Browsing by Subject "Primary immune deficiency"

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Open Access
Characterization of altered innate and adaptive immune responses of primary immune deficient patients
(2021-05) İbibik, Bilgehan
The immune system dysregulations led to primary immune deficiencies (PIDs). Immune deficiencies can be divided into two groups, primary and secondary immune deficiencies. The reasons for primary immune deficiencies could be inherited immune dysfunctions originated from autosomal recessive or autosomal dominant mutations. Combinations of ongoing infections, lymphoproliferation, atopies, malignancies, autoimmunity and granulomatous processes are seen in primary immune deficiency disorders. To establish distinctive therapies, characterization of immune deficiencies along with the understanding the course of impaired mechanisms are very critical to offer robust means of cure. In this study, the effects of different mutations (RLTPR, RLTPR-TLR1, and CTLA-4) on innate and adaptive immune systems were investigated. RLTPR (CARMIL2) is a cytosolic scaffold protein which facilitates CD28 co-stimulation for T-cell activation. RLTPR facilitates recruitment of CARMA1, a cytosolic adaptor, along with the BCL10 and MALT1 which form CBM complex to CD28 site to activate NF-κB signaling pathway. Besides facilitating CD28 co-stimulation, RLTPR takes place in cell shape control, phagocytosis and endocytosis movements by promoting actin polymerization. Reduced CD4+ T-cells, memory B-cells and antibody response along with the impaired B-cell receptor mediated NF-κB activation were mainly observed in RLTPR deficient patients. Moreover, polarity and migration of the T-cells were affected by RLTPR deficiency in patients. Herein, immunological phenotypes of RLTPR deficient patient with and without CMV infection were described. As expected, Treg counts of the patient were reduced as compared to healthy controls both with and without infection. Although Treg cell counts of patient were decreased, IL-10 secretion upon cytosolic nucleic acid ligands was increased in infection due to ongoing defense and homeostasis. Under CMV infection, patient showed elevated IL-1β and TNF-α responses upon activation of TLRs and cytosolic nucleic acid sensors. However, without any viral infections, TLR7 and TLR8 mediated IL-1β and TNF-α responses were impaired in patient. Moreover, under CMV infection, TLR and cytosolic nucleic acid sensors mediated antiviral IFN-α, IFN-γ and IL-12 responses were substantially increased compared to healthy subjects. Unexpectedly, when patient PBMCs were assessed following infection, we detect that IFN-α and IFN-γ levels of the patient in response to endosomal TLRs and cytosolic nucleic acids stimulations were reduced. Throughout the course of viral infection due to ongoing defense by innate immune cells, one would predict to detect higher type I and II IFN responses. When infection was cleared with medical treatment, it is observed that not only endosomal TLRs but also the cytosolic nucleic acid sensors were impaired in RLTPR patient which makes the patient vulnerable to viral infections. Thereafter, altered immune responses of RLTPR-TLR1 deficient patient was investigated. Decreased number of Treg cells from whole blood of the patient was confirmed. IL-1β response from PBMCs of patient was elevated by stimulation with the TLR and cytosolic nucleic acid ligands. Especially, TLR2-6 response was elevated which could be the result of TLR1 deficiency because immune response could be compensated when TLR1 is deficient but TLR6 is not. Also, remaining improved proinflammatory cytokine response to different PRR ligands could be reasoned by ongoing infection. IFN-α secretion was increased by endosomal TLR and cytosolic nucleic acid ligands while IL-12 secretion of patient showed ligand specific modulated responses which suggest an ongoing infection. As expected, there was no detectable TLR1 mediated IL-12 response due to TLR1 deficiency. However, TLR2-6 mediated IL-12 secretion was elevated in patient compared to healthy subjects which could be regarded as a compensatory response against TLR1 deficiency. RLTPR-TLR1 deficient patient PBMCs elicited elevated IL-10 response to TLR and cytosolic nucleic acid ligand triggering even though Treg cells were reduced., implying that other suppressor cells could be involved in this response. CTLA-4, a negative regulator of T-cells, is expressed on activated T-cells and Treg cells. CTLA-4 binds to CD80/86 molecules on APCs which prevents CD28 co-stimulation. Without CD28 co-stimulation, the T-cell cannot be activated. Patients with insufficient CTLA-4 receptors could have an increased number of Treg cells with reduced function. CTLA-4 haploinsufficiency leads to hyperactive T-cells, enhanced autoreactive B-cells and reduced numbers of circulating B-cells. In this work, immune responses of four CTLA-4 patients were also studied. Patient #1 showed higher CD4+/CD8+ ratio, elevated LDG frequency, as well as reduced TCR expression on CD3+ cells and elevated pAKT protein levels. Patient #2 had increased LDG count, reduced pDC and Treg population in whole blood. Similar blood cell profile to Patient #2, Patient #3 additionally had increased monocyte percentage and lower CD4+/CD8+ ratio which could indicate an ongoing infection. Patient #4 showed decreased pDC, Treg counts, increased levels of PD-L1 on CD8+ cells, Treg cells and B-cells, reduced TCR expression on CD3+ T-cells, increased pAKT and p4EBP1 levels which all may contribute to compensate the autoimmune status of the CTLA-4 mutation. B-cell percentages and CTLA-4 expression levels of all patients were not altered while mTOR, pmTOR, STAT3, pSTAT3, AKT, p4EBP1 and HIf-1α expression levels were impaired in all patients. Collectively, our findings imply that the complexity of the dysregulation of these deficiencies, and point-out to an unappreciated immune functional status of these patients. We propose that investigation of the innate immune arm of these individuals which were perceived as solely related to and impacting only adaptive immune system is necessary to offer more robust therapies to these patients.
