Utilizing principal component analysis in the identification of clinically relevant changes in patient HLA single antigen bead solid phase testing patterns.

BACKGROUND: HLA antibody testing is essential for successful solid-organ allocation, patient monitoring post-transplant, and risk assessment for both solid-organ and hematopoietic transplant patients. Luminex solid-phase testing is the most common method for identifying HLA antibody specificities, making it one of the most complex immunoassays as each panel contains over 90 specificities for both HLA class I and HLA class II with most of the analysis being performed manually in the vendor-provided software. Principal component analysis (PCA), used in machine learning, is a feature extraction method often utilized to assess data with many variables. METHODS & FINDINGS: In our study, solid organ transplant patients who exhibited HLA donor-specific antibodies (DSAs) were used to characterize the utility of PCA-derived analysis when compared to a control group of post-transplant and pre-transplant patients. ROC analysis was utilized to determine a potential threshold for the PCA-derived analysis that would indicate a significant change in a patient's single antigen bead pattern. To evaluate if the algorithm could identify differences in patterns on HLA class I and HLA class II single antigen bead results using the optimized threshold, HLA antibody test results were analyzed using PCA-derived analysis and compared to the clinical results for each patient sample. The PCA-derived algorithm had a sensitivity of 100% (95% CI, 73.54%-100%), a specificity of 75% (95% CI, 56.30%-92.54%), with a PPV of 65% (95% CI, 52.50%-83.90%) and an NPV of 100%, in identifying new reactivity that differed from the patients historic HLA antibody pattern. Additionally, PCA-derived analysis was utilized to assess the potential over-reactivity of single antigen beads for both HLA class I and HLA class II antibody panels. This assessment of antibody results identified several beads in both the HLA class I and HLA class II antibody panel which exhibit over reactivity from 2018 to the present time. CONCLUSIONS: PCA-derived analysis would be ideal to help automatically identify patient samples that have an HLA antibody pattern of reactivity consistent with their history and those which exhibit changes in their antibody patterns which could include donor-specific antibodies, de novo HLA antibodies, and assay interference. A similar method could also be applied to evaluate the over-reactivity of beads in the HLA solid phase assays which would be beneficial for lot comparisons and instructive for transplant centers to better understand which beads are more prone to exhibiting over-reactivity and impact patient care.

Next-generation sequencing and clinical histocompatibility testing.

Histocompatibility testing is essential for donor identification and risk assessment in solid organ and hematopoietic stem cell transplant. Additionally, it is useful for identifying donor specific alleles for monitoring donor specific antibodies in post-transplant patients. Next-generation sequence (NGS) based human leukocyte antigen (HLA) typing has improved many aspects of histocompatibility testing in hematopoietic stem cell and solid organ transplant. HLA disease association testing and research has also benefited from the advent of NGS technologies. In this review we discuss the current impact and future applications of NGS typing on clinical histocompatibility testing for transplant and non-transplant purposes.

Suppressor of cytokine signaling-1 mimetic peptides attenuate lymphocyte activation in the MRL/lpr mouse autoimmune model.

Autoimmune diseases are driven largely by a pathogenic cytokine milieu produced by aberrantly activated lymphocytes. Many cytokines, including interferon gamma (IFN-γ), utilize the JAK/STAT pathway for signal propagation. Suppressor of Cytokine Signaling-1 (SOCS1) is an inducible, intracellular protein that regulates IFN-γ signaling by dampening JAK/STAT signaling. Using Fas deficient, MRL/MpJ-Faslpr/J (MRL/lpr) mice, which develop lupus-like disease spontaneously, we tested the hypothesis that a peptide mimic of the SOCS1 kinase inhibitory region (SOCS1-KIR) would inhibit lymphocyte activation and modulate lupus-associated pathologies. Consistent with in vitro studies, SOCS1-KIR intraperitoneal administration reduced the frequency, activation, and cytokine production of memory CD8+ and CD4+ T lymphocytes within the peripheral blood, spleen, and lymph nodes. In addition, SOCS1-KIR administration reduced lymphadenopathy, severity of skin lesions, autoantibody production, and modestly reduced kidney pathology. On a cellular level, peritoneal SOCS1-KIR administration enhanced Foxp3 expression in total splenic and follicular regulatory T cells, reduced the effector memory/naïve T lymphocyte ratio for both CD4+ and CD8+ cells, and reduced the frequency of GL7+ germinal center enriched B cells. Together, these data show that SOCS1-KIR treatment reduced auto-reactive lymphocyte effector functions and suggest that therapeutic targeting of the SOCS1 pathway through peptide administration may have efficacy in mitigating autoimmune pathologies.

T cells expressing the lupus susceptibility allele Pbx1d enhance autoimmunity and atherosclerosis in dyslipidemic mice.

