Circulating T lymphocyte subsets, cytokines, and immune checkpoint inhibitors in patients with bipolar II or major depression: a preliminary study

This study aimed to investigate the less known activation pattern of T lymphocyte populations and immune checkpoint inhibitors on immunocytes in patients with bipolar II disorder depression (BD) or major depression (MD). A total of 23 patients with BD, 22 patients with MD, and 20 healthy controls (HCs) were recruited. The blood cell count of T lymphocyte subsets and the plasma level of cytokines (IL-2, IL-4, IL-6, IL-10, TNF-α, and IFN-γ) were selectively investigated. The expression of T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), programmed cell death protein 1 (PD-1) and its ligands, PD-L1 and PD-L2, on T lymphocytes and monocytes, was detected. In results, blood proportion of cytotoxic T cells significantly decreased in BD patients than in either MD patients or HCs. The plasma level of IL-6 increased in patients with BD and MD. The expression of TIM-3 on cytotoxic T cells significantly increased, whereas the expression of PD-L2 on monocytes significantly decreased in patients with BD than in HCs. These findings extended our knowledge of the immune dysfunction in patients with affective disorders.

cell death protein 1 (PD-1) also functions as an inhibitory immune checkpoint and negatively regulates immune response 15,16 . Moreover, TIM-3 and PD-1 both may weaken CD8+ T-cell function and induce T-cell exhaustion 17 . PD-L1 and PD-L2, two ligands of PD-1, and their interaction with PD-1 are correlated with T cell activation and tolerance 18,19 . However, no literature has reported the possible role of these immune checkpoints in the inflammatory challenge in patients with affective disorders hitherto, and their relationship with known blood level change of cytokines.
This preliminary study investigated the plasma levels of cytokines, proportion of T lymphocytes, and the expression of immune checkpoint inhibitors, including TIM-3, PD-1, PD-L1 and PD-L2, on T lymphocytes and monocytes, in patients with affective disorders.

Results
Demographic and clinical characteristics. As shown in Table 1, no significant difference was found in age, gender, marriage, education year, and handedness (P > 0.05). However, the age of onset was significantly smaller in patients with BD patients than in patients with MD (22.43 ± 4.50 vs. 29.95 ± 7.79, P < 0.001). No significant difference was found in the total course of illness, number of episodes, and total scores of HDRS-17, MADRS and YMRS between these two groups.

Activation pattern of T lymphocytes and NK cells.
The total blood proportion of CD3+ , CD4+ , and CD8 + T-cell subsets and CD16 + NK cells was analyzed. No significant difference was found in the percentages of CD3+ and CD4 + T cells and CD16 + NK cells in total lymphocyte populations ( Table 2). Of note, the proportion of cytotoxic T cells (CD3 + CD8+ ) was significantly lower in BD patients than in MD patients and HCs (P = 0.004 and, P = 0.002, respectively) ( Fig. 1).

Expression of immune checkpoint inhibitors on T lymphocytes and monocytes.
In terms of the expression of TIM-3 and PD-1 expression on T-cell subsets, no significant alterations were indicated in the total expression of TIM-3 and PD-1 on T cells (CD3+ ) or helper T cells (CD3 + CD4+ ). However, the expression of TIM-3 on cytotoxic T cells (CD3 + CD8+ ) significantly increased ( Fig. 2 and Table 3), but no significant change was revealed in the expression of PD-1. As for CD14+ monocytes, although no significant changes were revealed in the expression of TIM-3, PD-1, and PD-L1 in the three groups, the expression of PD-L2 significantly reduced in patients with BD compared with HCs (24.90 ± 10.84 vs. 36.74 ± 14.29, P = 0.029, Fig. 3 and Table 3 Table 2. Lymphocytes subsets presented as percentages of total lymphocytes gated (mean ± SD). * P < 0.05 (two-tailed). Abbreviations: Th, helper T cell; Tc, T cytotoxic cell; NK, Natural Killer cell. P 1 : BD and HC; P 2 : MD and HC; P 3 : BD and MD; P 4 : BD, MD and HC.
participants were lower than the set limit of detection, and no significant difference was found among the three groups.

