A single-cell atlas characterizes dysregulation of the bone marrow immune microenvironment associated with outcomes in multiple myeloma.

Multiple Myeloma (MM) remains incurable despite advances in treatment options. Although tumor subtypes and specific DNA abnormalities are linked to worse prognosis, the impact of immune dysfunction on disease emergence and/or treatment sensitivity remains unclear. We established a harmonized consortium to generate an Immune Atlas of MM aimed at informing disease etiology, risk stratification, and potential therapeutic strategies. We generated a transcriptome profile of 1,149,344 single cells from the bone marrow of 263 newly diagnosed patients enrolled in the CoMMpass study and characterized immune and hematopoietic cell populations. Associating cell abundances and gene expression with disease progression revealed the presence of a proinflammatory immune senescence-associated secretory phenotype in rapidly progressing patients. Furthermore, signaling analyses suggested active intercellular communication involving APRIL-BCMA, potentially promoting tumor growth and survival. Finally, we demonstrate that integrating immune cell levels with genetic information can significantly improve patient stratification.

Th17-inducing autologous dendritic cell vaccination promotes antigen-specific cellular and humoral immunity in ovarian cancer patients.

In ovarian cancer (OC), IL-17-producing T cells (Th17s) predict improved survival, whereas regulatory T cells predict poorer survival. We previously developed a vaccine whereby patient-derived dendritic cells (DCs) are programmed to induce Th17 responses to the OC antigen folate receptor alpha (FRα). Here we report the results of a single-arm open-label phase I clinical trial designed to determine vaccine safety and tolerability (primary outcomes) and recurrence-free survival (secondary outcome). Immunogenicity is also evaluated. Recruitment is complete with a total of 19 Stage IIIC-IV OC patients in first remission after conventional therapy. DCs are generated using our Th17-inducing protocol and are pulsed with HLA class II epitopes from FRα. Mature antigen-loaded DCs are injected intradermally. All patients have completed study-related interventions. No grade 3 or higher adverse events are seen. Vaccination results in the development of Th1, Th17, and antibody responses to FRα in the majority of patients. Th1 and antibody responses are associated with prolonged recurrence-free survival. Antibody-dependent cell-mediated cytotoxic activity against FRα is also associated with prolonged RFS. Of 18 patients evaluable for efficacy, 39% (7/18) remain recurrence-free at the time of data censoring, with a median follow-up of 49.2 months. Thus, vaccination with Th17-inducing FRα-loaded DCs is safe, induces antigen-specific immunity, and is associated with prolonged remission.

Longitudinal relationships between rheumatoid factor and cytokine expression by immunostimulated peripheral blood lymphocytes from patients with rheumatoid arthritis: New insights into B-cell activation.

To identify associations between immunostimulated cytokine production and disease characteristics, peripheral blood lymphocytes were collected from 155 adult patients with rheumatoid arthritis (RA) before and after a 5-year interval. The lymphocytes were activated in vitro with T-cell stimulants, cytosine-phosphate-guanine (CpG) oligonucleotide, and medium alone (negative control). Expression of 17 cytokines was evaluated with immunoassays, and factor analysis was used to reduce data complexity and identify cytokine combinations indicative of cell types preferentially activated by each immunostimulant. The findings showed that the highest numbers of correlations were between cytokine levels and rheumatoid factor (RF) positivity and between cytokine levels and disease duration. Scores for cytokines driven by CpG and medium alone were negatively associated with RF positivity and disease duration at baseline but positively associated with both at 5 years. Our findings suggest that RF expression sustained over time increases activation of B cells and monocytes without requirements for T-cell functions.

Interferon Chemokine Score and Other Cytokine Measures Track With Changes in Disease Activity in Patients With Juvenile and Adult Dermatomyositis.

