C-X-C chemokine receptor type 5+CD8+ T cells as immune regulators in hepatitis Be antigen-positive chronic hepatitis B under interferon-alpha treatment

Abstract

BACKGROUND

C-X-C chemokine receptor type 5 (CXCR5)⁺CD8⁺ T cells represent a unique immune subset with dual roles, functioning as cytotoxic cells in persistent viral infections while promoting B cell responses. Despite their importance, the specific role of CXCR5⁺CD8⁺ T cells in chronic hepatitis B (CHB), particularly during interferon-alpha (IFN-α) treatment, is not fully understood. This study aims to elucidate the relationship between CXCR5⁺CD8⁺ T cells and sustained serologic response (SR) in patients undergoing 48 weeks of pegylated IFN-α (peg-IFN-α) treatment for CHB.

AIM

To elucidate the relationship between CXCR5⁺CD8⁺ T cells and sustained SR in patients undergoing 48 weeks of peg-IFN-α treatment for CHB.

METHODS

This study enrolled 60 patients with hepatitis Be antigen (HBeAg)-positive CHB undergoing 48 weeks of peg-IFN-α treatment. Participants were assessed for eligibility based on criteria such as persistent HBsAg-positive status for at least six months, HBeAb-negative, hepatitis B virus DNA levels exceeding 2 × 10⁴ copies/mL, and alanine aminotransferase (ALT) levels between 2 and 10 times the upper limit of normal. Blood samples were collected at baseline and at weeks 12, 24, 48, and a 24-week treatment-free follow-up (week 72) to measure serum interleukin (IL)-21 concentration via ELISA and to analyze CXCR5 and programmed death-ligand 1 (PD-L1) expression on CD8⁺ T cells by flow cytometry, CXCR5 is a chemokine receptor that directs immune cells to specific tissues, while PD-L1 is a protein that regulates immune responses by inhibiting T cell activity.

RESULTS

Patients with CHB exhibited significantly lower levels of circulating CXCR5⁺CD8⁺ T cells compared to healthy controls (P < 0.01). Notably, CXCR5+CD8+ T cells were prominently expressed in patients who achieved sustained SR compared to non-SR (NSR). A significant correlation was observed between CXCR5 and PD-L1 expression (r = -0.189, P = 0.002). However, there was no significant correlation between serum IL-21 levels and CXCR5⁺CD8⁺ lymphocytes (r = -0.03, P = 0.625) or serum ALT levels (r = 0.026, P = 0.678).

CONCLUSION

The enhanced expression of CXCR5⁺CD8⁺ T cells in patients achieving HBeAg seroconversion during IFN-α treatment suggests that these cells play a crucial role in antiviral immune responses against hepatitis B. This study highlights the potential of CXCR5⁺CD8⁺ T cells as immune regulators in CHB, which may inform future therapeutic strategies to optimize antiviral treatments.

Key Words: C-X-C chemokine receptor type 5; Programmed death-ligand 1; Interleukin -21; Pegylated interferon-alpha; Chronic hepatitis B

Core Tip: This study highlights the pivotal role of C-X-C chemokine receptor type 5 (CXCR5)⁺CD8⁺ T cells in chronic hepatitis B (CHB) during pegylated interferon-alpha treatment. Our findings revealed that patients who achieved sustained serologic response exhibited higher baseline and treatment levels of CXCR5⁺CD8⁺ T cells, suggesting a connection between CXCR5 expression and IFN-induced antiviral immune responses. This research emphasizes the importance of CXCR5⁺CD8⁺ T cells as a potential biomarker for monitoring treatment efficacy in CHB patients.

INTRODUCTION

Hepatitis B virus (HBV) establishes a persistent infection, leading to a significant health burden worldwide[1]. Approximately 240 million individuals are living with chronic HBV infection, and HBV-related liver diseases claim around 780000 lives annually[2]. Pegylated interferon-alpha (peg-IFN-α), which mediates both immunomodulatory and direct antiviral effects, represents a preferred treatment option for chronic hepatitis B (CHB), which includes both the innate and adaptive immune responses[3]. This activation enhances the synthesis of antiviral proteins that inhibit viral replication and facilitate the elimination of the virus.

The intracellular JAK/STAT signaling pathway is activated by interferon-alpha (IFN-α) through the IFN-α/β receptor complex, influencing the transcription of numerous genes known as IFN-stimulated genes. It is well-established that pattern recognition receptors in various cell types can efficiently activate interferon (IFN) production. A subset of CD8⁺ T lymphocytes expressing the chemokine receptor C-X-C chemokine receptor type 5 (CXCR5) plays a crucial role in regulating viral replication. Circulating CXCR5⁺CD8⁺ T cells maintain robust antiviral capabilities and are associated with treatment response in patients with CHB[4].

