Ti) implants have shown good prospects for personalized clinical application, but further research is necessary to develop stabilized long-term properties. Since surface modification has been proven bioactive for osseointegration, conventional Ti surface treatment technologies, including sandblasting/acid-etching (SLA) and sandblasting/alkali-heating (SAH), were applied to construct micro and micro/nano surfaces. The SAH group with netlike nano-structure topography exhibited appropriate surface roughness and high hydrophilicity, and as expected, the osseointegration capacities in vivo of the three groups were in order of SAH > SLA > SLM. Besides, both in vivo and in vitro studies revealed that the SLA- and SAH-treated SLM Ti implants significantly inhibited osteoclast activity of peri-implants. Considering the close associations between osteoclasts and macrophages, the effects of Ti surface topography on macrophage polarization were detected. The results showed that the SLA- and SAH-treated SLM Ti implants, especially the latter, had the capacity to promote macrophage polarization to the M2 phenotype. Moreover, the cell culture supernatants of M2 macrophages and RAW264.7 cells seeded on SLA- and SAH-treated SLM Ti surfaces had an adverse effect on osteoclastogenesis. Collectively, this study demonstrated that micro/nano topographies of SLM Ti implants were effective for osseointegration promotion, and their inhibition of osteoclastogenesis might be attributed to macrophage polarization. Our findings shed some light on clinical application of SLM Ti implants and also prove a specific association between macrophage polarization and osteoclastogenesis.

Characterization and immunomodulatory activity of sulfated galactan from the red seaweed Gracilaria fisheri.

Polysaccharides from the red seaweed Gracilaria fisheri possess many functions, which include antioxidant, antiviral, and antibacterial activities. However, detailed data on their immunomodulatory activities are scarce. Here, we isolated sulfated galactans (SG) from G. fisheri. We found that the predominant SG from G. fisheri, termed SG-1, had an estimated molecular mass of 100 kDa and activated murine J774A.1 macrophages via the dectin-1 signaling pathway. Furthermore, we observed enhancement of nitric oxide (NO) secretion, increased expression of inducible nitric oxide synthase (iNOS) mRNA, and increased mRNA levels of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukins IL-1ß and IL-6 by SG-1 in macrophages. Moreover, there was higher expression of intercellular adhesion molecule 1 (ICAM-1) and co-stimulatory molecules (B7-1 and B7-2) mRNA. Treatment with G. fisheri SG-1 at 50 µg/mL generally achieved or exceeded the pro-inflammatory activities of 100 ng/mL lipopolysaccharide. Our study demonstrates immune-stimulatory activities of G. fisheri SG that may be of value for immune-potentiating treatment in humans or livestock.

The myeloid cell biomarker EMR1 is ectopically expressed in colon cancer.

OBJECTIVE: The microenvironment of colon cancer (CC) is heterogeneous including cells of myeloid lineage affecting tumor growth and metastasis. Two functional subtypes of myeloid cells have been identified; one (M1) is tumor-inhibitory and the other one (M2) is tumor-promoting. Whether the three myeloid markers EMR1, CD206 and CD86 are expressed only in the infiltrating myeloid cells or also in the tumor cells was investigated. METHODS: Expression of the myeloid markers was investigated in CC at the mRNA and protein levels in primary tumors and lymph nodes. mRNA expression was also determined in 5 CC cell lines. Protein expression was investigated by two-color immunofluorescence and consecutive-sections-immune-staining combined with morphometry using specific antibodies for the myeloid cell markers and the epithelial cell markers CEACAM5 and EpCAM. RESULTS: EMR1 and CD86, but not CD206, mRNA levels were significantly higher in CC primary tumors compared to apparently normal colon tissue (P <  0.0001). EMR1 mRNA levels were significantly higher in both hematoxylin-eosin positive (H&E(+)) and H&E(-) lymph nodes of CC patients compared to control nodes (P = 0.03 and P = 0.01, respectively). EMR1 and CD206 mRNAs were expressed in 4/5 and 5/5 CC cell lines, respectively, while CD86 mRNA was not expressed. Immuno-morphometry revealed that about 20% of the tumor cells expressed EMR1 and CD206. Positive cells were tumor cells as revealed by anti-CEACAM5 and anti-EpCAM staining. The number of EMR1, CD206 and CD86 positive cells were significantly increased in CC primary tumors compared to normal colon tissue (P <  0.0001). However, CD206 was also expressed in normal colonocytes. Only EMR1 showed significantly increased numbers of positive tumor cells in H&E(+) nodes compared to H&E(-) nodes (P = 0.001). EMR1 expression in CC tumor cells correlated with CXCL17 expressing tumor cells. CONCLUSION: EMR1, like the chemokine CXCL17, is ectopically expressed in colon cancer possibly in the same cancer cells.

Cutting Edge: A Threshold of B Cell Costimulatory Signals Is Required for Spontaneous Germinal Center Formation in Autoimmunity.