OBJECTIVE: Our aim was to identify cytokines and chemokines in patients with adult dermatomyositis (DM) and juvenile dermatomyositis (JDM) that predict changes in disease activity. METHODS: Multiplexed immunoassays (Meso Scale Discovery) enabled simultaneous measurement of interferon (IFN)-regulated chemokines and other pro- and anti-inflammatory cytokines specific to differentiation of specific T-cell and innate pathways. Cytokine scores were computed for IFNCK (IP-10, MCP-1), Th1 (IFNÉ£, TNFα, and IL2), Th2 (IL4, IL10, IL12, and IL 13), Th17 (IL6, IL17, IL1ß), macrophage (MIP-1α, MIP-1ß, IL8), and regulatory (IL10, TNFα) factors. Spearman correlation and mixed models were used to examine whether cytokines at a previous visit predict change in disease activity at the next visit. RESULTS: The study included 36 patients (16 DM and 20 JDM) with at least two visits (87 patient intervals between two visits). Mean age (SD) at inclusion was 56.9 (18.4) years for DM and 10.8 (6.6) years in JDM, 67% of patients were female, 89% Caucasian. The mean (SD) physician global, muscle and extra-muscular disease activity Visual Analog Scale scores at inclusion were 41 (26), 36 (30), and 34 (21) mm, respectively. The change in IFN score from one visit to the next was associated with the change in physician global (P = 0.010) and extramuscular (P < 0.001) disease activity scores. Preliminary results revealed significant correlations of previous IFNCK score and IL-6 with subsequent disease activity measures, but after adjustment for multiple visits per patient, these associations did not reach statistical significance. CONCLUSION: There is a potential relationship between IFNCK and other cytokine scores seen in adult and juvenile DM with future disease states.

Gene Expression Profiling in Blood and Affected Muscle Tissues Reveals Differential Activation Pathways in Patients with New-onset Juvenile and Adult Dermatomyositis.

OBJECTIVE: To identify shared and differential molecular pathways in blood and affected muscle between adult dermatomyositis (DM) and juvenile DM, and their association with clinical disease activity measures. METHODS: Gene expression of transcription factors and cytokines involved in differentiation and effector function of T cell subsets, regulatory T cells and follicular Th cells, were analyzed in the blood from 21 newly diagnosed adult and 26 juvenile DM subjects and in 15 muscle specimens (7 adult and 8 juvenile DM) using a custom RT2 Profiler PCR Array. Disease activity was determined and measured by established disease activity tools. RESULTS: The most prominent finding was the higher blood expression of Th17-related cytokines [retinoic acid-related orphan receptor-γ, interferon regulatory factor 4, interleukin (IL)-23A, IL-6, IL-17F, and IL-21] in juvenile DM at baseline. In contrast, adult patients with DM showed increased blood levels of STAT3 and BCL6 compared with juvenile DM. In muscle, GATA3, IL-13, and STAT5B were found at higher levels in juvenile patients with DM compared with adult DM. Among 25 patients (11 adult and 14 juvenile DM) who had blood samples at baseline and at 6 months, increased expression of IL-1ß, STAT3, STAT6, STAT5B, and BCL6 was associated with an improvement in global extramuscular disease activity. CONCLUSION: We observed differences in gene expression profiling in blood and muscle between new-onset adult and juvenile DM. Cytokine expression in the blood of juvenile patients with new-onset DM was dominated by Th17-related cytokines compared with adult patients with DM. This may reflect the activation of different Th pathways between muscle and blood.

Interferon-regulated chemokine score associated with improvement in disease activity in refractory myositis patients treated with rituximab.

OBJECTIVES: The purpose of this study was to investigate whether serum interferon (IFN)-regulated chemokine and distinct cytokine response profiles are associated with clinical improvement in patients with refractory inflammatory myopathy treated with rituximab. METHODS: In a randomised, placebo-phase trial Rituximab in Myositis Trial (RIM), 200 refractory adult and paediatric myositis subjects received rituximab. Following rituximab, clinical response and disease activity were assessed. Serum samples and clinical data were collected at baseline and several time-points after rituximab treatment. Multiplexed sandwich immunoassays quantified serum levels of IFN-regulated chemokines and other pro-inflammatory cytokines. Composite IFN-regulated chemokine and Th1, Th2, Th17 and regulatory cytokine scores were computed. RESULTS: Baseline IFN-regulated chemokine, Th1, Th2, Th17 and regulatory cytokine scores correlated with baseline physician global VAS, whereas the baseline Th1, Th2 and Th17 cytokine scores correlated with baseline muscle VAS. We also found baseline IFN-regulated chemokine scores correlated with specific non-muscular targets such as baseline cutaneous (r=0.29; p=0.002) and pulmonary (r=0.18; p=0.02) VAS scores. Among all cytokine/chemokines examined, the baseline score of IFN-regulated chemokines demonstrated the best correlation with changes in muscle VAS at 8 (r=-0.19; p=0.01) and 16 weeks (r=-0.17; p=0.03) following rituximab and physician global VAS at 16 weeks (r=-0.16; p=0.04). In vitro experiments showed increased levels of IL-8 (p=0.04), MCP-1 (p=0.04), IL-6 (p=0.03), IL-1ß (p=0.04), IL-13 (p=0.04), IL-10 (p=0.02), IL-2 (p=0.04) and IFN-γ (p=0.02) in supernatants of TLR-3 stimulated PBMCs from non-responder compared to patients responders to rituximab. CONCLUSIONS: IFN-regulated chemokines before treatment is associated with improvement in disease activity measures in refractory myositis patients treated with rituximab.