Furthermore, the expression of CXCR5 on the surface of CD8⁺ T cells serves as an indicator of memory stem cell-like properties in progenitor cells, enhancing responsiveness to immune checkpoint blockade therapy in cancer[5]. This phenomenon has also been demonstrated in certain viral infections, underscoring the dual roles of the cellular immune response mediated by CXCR5⁺ CD8⁺ T cells.

In contrast to other CXCR5 subsets, CD8⁺CXCR5⁺ T cells express higher levels of PD-1 and produce greater amounts of HBV-specific cytokines, such as IFN and interleukin-21 (IL-21)[6], and are associated with decreased levels of HBsAg and HBV-DNA. These CXCR5⁺CD8⁺ T cells can migrate into B-cell follicles and exhibit enhanced cytotoxicity. Moreover, they have demonstrated improved therapeutic responses when combined with anti- programmed death-ligand 1 (PD-L1) treatment. However, the role of CXCR5-expressing CD8⁺ T cells in the context of anti-HBV treatment using peg-IFN-α remains unclear. Therefore, this study aims to investigate the dynamic changes in CXCR5-expressing CD8⁺ T cells in hepatitis Be antigen (HBeAg)-positive patients receiving peg-IFN-α-2b treatment from baseline to 72 weeks. Additionally, we will examine the relationships between CXCR5⁺CD8⁺ T cells and PD-L1, as well as IL-21 in peripheral blood.

MATERIALS AND METHODS

Statement

This trial was registered at http://clinicaltrials.govas (NCT01760122) on 03-01-2013 and China Drug Trials.org (ID: TB1211IFN). The study was conducted according to the Declaration of Helsinki and was approved by the Ethics Committee of the Second Xiangya Hospital. All methods were carried out in accordance with relevant guidelines and regulations.

Study subjects

Sixty patients with HBeAg-positive CHB from The Second Xiangya Hospital of Central South University (Changsha, China) participated in a phase III, multi-center, open-label clinical trial of peg-IFN-α-2b (PegBeron^®; Amoytop Biotechnology, Xiamen, China) at a dosage of 180 µg/week (Clinical trial registration: NCT03903796, registered on 03-01-2013 at (http://clinicaltrials.gov). Five milliliters of serum were collected for cytokine detection, and twenty milliliters of heparinized blood were obtained for flow cytometry analysis at The Second Xiangya Hospital. Samples were taken at baseline and at 12, 24, and 48 weeks after initiating peg-IFN treatment, with a treatment-free follow-up at 24 weeks. Samples were transported on dry ice and stored at -80 °C in the Central South University Liver Disease Research Center.

The study population was divided into two groups: A sustained seroconversion (SR) group, which showed HBeAg loss with anti-HBe antibody seroconversion by week 72, and a non-sustained seroconversion (NSR) group, in which HBeAg remained positive or HBeAb did not appear by week 72. Additionally, twenty HBsAg-negative individuals with normal alanine aminotransferase (ALT) levels from laboratory staff and students served as healthy controls (HC). This study was conducted in compliance with the Declaration of Helsinki and approved by the Ethics Committee of the Second Xiangya Hospital. Informed consent was obtained from each participant.

Inclusion criteria

(1) Diagnosis of HBeAg positive CHB; (2) HBsAg positivity for at least six months prior to study enrollment; (3) HBV DNA > 2 × 10⁴ copies/mL; (4) ALT levels between 2 and 10 times the upper limit of normal; and (5) Treatment naive to peg-IFN-α-2b.

Exclusion criteria

(1) History of liver transplantation; (2) Coinfection with hepatitis C virus, hepatitis D virus, or human immunodeficiency virus (HIV); (3) Severe liver dysfunction or cirrhosis; (4) Presence of autoimmune or immune related disorders; (5) Pregnancy or breastfeeding; and (6) Current or recent use of immunosuppressive medications.

Sample collection and storage

Blood samples were collected at baseline and at 12, 24, and 48 weeks after initiating peg-IFN treatment, with an additional treatment-free follow-up at 24 weeks. A total of five milliliters of serum was collected for cytokine detection, and twenty milliliters of heparinized blood were obtained for flow cytometry analysis. Samples were transported on dry ice and stored at -80 °C in the Central South University Liver Disease Research Center.

Group classifications

Participants were classified into two groups: The SR group, which demonstrated HBeAg loss with anti-HBe antibody seroconversion by week 72, and the NSR group, in which HBeAg remained positive or HBeAb did not appear by week 72. Additionally, twenty HBsAg-negative individuals with normal ALT levels from laboratory staff and students served as HCs.

