© 2019 Cedars-Sinai

Type 1 Interferon Gene Signatures in RBC Alloimmunized and Non-Alloimmunized Patients with Sickle Cell Disease

Background:RBC transfusion can lead to alloimmunization against RBC antigens. Patients with sickle cell disease (SCD) have the highest frequency of alloimmunization. However, factors that promote alloimmunization in SCD are poorly understood. Inflammation in the recipient promotes alloimmunization in mice and humans; and type 1 interferons (IFNα/β) and Interferon Stimulated Genes (ISGs) promote alloimmunization in mice. We hypothesized that an IFNα/β gene signature may contribute to the increased frequency of alloimmunization in SCD..

Objectives

To determine if there is an increased expression of Type 1 Interferon and activation of related pathways in SCD.

To determine to what degree does it correlate with RBC alloimmunization.

MethodsUsing blood from previously transfused SCD (SS genotype) patients with (n=9) or without alloimmunization (n=7) and race-matched healthy controls (ββ, n=5), we measured the expression of ISGs, including CD38 and Siglec-1, and calculated IFNα/β gene scores from expression of 7 ISGs as previously described. Whole blood or Peripheral Blood Mononuclear Cells (PBMCs) were stimulated with or without IFNβ and ISGs were measured by ELISA, flow cytometry and qPCR. ISGs and IFNα/β related cytokines were measured in plasma. Expression of ISGs and IFNα/β gene scores in alloimmunized patients was compared to expression in patients without alloantibodies and healthy controls. Statistical significance was determined by a Mann-Whitney U test or a Kruskal-Wallis test with a Dunn’s post-test.

Results Cont.

There were no significant differences in the age of SCD patients and controls, and no difference in transfusion burden between alloimmunized and non-alloimmunized patients. IFNα/β gene scores of SCD blood leukocytes and plasma IFNα/β-related cytokines (IL-10, IP-10, IL-6) were elevated, compared to controls (IFNα/β score SCD= 145±44, ββ= 0±2.65, p<0.01; IP-10 SCD= 148±13, ββ= 46±15, p<0.001). Upon stimulation with IFNβ, isolated PBMCs, but not whole blood, from SCD patients had elevated IFNα/β gene scores, compared to controls (Stimulated PBMC IFNα/β score SS= 16±4.0, ββ= 0±2.4, p< 0.05). Trends of increased IL-10, TNFα, and IL-8 were noted in plasma from alloimmunized patients with SCD, though IFNβ-stimulated and unstimulated ISG expression did not significantly differ between alloimmunized and non-alloimmunized patients.

Conclusions

Patients with SCD have increased IFNα/β gene scores and related cytokines, compared to controls. Differing results in PBMC and whole blood IFNβ stimulation experiments indicate that serum cytokines of SCD blood may regulate IFNβ responses. In this small sample, there are trends of an increase in ISGs and IFNα/β related cytokines in patients with alloimmunization compared to those without alloimmunization. Differing time points of antibody formation and sample collection may contribute to the lack of statistical significance. Larger studies are needed to determine the role of the SCD IFNα/β gene signature in RBC alloimmunization.

AcknowledgementsThe Cedars-Sinai Graduate Program

National Blood Foundation (R13672)

American Society of Hematology (5283)

NIH/NHLBI (5 K08 HL141446)

Emaan Madany, PhD, June Lee, MS, Chelsea Halprin DO, Chelsea Hayes, M.D., Sam Pepkowitz, M.D., Ellen Klapper M.D., Jeanne Hendrickson, M.D., and David Gibb, M.D., PhDDepartment of Pathology & Laboratory MedicineCedars Sinai Medical Center, Los Angeles

Results

Figure 1. Whole Blood ELISA Showed an Increase in MxA Levels for SS Patients Compared to Healthy Controls. Whole blood Immunoassay for MxA was performed on samples from SS patients (Hb SS) or healthy controls (AA, Hb ββ). p=0.05 by Mann-Whitney U-test. (B) MxA levels in whole blood of AA healthy controls, alloimmunized SS and non-alloimmunized SS patients.

Figure 6. Elevated plasma cytokine levels in alloimmunized SS patients compared to controls. Plasma cytokine levels were measured by a multiplex bead assay. (A) Cytokine levels of AA controls and SS patients. (B) Cytokine levels of AA controls, alloimmunized and non-alloimmunized SS patients. *p<0.05, **p<0.01.

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Figure 2. Interferon-stimulated genes in blood leukocytes of SS patients compared to AA controls. (A) Relative expression of interferon-stimulated genes (MX1, Ly6E, IFIT3, IFI44, IFI44L, ISG15, and IFI27) by qPCR using RNA from whole blood of SS patients and AA controls. (B) Equation used to calculate IFNα/β gene scores. (C) IFNα/β gene scores calculated from expression of ISGs in (A). (D) IFNα/β gene scores of AA controls, alloimmunized, and non-alloimmunized SS patients. *p<0.05, ** p<0.01.

Figure 3. IFNβ induces interferon-stimulated genes in PBMCs of patients with SCD. (A) Relative expression of interferon-stimulated genes (MX1, Ly6E, IFIT3, IFI44, IFI44L, IFI27, and ISG15) by qPCR. PBMCs from SS patients and AA controls were stimulated with 2ng/mL IFNβ. (B) IFNα/β gene score calculated from expression of ISGs in (A). (C) IFNα/β gene score of AA controls, alloimmunized, and non-alloimmunized SS patients. * p<0.05, **p<0.01.

Figure 4. Unstimulated monocytes of patients with SCD show a trend towards an increase in CD86 a marker of monocyte activation Peripheral blood leukocytes from AA controls and patients with SCD (SS) without IFNβ. (A) Representative histograms and (B) quantification of CD86 in AA controls and SS patients (C). quantification of CD86 in AA controls and alloimmunized and non-alloimmunized SS patients.

Figure 5. Reduced IFNβ-induced expression of ISGs by monocytes from whole blood of patients with SCD. Peripheral blood leukocytes from AA controls and patients with SCD (SS) were stimulated in whole blood with or without IFNβ. (A) Representative flow cytometric analysis of CD14+CD64+ monocytes, gated on HLADR+ non-lymphocytes. Numbers on plots indicate the percent of cells within the drawn gate. (B) Representative histograms and (C) quantification of Siglec-1 and CD38 expression by unstimulated and IFNβ-stimulated CD14+CD64+ monocytes gated in (A). **p<0.01, ***p<0.001.