Short communication: CD4 T cell declines occurring during suppressive antiretroviral therapy reflect continued production of Casp8p41.

Most patients on suppressive antiretroviral therapy (ART) experience improvements in CD4 T cell count. However, some patients with undetectable viral load continue to lose CD4 T cells for unknown reasons. Casp8p41 is a host-derived protein fragment that is present only in productively infected cells and that causes the death of HIV-infected cells. We questioned whether ongoing CD4(+) T cell losses while on suppressive ART were associated with subclinical HIV replication causing production of Casp8p41. We analyzed the association of Casp8p41 content with subsequent CD4 losses in patients on continuous suppressive ART and in patients who discontinued ART after Casp8p41 content was determined, adjusting for age, baseline CD4(+) T cell count, and baseline HIV RNA level. Casp8p41 expression in memory CD4(+) T cells was measured by intracellular flow cytometry and was correlated with viral load and CD4(+) T cell change over time. In patients who stopped therapy after Casp8p41 content was determined, baseline Casp8p41 content did not predict CD4(+) T cell change. However, in patients on continuous ART, higher baseline Casp8p41 content was associated with a greater odds of a CD4(+) T cell decline at 6 months (p=0.01). Therefore, patients on suppressive ART, who have ongoing production of Casp8p41, have an increased risk of CD4 T cell losses, suggesting that subclinical HIV replication is driving both Casp8p41, which in turn causes a CD4(+) T cell decline.

Immune response profiling in early rheumatoid arthritis: discovery of a novel interaction of treatment response with viral immunity.

INTRODUCTION: It remains challenging to predict the outcomes of therapy in patients with rheumatoid arthritis (RA). The objective of this study was to identify immune response signatures that correlate with clinical treatment outcomes in patients with RA. METHODS: A cohort of 71 consecutive patients with early RA starting treatment with disease-modifying antirheumatic drugs (DMARDs) was recruited. Disease activity at baseline and after 21 to 24 weeks of follow-up was measured using the Disease Activity Score in 28 joints (DAS28). Immune response profiling was performed by analyzing multi-cytokine production from peripheral blood cells following incubation with a panel of stimuli, including a mixture of human cytomegalovirus (CMV) and Epstein-Barr virus (EBV) lysates. Profiles identified via principal components analysis (PCA) for each stimulus were then correlated with the ΔDAS28 from baseline to follow-up. A clinically meaningful improvement in the DAS28 was defined as a decrease of ≥1.2. RESULTS: A profile of T-cell cytokines (IL-13, IL-4, IL-5, IL-2, IL-12, and IFN-γ) produced in response to CMV/EBV was found to correlate with the ΔDAS28 from baseline to follow-up. At baseline, a higher magnitude of the CMV/EBV immune response profile predicted inadequate DAS28 improvement (mean PCA-1 scores: 65.6 versus 50.2; P = 0.029). The baseline CMV/EBV response was particularly driven by IFN-γ (P = 0.039) and IL-4 (P = 0.027). Among patients who attained clinically meaningful DAS28 improvement, the CMV/EBV PCA-1 score increased from baseline to follow-up (mean +11.6, SD 25.5), whereas among patients who responded inadequately to DMARD therapy, the CMV/EBV PCA-1 score decreased (mean -12.8, SD 25.4; P = 0.002). Irrespective of the ΔDAS28, methotrexate use was associated with up-regulation of the CMV/EBV response. The CMV/EBV profile was associated with positive CMV IgG (P <0.001), but not EBV IgG (P = 0.32), suggesting this response was related to CMV exposure. CONCLUSIONS: A profile of T-cell immunity associated with CMV exposure influences the clinical response to DMARD therapy in patients with early RA. Because CMV latency is associated with greater joint destruction, our findings suggest that changes in T-cell immunity mediated by viral persistence may affect treatment response and possibly long-term outcomes of RA.