Patients with systemic lupus erythematosus (SLE) present a high incidence of atherosclerosis, which contributes significantly to morbidity and mortality in this autoimmune disease. An impaired balance between regulatory (Treg) and follicular helper (Tfh) CD4+ T cells is shared by both diseases. However, whether there are common mechanisms of CD4+ T cell dysregulation between SLE and atherosclerosis remains unclear. Pre-B cell leukemia transcription factor 1 isoform d (Pbx1d) is a lupus susceptibility gene that regulates Tfh cell expansion and Treg cell homeostasis. Here, we investigated the role of T cells overexpressing Pbx1d in low-density lipoprotein receptor-deficient (Ldlr-/-) mice fed with a high-fat diet, an experimental model for atherosclerosis. Pbx1d-transgenic T cells exacerbated some phenotypes of atherosclerosis, which were associated with higher autoantibody production, increased Tfh cell frequency, and impaired Treg cell regulation, in Ldlr-/- mice as compared with control T cells. In addition, we showed that dyslipidemia and Pbx1d-transgenic expression independently impaired the differentiation and function of Treg cells in vitro, suggesting a gene/environment additive effect. Thus, our results suggest that the combination of Pbx1d expression in T cells and dyslipidemia exacerbates both atherosclerosis and autoimmunity, at least in part through a dysregulation of Treg cell homeostasis.

Influence of Biological Therapeutics, Cytokines, and Disease Activity on Depression in Rheumatoid Arthritis.

PURPOSE: Rheumatoid arthritis (RA) is an often debilitating autoinflammatory disease. Patients with rheumatoid arthritis are often troubled by co-occurring depression or other psychological manifestations. RA patients have a variety of treatment options available, including biologicals that inhibit cytokines or immune cells. If these cytokines influence the psychological symptoms, then the use of cytokine inhibitors should modulate these symptoms. METHODS: A cohort of 209 individuals was recruited. This group included 82 RA patients, 22 healthy subjects, 32 depressed control subjects, and 73 subjects with systemic lupus erythematosus. Of the RA patients, 51% were on a biological therapeutic. ELISA was used to measure cytokine levels. A variety of psychological assessments were used to evaluate depression, anxiety, sleep, fatigue, and relationship status. Clinical values were obtained from medical records. RESULTS: IL-10 concentration was associated with depressive symptoms in the RA patients, healthy controls, and the lupus patients. In the patients with primary depression, depressive symptoms were associated with IL-6 and TNF-alpha. In RA patients, Tocilizumab use was associated with decreased depressive symptoms. 14 RA patients who were not using biologicals began using them by a one-month follow-up. In these patients, there was no significant change to any value except for fatigue. CONCLUSIONS: A variety of both biological and social factors influences depressive symptoms in RA. IL-10 and IL-6 are likely to be involved, since IL-10 concentration was associated with depression and Tocilizumab decreased depressive symptoms in the RA patients. The roles of these cytokines are different in RA and lupus, as high IL-10 in RA is associated with increased depressive symptoms, but high IL-10 in the lupus patients is associated with decreased depression. IL-6 was also associated with depressive symptoms in the patients with primary depression. These results strongly indicate that disease activity, including cytokine levels, has a strong impact on depressive symptoms.

EBI2 expression in B lymphocytes is controlled by the Epstein-Barr virus transcription factor, BRRF1 (Na), during viral infection.

Epstein-Barr virus-induced gene 2 (EBI2) is an important chemotactic receptor that is involved in proper B-cell T-cell interactions. Epstein-Barr virus (EBV) has been shown to upregulate this gene upon infection of cell lines, but the timing and mechanism of this upregulation, as well as its importance to EBV infection, remain unknown. This work investigated EBV's manipulation of EBI2 expression of primary naive B cells. EBV infection induces EBI2 expression resulting in elevated levels of EBI2 after 24 h until 7 days post-infection, followed by a dramatic decline (P=0.027). Increased EBI2 expression was not found in non-specifically stimulated B cells or when irradiated virus was used. The EBV lytic gene BRRF1 exhibited a similar expression pattern to EBI2 (R2=0.4622). BRRF1-deficient EBV could not induce EBI2. However, B cells transduced with BRRF1 showed elevated expression of EBI2 (P=0.042), a result that was not seen with transduction of a different EBV lytic transfection factor, BRLF1. Based on these results, we conclude that EBI2 expression is directly influenced by EBV infection and that BRRF1 is necessary and sufficient for EBI2 upregulation during infection.

Piracy on the molecular level: human herpesviruses manipulate cellular chemotaxis.

Cellular chemotaxis is important to tissue homeostasis and proper development. Human herpesvirus species influence cellular chemotaxis by regulating cellular chemokines and chemokine receptors. Herpesviruses also express various viral chemokines and chemokine receptors during infection. These changes to chemokine concentrations and receptor availability assist in the pathogenesis of herpesviruses and contribute to a variety of diseases and malignancies. By interfering with the positioning of host cells during herpesvirus infection, viral spread is assisted, latency can be established and the immune system is prevented from eradicating viral infection.