Characterization of innate and adaptive immune responses of two rare primary immune deficiencies: CTPS1 and CD55

Abstract

Primary Immune deficiencies (PIDs) are disorders of immune system caused by mutated genes. There are approximately 350 different disorders and each day novel ones are being defined. They can be categorized based on part of immune system harboring mutation; that is, they can be divided into disorders of innate and adaptive immune system. Each of them represents itself distinctly. In that perspective, studies based on characterization of PIDs enable us to comprehend how immune system works. Herein, we characterized innate and adaptive immune responses of two rare immune deficiencies: CTPS1 and CD55 which are novel examples of disorders of adaptive and innate immune system, respectively. CTPS1 is an enzyme functioning in de novo synthesis of nucleotide, CTP. Defective CTPS1 enzyme impairs lymphocytes to proliferate, however, other aspects of this deficiency still remain elusive. Since patients are prone to viral infections, we first explored functionality of cytotoxic T-cells through assessing STAT1 phosphorylation levels and expression of activation markers. Even though flow cytometry analyses revealed that CTPS1 deficient CD8+ T-cells had normal phospho-STAT1 levels, degranulation marker confined to surface of CD8+ Tcells were found to be elevated. Next, we investigated CD4+ T-cells with cytokines that are crucial for differentiation and fate. We detected that patient CD4+ T-cells had low phospho-STAT3 and phospho-STAT5 levels. Then, we checked the cytokine production profiles of CD4+ T-cells. Data indicated that percentages of IL-17a and IL 10 secreting cells are reduced in patient whereas Th1 and Th2 signatures were similar to healthy controls. Moreover, IFN-a levels of PBMCs upon TLR3, TLR7 and TLR9 ligand stimulations were found to be similar to healthy responses. Notably, patient had slightly reduced TLR7 and IFI16-STING mediated type II IFN secretion. We further showed that CTPS1 PBMCs had normal IL-12 levels, implying that the reduction in IFN-g was not due to either dysfunction of innate immune cells or by aberrant APC function. Surprisingly, patient PBMCs had higher number of granulocytes and flow cytometry analyses revealed that these granulocytes were CD14- CD15+ low-density granulocytes. This prompted us to assess the NETotic tendencies of CTPS1 neutrophils and we observed via microscopic and spectrofluorometric investigations that they underwent spontaneous NETosis. In the second part of this study, we worked with CD55 deficient patient PBMCs. CD55 is a complement regulatory protein and it inhibits formation of C3-convertase in classical and alternative complement pathways. Thus, patients suffer from aberrant complement activation in its absence as well as severe bowel inflammation and recurrent infections along with nutrient loss leading to malnutrition and growth deprivation. Eculizumab therapy was initiated to these patients in order to neutralize their pathologic C5 levels. We attempted to investigate effect of CD55 deficiency on PRR-complement cross-talk, recurrent infections and checked the contribution of Eculizumab therapy to their immune status. PBMCs of 4 patients, i) before (BT) and ii) after (AT) a single dose of Eculizumab administration were isolated. BT PBMCs had significantly reduced IFN-a and IP-10 secretions upon endosomal (TLR3, TLR7 and TLR9) TLRs and nucleic acid sensors (STING, DAI, RIG-I & MAVS) stimulations. Moreover, single Eculizumab therapy did not alter this innate immune dysfunction. Furthermore, we assessed levels of TNF-a, IL-6 productions from PBMCs stimulated with same ligands and observed that IL-6 but not TNF-a was reduced after PRR stimulations. Next, immunomodulatory effects of CD55 EVs before and after Eculizumab therapy was sought. ELISA results demonstrated that AT EV incubation on CD55-/- PBMCs lead to reduced TNF-a, IL-1b and IFN-g production. Meanwhile, AT EVs increased IL-10 production from patient PBMCs. Lastly, we assessed cytokine levels after healthy PBMCs were incubated with BT and AT EVs and found that PBMCs that incubated with BT EVs had elevated levels of IP-10 cytokine. When taken together, progression of PIDs might have been contributed by extracellular vesicles.