Discussion
The present study showed a decreased level of cytotoxic T cells (CD3+ CD8+ ) in patients with BD, accompanied by an elevated concentration of plasma IL-6. Besides, upregulated expression of TIM3 on cytotoxic T cells and downregulated expression of PD-L2 on monocytes were observed in patients with BD, but not in patients with MD. These findings support and extend the understanding of the inflammatory burden in patients with affective disorders, indicating the involvement of immune checkpoint inhibitors in the pathogenesis of BD. In this respect, this work was of importance in extending the research focus from foregone cytokines to immune checkpoints.
In depressive episodes, patients with BD and MD both had an elevated plasma level of IL-6. This was in accordance with the previous evidence the changes of proinflammatory cytokines in patients with affective disorders 20 . As a proinflammatory cytokine, IL-6 was hypothesized to drive the development of depression by impairing serotonin production, enhancing monoamine reuptake, activating the hypothalamic-pituitary-adrenocortical axis 21 . In a STAT3-dependent pattern, IL-6 can directly affect the function of serotonin transporter and consequently induces depression-like behaviour 22 . Moreover, IL-6 and its downstream JAK/STAT pathway participate in facilitating cognitive flexibility 23 . Increased level of IL-6 was also associated with impaired function in reward circuit and other brain regions involved in mood regulation 24,25 . However, the exploration of cytokine network is far from enough to uncover the inflammatory changes in patients with MD and BD.
With regard to the T lymphocyte subsets, the proportion of cytotoxic T cells (CD3+ CD8+ ) was reduced in patients with BD, which was negatively associated with the age of onset. These findings indicated that patients with lower number of cytotoxic T cells possibly had an older onset age. In a recent study, a decreased proportion of cytotoxic T cells was also observed in patients with BD 13 , while other studies reported no significant changes 10,26 . The primary role of cytotoxic T cells is to mediate cellular immunity against foreign pathogens. A decreased proportion of peripheral cytotoxic T cells might be related to weakened immune defense and increased risk of infection. To date, no research has ever documented whether an increased risk of infection exists in BD patients and its correlation with suppressed activation of cytotoxic T cells. Interestingly, this study was the first to reveal that the expression of TIM-3 on cytotoxic T cells significantly increased in patients with BD, which tented to negatively associate with the score of MADRS. As documented, TIM-3 not only plays a crucial role in mediating the exhaustion of cytotoxic T cells 14,17 , but also presents as a potent suppressor of cytotoxic T cell inflammation 27 . Blockade of TIM-3 pathway can help to restore the adaptive immunity 28 . For BD patients, therefore, we speculate that an elevated expression of TIM-3 can lead to decreased number of cytotoxic T cells, suppressed secretion of proinflammatory cytokines from cytotoxic T cells, and eventually play a protective role against the great inflammatory burden during depressive episodes. In this regard, BP patients with reduced number of cytotoxic T cells had a lower overall inflammatory level, which may help to delay the onset of disease.
In addition, the expression of PD-L2 on CD14+ monocytes was downregulated in patients with BD. As another ligand of PD-1, the expression of PD-L2 was more restricted and less extensive than the expression of PD-L1. Similar to PD-L1, engagement PD-1 by PD-L2 was correlated to the downregulation of T-cell proliferation and cytokine production 19 , while knockdown of both PD-L1 and PD-L2 can improve the proliferation and cytokine production of CD4+ T cells 29 . Therefore, the downregulation of PD-L2 on monocytes was possibly correlated with enhanced CD4+ T-cell function, which in turn promoted the production and secretion of proinflammatory cytokines, such as IL-6. However, recent researches have characterized an opposing and competitive role of PD-L1 and PD-L2 in modulating T cell functions during certain pathologic processes, such as allergic asthma 30 . Meanwhile, the essential role of PD-L2 in establishing CD4+ T cell immunity by interfering the PD-1-PD-L1 axis has also been reported 31 . Hence, although the effect of reduced expression of PD-L2 on CD14+ monocytes in patients with BD remains unknown, it will inevitably disrupt the cellular interaction between monocytes and T cells, and as a consequence, cause disturbance in T cell homeostasis. The aforementioned findings would be critical if they are further verified in patients with BD.
The present study had several limitations. One major limitation was the relatively small sample size, which inevitably weakened the statistical power. Another is the detection limit of cytokine level in this study, which appeared to be too high for most cytokines explored and made the statistical results of the cytokine levels less persuasive. In the present study, the role of altered expression of TIM-3 and PD-L2 was not further investigated, which hindered the comprehensive interpretation of our findings.
Above all, the present study favored the immune dysfunction in patients with affective disorders, and called for more attention to be paid to the role of immune checkpoint inhibitors in future studies.   Table 3. Levels of immune checkpoint inhibitors on immune cells in patients with BD, MD and controls (mean ± SD). * P < 0.05 (two-tailed). P 1 : BD and HC; P 2 : MD and HC; P 3 : BD and MD; P 4 : BD, MD and HC. † One-way ANOVA test; Δ Kruskall-Wallis test. (e) history of pregnancy or lactation 6 months before commencement of the study; and (f) use of immune-regulatory drugs or alcohol/drug dependence history 6 months before commencement of the study. This study was approved by the Hospital Ethical Committee of the First Affiliated Hospital, Zhejiang University School of Medicine, and was performed in accordance with the principles of the Declaration of Helsinki. All participants, or their guardians, signed informed consent before enrollment. The frequency of different types of immunocompetent cells was characterized by flow cytometry as previously described (PMID: 8404602). Briefly, at least 1 × 10 5 cells were analyzed using BD FACS Canto TM II flow cytometer (BD Bioscience, CA, USA). The staining of T cells or monocytes was performed with a single antibody or a combination of the following antibodies: anti-Tim-3-APC (R&D Systems), anti-PD-1-PE (eBiosciences), anti-PD-L1-BrilliantViolet421 (Biolegend), and anti-PD-L2-APC (Biolegend). The isotype-matched immunoglobulins were used as controls. The tests were performed at least in triplicate.

Plasma cytokine determination.
In the present study, the levels of plasma cytokines, including IFN-γ , TNF-α , IL-2, IL-4, IL-6, and IL-10, were also determined using BD cytometric bead array human Th1/Th2 cytokine kit (BD Bioscience), according to the manufacturer instructions. The concentrations of plasma cytokines were determined using standard curves established with the individual recombinant cytokines provided. The limitation of detection was 7.1 pg/mL for IFN-γ , 2.8 pg/mL for TNF-α , 2.6 pg/mL for IL-2, 2.6 pg/mL for IL-4, 3.0 pg/ mL for IL-6, and 2.8 pg/mL for IL-10. All these tests were carried out in triplicate.

Statistical interpretation.
Statistical analysis was conducted using the SPSS 17.0 statistical software (IBM, IL, USA). When data were not qualified for normal distribution even after transformation, they were analyzed using Kruskal-Wallis test, or otherwise, one-way analysis of variance and t test were used. The difference between proportions was analyzed using the chi-square test. Correlation analyses between the expression of cell surface markers, plasma cytokines and clinical parameters were performed by Spearman's rho test. The significance level was set at two-tailed P value < 0.05.