A profile of immune response to herpesvirus is associated with radiographic joint damage in rheumatoid arthritis.

INTRODUCTION: Progression of joint damage despite appropriate therapy remains a significant problem for patients with rheumatoid arthritis (RA). This study was undertaken to identify profiles of immune response that correlate with radiographic joint damage as a first step toward the discovery of new pathogenic mechanisms of joint destruction in RA. METHODS: The study included 58 patients with RA and 15 healthy controls. The profiles of cytokine release from peripheral blood mononuclear cells (PBMC) in response to stimulation for 48 hours with one of six stimuli, or in media alone, were measured. Immune response profiles identified for each stimulus were correlated with radiographic joint damage as defined by the Sharp-van der Heijde score (SHS), before and after multivariable adjustment. For profiles correlated with the SHS, the distributions of individual cytokines were evaluated in patients according to the severity of joint damage and compared to healthy controls. RESULTS: The immune response profile for cytomegalovirus (CMV)/Epstein-Barr virus (EBV) stimulation was correlated with both the SHS total and erosion scores (r = 0.31, P = 0.018 and r = 0.33, P = 0.011, respectively). After adjusting for age, sex, disease duration, autoantibody status, CMV/EBV serological status, current disease activity, disability and treatments, the correlation of the CMV/EBV immune response and the SHS erosion score became stronger (r = 0.43, P < 0.003). The CMV/EBV immune response correlated with CMV IgG (r = 0.44, P < 0.001), but not with EBV IgG. The most important cytokines for the CMV/EBV immune response profile were IFN-γ, IL-2, IL-4, IL-5, IL-13 and IL-17A, all of which are associated with T-cell immunity. Both the summary immune response score and the individual responses of IFN-γ and IL-13 to CMV/EBV stimulation were associated with greater joint damage. CONCLUSIONS: A profile of immune response to purified CMV/EBV lysates is associated with radiographic joint damage. The correlation of this immune response to CMV serology implies possible involvement of latent CMV infection. Therefore, the findings suggest that the immune response to latent CMV infection could play a fundamental role in the progression of inflammation and structural joint damage in patients with RA.

Assessing immune function by profiling cytokine release from stimulated blood leukocytes and the risk of infection in rheumatoid arthritis.

Persons with rheumatoid arthritis (RA) suffer a high burden of infections, but currently no biomarkers are available to identify individuals at greatest risk. A prospective longitudinal study was therefore conducted to determine the association between the responsiveness of ex vivo cytokine production and 6-month risk of infections. Infections were identified by billing codes and validated by medical record review. At baseline, the release of 17 cytokines by peripheral blood mononuclear cells in response to stimulation, or media alone, was measured using multiplexed cytokine analysis. Production of IL-2, IL-8, IL-10, IL-17, TNF-α, IFN-γ, and GM-CSF, induced by various conditions, was significantly associated with the occurrence of infections. A multivariable prediction model based on these data provided new information on the risk of infection beyond standard assessments of disease activity, severity, and treatment. Future studies could utilize this information to devise new biomarkers for the prediction of infection in patients with RA.

A signature of aberrant immune responsiveness identifies myocardial dysfunction in rheumatoid arthritis.

OBJECTIVE: Heart failure is an important cause of death in patients with rheumatoid arthritis (RA). Evidence suggests that immune mechanisms contribute to myocardial injury and fibrosis, leading to left ventricular diastolic dysfunction (LVDD). The purpose of this study was to identify a signature of LVDD in patients with RA by analyzing the responsiveness of the innate and adaptive immune systems to stimulation ex vivo. METHODS: RA patients (n=212) enrolled prospectively in a population-based cohort underwent echocardiography, and LV function was classified as normal, mild LVDD, or moderate-to-severe LVDD. The release of 17 cytokines by blood mononuclear cells in response to stimulation with a panel of 7 stimuli or in media alone was analyzed using multiplex immunoassays. Logistic regression models were used to test for associations between a multicytokine immune response score and LVDD, after adjusting for clinical covariates. RESULTS: An 11-cytokine profile effectively differentiated patients with moderate-to-severe LVDD from those with normal LV function. An immune response score (range 0-100) was strongly associated with moderate-to-severe LVDD (odds ratio per 10 units 1.5 [95% confidence interval 1.2-2.1]) after adjusting for serum interleukin-6 levels, brain natriuretic peptide values, and glucocorticoid use, as well as other RA characteristics and LVDD risk factors. CONCLUSION: The major finding of this study was that aberrant systemic immune responsiveness is associated with advanced myocardial dysfunction in patients with RA. The unique information added by the immune response score concerning the likelihood of LVDD warrants future longitudinal studies of its value in predicting future deterioration in myocardial function.