Open Access
Investigation of immune impairment status in primary immune deficiency and spinal cord injury
(2021-02) Evcili, İrem
Primary Immunodeficiencies (PIDs) are raised by immune impairment causing disorders. In the patients carrying deficiency in any part of immune system, various defects and symptoms are observed. Generally, abnormality against microbial agents are seen in patients and different therapies are applied depending on defect. That’s why it is crucial to characterize how immune system works in PIDs for better understating of disease and treatment options. In this study, we investigated two different mutations (CD27 and STAT1 GOF) and their effects on innate and adaptive arm of immune system. A trans-membrane receptor, CD27, is a one of the leukocyte differentiation markers for T, B and NK cells. It is involved in the activation of these cells by binding to the co-stimulatory molecule CD70 and mediates cell survival. In CD27 deficiency, loss of T cell dependent B cell responses and antibody production along with T cell dysfunction are mainly seen. Herein, immunological phenotypes of two patients with genetic CD27 deficiency were described. CD27 expression of the PBMCs, CD3+ T and CD19+ B cells obtained from the patients were absent in patients. Frequency of CD19+/CD24hi/CD38hi transitional B cells were higher compared to healthy controls. Reversed CD4+/CD8+ T cell ratio was observed in one patient. After IFN-β stimulation, the phosphorylation level of STAT1 in CD4+ T cells was increased in CD27-/- patients as compared to healthy group. There was no difference in phospho-STAT3 levels between CD27-/- and healthy donor CD4+ T cells upon IL-6 stimulation. However, decreased STAT5 phosphorylation levels were observed upon IL-2 stimulation of CD4+ T cells of CD27-/-. When PBMCs from healthy and CD27 deficient patients were stimulated with PMA/Ionomycin and analyzed for cytokine secretions, increased IFN-, IL-4 and IL-17a secretion were investigated. Through activation of different TLR and inflammasome pathways, normal IFN- and IL-1β responses were observed while increased TNF-α and IL-10 was present in patients. Results suggest that patients’ both humoral and cellular immune responses could be impaired in CD27 deficiency. STAT1 is a member of the STAT protein family whose mutations can cause gain-of-function (GOF) or loss of function (LOF). Recurrent widespread infections are common in both mutations. In this study, the responses of innate and adaptive immune systems of 6 patients from different hospitals known to have STAT1 GOF were examined. To determine the STAT1 phosphorylation and dephosphorylation levels, cells were treated with IFN-β and CD4+ T cell phosphorylation levels were analyzed by flow cytometry. Th1 and Th17 responses were examined since imbalanced Th1 and Th17 response is known in STAT1 GOF patients. To identify defects in innate immunity, cytokine ELISA was performed through different PRR ligands. For neutrophil activities, cells were examined as untreated and treated form by microscopically and fluorometrically. As a result, it was found that p-STAT1 levels and p-STAT1 positive CD4+ T cells of all patients were higher than healthy controls which approves STAT1 GOF mutation in patients. Although the STAT1 phosphorylation level was high, it was determined that IFN- released due to Th1 response was low in patients. Th17 response were low in patients which is supported by current infection of Chronic Mucocutaneous Candidiasis. Type II interferon responses of the patients to the ligands of the endosomal and inflammatory pathways were lower than healthy controls while normal IL-12 secretion was observed. Some patients had increased NETotic activities at the basal levels supported by spontaneous NET formation, while some patients were not able to induce NETosis through PMA stimulation. Impaired immune status was observed in STAT1 GOF patients as well. Spinal cord injury (SCI) is damage occurring in the spinal cord and nerves within the spinal canal that causes completely or partially loss of sensation around injury site. Since uncontrolled immune response occurs at the post-traumatic injury site in SCI patients and this situation increases the damage caused by the lesion, it is important to examine the immune system responses in these patients. In this study, we tried to determine how innate and adaptive immune system responses of SCI patients with and without infection differ. Percentages of T, B, NK cells and pDCs were determined from whole blood. Reversed CD4/CD8 ratio was observed in two patients. Increased Treg and pDCs cell percentage was seen in patients while B and NK cell percentages are different from patient to patient. Released IFN- , IL-17 and IL-10 levels were determined by flow cytometry upon PMA/Ionomycin. Normal Th17, increased Th1 and Treg was investigated in SCI patients. NETotic activities were examined microscopically and fluorometrically and via ROS production. Although NETotic activities of neutrophils of all patients were similar in healthy subjects, fluorometric results showed that some patients had more NETotic activities compared to healthy controls. It was also found that patients produced lower amounts of ROS as a result of incubation with PMA. These results suggest that SCI patients become more susceptible to infection due to the low response to the pathogen despite increased neutrophil activity and the immunosuppressive effect of the amount of IL-10 caused by excess Treg activity.