Cognate interactions between autoreactive B and T cells promote systemic lupus erythematosus pathogenesis by inter alia facilitating spontaneous germinal center (GC) formation. Whereas both myeloid and B cell APCs express B7 ligands (CD80 and CD86), the prevailing model holds that dendritic cell costimulation is sufficient for CD28-dependent T cell activation. In this study, we report that B cell-intrinsic CD80/CD86 deletion unexpectedly abrogates GCs in murine lupus. Interestingly, absent GCs differentially impacted serum autoantibodies. In keeping with distinct extrafollicular and GC activation pathways driving lupus autoantibodies, lack of GCs correlated with loss of RNA-associated autoantibodies but preserved anti-dsDNA and connective tissue autoantibody titers. Strikingly, even heterozygous B cell CD80/CD86 deletion was sufficient to prevent autoimmune GCs and RNA-associated autoantibodies. Together, these findings identify a key mechanism whereby B cells promote lupus pathogenesis by providing a threshold of costimulatory signals required for autoreactive T cell activation.

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.

Association of Polymorphisms in T-Cell Activation Costimulatory/Inhibitory Signal Genes With Allograft Kidney Rejection Risk.

The genes CD28, CD86 and CTLA-4 conform the costimulatory (CD28-CD86) or inhibitory (CTLA-4-CD86) signal in T-cell activation. T-cell immune response has a critical role in allograft rejection, and single nucleotide polymorphisms (SNPs) located in these genes have been widely analyzed with controversial results. We analyzed a group of SNPs located in the three genes: CD28: rs3116496; CD86: rs1129055; and CTLA-4: rs231775 and rs3087243 in a cohort of 632 consecutively recruited kidney transplanted subjects. All polymorphisms were genotyped by TaqMan chemistry and the diagnosis of rejection was confirmed by biopsy and categorized according to the Banff classification. The analyses showed a statistically significant protective effect to T cell-mediated rejection (TCMR) in carriers of the CTLA-4 rs3087243*G allele, especially in patients with TCMR Banff ≥2 in the overall cohort and in patients without thymoglobulin induction therapy. Both associations were corroborated as independent factors in the multivariate analysis. Interestingly, associations with rejection were not found for any SNP in patients with thymoglobulin induction therapy. As expected, considering the major role of these genes in T-cell activation, no effect was observed for antibody-mediated rejection (ABMR). In conclusion, the SNP rs3087243 located in the CTLA-4 gene may be considered a useful independent biomarker for TCMR risk especially for severe TCMR in patients who did no received thymoglobulin induction therapy.

Human and viral membrane-associated E3 ubiquitin ligases MARCH1 and MIR2 recognize different features of CD86 to downregulate surface expression.

Immune-stimulatory ligands, such as major histocompatibility complex molecules and the T-cell costimulatory ligand CD86, are central to productive immunity. Endogenous mammalian membrane-associated RING-CHs (MARCH) act on these and other targets to regulate antigen presentation and activation of adaptive immunity, whereas virus-encoded homologs target the same molecules to evade immune responses. Substrate specificity is encoded in or near the membrane-embedded domains of MARCHs and the proteins they regulate, but the exact sequences that distinguish substrates from nonsubstrates are poorly understood. Here, we examined the requirements for recognition of the costimulatory ligand CD86 by two different MARCH-family proteins, human MARCH1 and Kaposi's sarcoma herpesvirus modulator of immune recognition 2 (MIR2), using deep mutational scanning. We identified a highly specific recognition surface in the hydrophobic core of the CD86 transmembrane (TM) domain (TMD) that is required for recognition by MARCH1 and prominently features a proline at position 254. In contrast, MIR2 requires no specific sequences in the CD86 TMD but relies primarily on an aspartic acid at position 244 in the CD86 extracellular juxtamembrane region. Surprisingly, MIR2 recognized CD86 with a TMD composed entirely of valine, whereas many different single amino acid substitutions in the context of the native TM sequence conferred MIR2 resistance. These results show that the human and viral proteins evolved completely different recognition modes for the same substrate. That some TM sequences are incompatible with MIR2 activity, even when no specific recognition motif is required, suggests a more complicated mechanism of immune modulation via CD86 than was previously appreciated.

Susceptibility of cyclin-dependent kinase inhibitor 1-deficient mice to rheumatoid arthritis arising from interleukin-1ß-induced inflammation.

We recently reported that cyclin-dependent kinase inhibitor 1 (p21) deficiency induces osteoarthritis susceptibility. Here, we determined the mechanism underlying the effect of p21 in synovial and cartilage tissues in RA. The knee joints of p21-knockout (p21-/-) (n = 16) and wild type C57BL/6 (p21+/+) mice (n = 16) served as in vivo models of collagen antibody-induced arthritis (CAIA). Arthritis severity was evaluated by immunological and histological analyses. The response of p21 small-interfering RNA (siRNA)-treated human RA FLSs (n = 5 per group) to interleukin (IL)-1ß stimulation was determined in vitro. Arthritis scores were higher in p21-/- mice than in p21+/+ mice. More severe synovitis, earlier loss of Safranin-O staining, and cartilage destruction were observed in p21-/- mice compared to p21+/+ mice. p21-/- mice expressed higher levels of IL-1ß, TNF-α, F4/80, CD86, p-IKKα/ß, and matrix metalloproteinases (MMPs) in cartilage and synovial tissues via IL-1ß-induced NF-kB signaling. IL-1ß stimulation significantly increased IL-6, IL-8, and MMP expression, and enhanced IKKα/ß and IκBα phosphorylation in human FLSs. p21-deficient CAIA mice are susceptible to RA phenotype alterations, including joint cartilage destruction and severe synovitis. Therefore, p21 may have a regulatory role in inflammatory cytokine production including IL-1ß, IL-6, and TNF-α.