A 62-year-old man attended a community hospital on April 16, 2012, due to a series of respiration symptoms such as cough and asthma with sputum. Chest X-rays showed a 2.8 cm anterior mediastinal mass and signs of pneumonia (Figures 1(a) and 1(b)). Afterwards, TBNA (transbronchial needle aspiration) failed to collect target tissues after two attempts. So he went to the First Affiliated Hospital of Nanjing Medical University for clear-cutting diagnosis on May 10, 2012. PET/CT scan was subsequently taken, and the SUV value of the lesion was as depicted by 2.4 (Figure 1(c)), which almost excluded the possibility of tumor. Considering his dissipating symptoms of pneumonia, the patient adopted the outpatient follow-up treatment for 7 months until November 27, 2012. In the meantime, the lesions showed no significant regression in computed tomography (CT) imaging during routine follow-ups. On December 4, 2012, the patient was admitted to the Department of Thoracic Surgery for lump resection surgery. The lesion was later pathologically diagnosed with thymoma, B2 type. On February 25, 2013, the patient was readmitted for recurrent respiratory infection. A series of detailed clinical analyses was further carried out, and the results were as follows: fasting blood glucose 7 mmol/l (3.9-6.1 mmol/L), HA1c 7.2% (4.0%-6.4%), positive autoantibodies of glutamic acid decarboxylase (GAD) and insulin autoantibody (IAA), globulin 19.7 g/L (20.0-40.0 g/L), and serum IgG 3.2 g/L (7.0-16.0 g/L), IgM 0.17 g/L (0.4-2.3 g/L), and IgA 0.37 g/L (0.7-4.0 g/L). Complement component 3 (C3) and complement component 4 (C4) results were within reference ranges. The results of routine blood test and blood biochemistry analysis were normal. Serum virus copy number detection including CMV, EBV, HIV, and HBV was negative. But his peripheral lymphocyte subsets were abnormal with low CD4+ T cells (16.2%; 30%-40%) and extremely low B cells (0.1%; 9%-14%). He also had slightly decreased muscle tension which improved after surgery. Moreover, this GS patient had mild diarrhea after thymoma resection, which improved without additional treatment. The combination of disrupted peripheral lymphocytes and immunoglobulin and a history of high blood sugar, as well as the diagnosis of thymoma, lead to the patient final diagnosis of GS. Immunoglobulin replacement was immediately and regularly applied to enhance postsurgery immunity supportive therapy until now. Between February 2012 and March 2017, the patient was readmitted several times to our hospital for recurrent respiratory infections due to high susceptibility to gram-negative bacilli or fungi of Candida albicans (July 7, 2013; August 19, 2013; May 14, 2015; and March 25, 2016). This GS patient often suffered from cough with sputum, fever of 39 C, and chest pain as well as signs of pneumonia identified via chest CT images. Doctors usually utilized combination therapy including antibiotics (levofloxacin tablets/imipenem/cefodizime) and antifungal (fluconazole) drug to prevent Candida albicans, as well as ambroxol for cough with sputum, which eliminated respiratory inflammation and prevented double infection (bacterial-fungal infections). Moreover, the routine monitoring of immunoglobulin, blood sugar, peripheral lymphocyte subsets, and immunoglobulin infusion is still ongoing every 3 months in our GS patient.
A peripheral blood sample was collected on November 22, 2016 during a routine outpatient examination of a GS patient 4 years after thymectomy. At the time of sample collection, the patient did not show any obvious signs of respiratory infection, but he had a history of recurrent respiratory infections. The proportions of his peripheral immune cells were evaluated by flow cytometry. As shown in Figure 2, this GS patient had an elevated proportion of CD8+ T cells (55.6%) and a reduced proportion of CD4+ T cells (12.1%) compared with the corresponding levels in healthy control subjects (HCs); furthermore, B cells (1%) were nearly absent in this patient, and he also had an inverted ratio of CD4+/CD8+ cells. gammadeltaT cells play a crucial role in preventing bacterial and fungal infection and act as the first protective barrier of innate immunity. These cells are split into two subgroups based on the classification of the delta ligand: Vdelta1 T cells, which are almost located in the epithelial tissue, and Vdelta2 T cells, which account for 70% of the total gammadeltaT cells in the peripheral blood. Although the relative proportion of gammadeltaT cells in our GS patient was similar to that in HCs, the percentage of Vdelta2 T cells was particularly low (12.5% versus 66.45 +- 20.79%) in our GS patient. Accordingly, the percentage of Vdelta1 T cells in this patient was higher than that in the HCs (43.9% versus 19.12 +- 17.20%) (Figure 2(c)). This different distribution of gammadeltaT cell subsets may be related to the patient's recurrent respiratory infections. It is possible that the altered distribution of peripheral immune cells partly caused an inefficient peripheral immune response.