Analysis of complex biomarkers for human immune-mediated disorders based on cytokine responsiveness of peripheral blood cells.

The advent of improved biomarkers promises to enhance the clinical care for patients with rheumatoid arthritis (RA) and other immune-mediated disorders. We have developed an innovative approach to broadly assess the cytokine responsiveness of human PBMCs using a multistimulant panel and multiplexed immunoassays. The objective of this study was to demonstrate this concept by determining whether cytokine profiles could discriminate RA patients according to disease stage (early versus late) or severity. A 10-cytokine profile, consisting of IL-12, CCL4, TNF-alpha, IL-4, and IL-10 release in response to stimulation with anti-CD3/anti-CD28, CXCL8 and IL-6 in response to CMV and EBV lysate, and IL-17A, GM-CSF, and CCL2 in response to human heat shock protein 60, easily discriminated the early RA group from controls. These data were used to create an immune response score, which performed well in distinguishing the early RA patients from controls and also correlated with several markers of disease severity among the patients with late RA. In contrast, the same 10-cytokine profile assessed in serum was far less effective in discriminating the groups. Thus, our approach lays the foundation for the development of immunologic "signatures" that could be useful in predicting disease course and monitoring the outcomes of therapy among patients with immune-mediated diseases.

Nelfinavir monotherapy increases naïve T-cell numbers in HIV-negative healthy young adults.

Although patients treated with HIV protease inhibitor (PI) containing regimens manifest increases in naïve T cell number, it is unclear whether this is due to reduction in viral replication or a direct drug effect. We questioned whether Nelfinavir monotherapy directly impacted naïve T-cell number in HIV-negative individuals. HIV-negative volunteers received Nelfinavir, 1250 mg orally, BID for 3 weeks, and T-cell receptor recombination excision circles (TREC) content in peripheral blood were assessed. Whereas TREC copies did not change over 3 weeks in untreated controls, TREC copies/copies CCR5 increased following Nelfinavir monotherapy in 8 patients (p < 0.02), and did not change in 7 patients (p = NS). Those patients who responded were younger than those who did not with a median age of 55 years for responders and 71 years for non-responders (p < 0.03). The increase in TREC was most pronounced in those patients less than 40-years old (p < 0.01). Moreover, the patients who did not increase TREC levels were more likely to have suffered a medical illness previously shown to reduce thymic function. In HIV-negative patients, monotherapy with the HIV PI Nelfinavir for 21 days increases TREC-positive naïve T cell number, particularly in individuals who are healthy and young.

Mitosis increases levels of secretory leukocyte protease inhibitor in keratinocytes.

Chronic wounds are a major health care burden. Multiple factors produced by healing wounds play important roles in efficient and orderly wound healing. Secretory leukocyte protease inhibitor (SLPI) is constitutively expressed in epithelial cells, and its expression is increased by inflammation. SLPI has antimicrobial activities and improves wound healing. We hypothesized that SLPI expression correlates with keratinocyte growth rate and is increased by epidermal growth factor (EGF). Keratinocytes were isolated from neonatal foreskin. Subconfluent and confluent culture conditions were used. SLPI-specific primers were designed for use in quantitative-competitive reverse-transcription polymerase chain reaction assays to detect and quantify SLPI mRNA expression levels. SLPI levels were increased 2-fold in subconfluent cultures and 1.6-fold in confluent cultures to which EGF had been added. Confluent cultures also demonstrated 7-fold (growth factor depleted) and 6-fold (EGF) increase in SLPI RNA copy number, compared to subconfluent cultures. The results indicate that EGF increases SLPI expression.