Tregs modulate immune homeostasis that induced immune suppression effect in the peripheral blood. We further analyzed the proportions of five subtypes of Tregs with GS patient via flow cytometry. As shown in Figure 3, the percentages of CD4+CD25+Foxp3+ Treg cells (5.54% versus 1.46 +- 0.71%) and CD4+CD25+CTLA-4+ Treg cells (3.18% versus 0.66%) were higher in our GS patient compared with HCs. Surprisingly, the percentage of CD4+CD39+ Treg cells was 21.8% in our GS patient, which was much higher than that in the HCs (8.034 +- 1.868%). Recent studies indicated that CD8+ Treg cells are increased and associated with tumor stages in human ovarian cancer. As shown in Figure 4, the proportions of CD8+CD28- Treg cells and CD8+Foxp3+ Treg cells in our GS patient were 88.8% (HCs 38.66 +- 6.93%) and 2.3% (HCs 0.71 +- 0.29%), respectively. Additionally, the CD28 expression in our GS patient's immune cells was significantly lower than that in HCs. Thus, the excessive populations of various Treg cells in our GS patient's peripheral blood highlight the severe immune suppression status of this GS patient.
Cell proliferation capacity is an essential index for assessing the presence of an effective immune response. We investigated the ex vivo proliferation of circuiting CD4+ T cells from our GS patient via flow cytometry. The result shows that CD4+ T cells from our GS patient underwent less proliferation than those from HCs (34.0% versus 80.8%) following stimulation by CD3/CD28; additionally, the cells from the GS patients had lower proliferation rates and fewer divisions (Figure 5). It has been widely reported that immune checkpoints, including programmed death receptor-1 (PD-1) and programmed death ligand-1 (PD-L1), participate in the progression of immune suppression, which also partly correlates with T cell activation and proliferation as well as cytokine secretion. In this study, our GS patient's peripheral CD4+ T cells expressed markedly higher levels of PD-1 (34.2% versus 4.2 +- 1.8%) but lower levels of PD-L1 (6.61% versus 10.1 +- 3.9%) than those reported for GS patients (Figure 6); this difference was similar to that reported by previous studies. These results suggest that both poor proliferation and increased PD-1 expression of circulating CD4+ T cells, along with a higher level of CTLA-4 expression, may be at least partly associated with the delayed immune reactions to infection in our GS patient.
We detected the cellular IFN-gamma levels produced by ex vivo CD4+ T cells, CD8+ T cells, and gammadeltaT cells from our GS patient, and cells from 10 healthy volunteers were included as controls (Figure 7). The amount of IFN-gamma produced by CD4+ T cells was similar between our GS patient and the HCs (32.5% versus 31.08 +- 12.50%). In contrast, the amount of IFN-gamma secreted by CD8+ T cells was higher in our GS patient than in the HCs (71.1% versus 44.15 +- 12.49%), and the opposite trend was observed in gammadeltaT cells (15.1% versus 33.98 +- 12.26%). Additionally, we also found that the circuiting CD4+ T cells from our GS patient secreted slightly more IL-17A compared with those from the HCs (4.3% versus 2.62 +- 1.38%) (Figure 8). However, the levels of cellular IL-17A produced by CD8+ T cells and gammadeltaT cells were not significantly different between our GS patient and the HCs (1.20% versus 1.37 +- 0.91%; 2.70% versus 1.42 +- 0.41%, resp.). These results illustrate the abnormality of cellular cytokine levels in our GS patient, which may be linked to the recurrent respiratory infections suffered by this patient.