Laboratory assessments

Routine physical examinations, biochemical, and hematologic assessments were conducted at weeks 0, 12, 24, 48, and 72. The presence of HBsAg, HBeAg, anti-HBs, and anti-HBe was determined using Elecsys tests (Roche Diagnostics GmbH, Germany), with detection limits of 1 COI (cut-off index) for HBeAg and 0.05 IU/mL for HBsAg. HBV DNA levels were measured using the Roche Cobas^® Amplicor HBV Test, version 2.0 (Roche Diagnostics, Germany), with a detection limit of 20 IU/mL.

Cytokine measurement

Serum IL-21 concentrations were measured in duplicate using a commercial human IL-21 ELISA kit (Human ELISA kit ab100554, Abcam Inc., Cambridge, MN, United States), with a lower detection limit of 20 pg/mL.

Preparation of peripheral blood mononuclear cells and flow cytometry

Peripheral blood mononuclear cells (PBMCs) were isolated and processed as previously described. A total of 1 × 10⁶ PBMCs were stained with fluorochrome-conjugated antibodies: Anti-CD3-PE, anti-CD8-PE-CY7, anti-CD185 (CXCR5)-preCP-CY5.5, and anti-CD274 (PD-L1)-FITC for 30 minutes. Isotype controls were also included. Following staining, cells were washed twice and analyzed using a FACScan flow cytometer (Beckman Coulter, Inc., Brea, CA, United States) with Cell Quest software (version 5.1; BD Biosciences).

Statistical analysis

Statistical analyses were performed using IBM SPSS Statistics 23.0, with P values < 0.05 considered statistically significant in two-tailed tests. Continuous data are expressed as mean ± SE. Group comparisons utilized independent samples t-tests or one-way ANOVA with post-hoc least significant difference tests. χ² tests were applied for categorical variables. Variance comparisons between groups employed a Folded F test. Spearman's rank correlation coefficient calculated associations among variables. Logistic binary regression analysis was conducted to identify factors associated with HBeAg seroconversion, reporting OR and 95%CI. Graphical representations and data visualizations were created using GraphPad Prism 9.

RESULTS

Baseline demographic profiles and characteristics

A total of 60 CHB patients receiving peg-IFN-α treatment (41.7% female, 58.3% male) and 20 HCs were studied (Figure 1). In the CHB cohort, 26.7% achieved sustained SR after 48 weeks of treatment, with baseline factors such as gender, HBeAg levels, and CXCR5⁺CD8⁺ T cells linked to HBeAg seroconversion (Table 1). Notably, CXCR5⁺CD8⁺ T cell levels were significantly higher in SR patients compared to non-SR (NSR), emphasizing their potential role in predicting response.

Figure 1 Research flowchart. ALT: Alanine aminotransferase; HBV: Hepatitis B virus; peg-IFN-α: Pegylated interferon-alpha; HBeAg: Hepatitis Be antigen; HCV: Hepatitis C virus; HDV: Hepatitis D virus; HIV: Human immunodeficiency virus.

Table 1 Baseline demographic profiles and characteristics related to pegylated interferon response.

HC (n = 20)		SR (n = 16)	NSR (n = 44)	P value
Age (years)	28.17 ± 0.25	26.00 ± 4.94	28.86 ± 8.32	0.260
Gender (male/female)	10/10	6/10	29/15	0.048a
Weight (kg)	56.26 ± 4.20	57.54 ± 9.12	63.51 ± 11.03	0.097
ALT (IU/mL)	24.97 ± 6.47	205.55 ± 69.38	185.5 ± 103.61	0.530
HBV DNA (log10 IU/mL)	n.d	7.73 ± 0.61	7.94 ± 1.15	0.530
HBsAg (log10 IU/mL)	Negative	4.13 ± 0.46	4.44 ± 0.56	0.087
Anti-HBs positive	12	0	0	n.d
HBeAg (log10 COI)	n.d	2.90 ± 0.62	3.26 ± 0.46	0.044a
IL-21 (pg/mL)	12.31 ± 5.42	91.27 ± 188.34	105.30 ± 379.75	0.901
CXCR5+ (%)	12.11 ± 4.00	17.39 ± 8.11	16.04 ± 8.68	0.639
PD-L1+ (%)	8.65 ± 1.94	12.57 ± 9.55	18.31 ± 10.97	0.113
CXCR5+CD8+ (%)	5.50 ± 2.43	3.41 ± 3.00	1.47 ± 1.34	0.042a
CXCR5+CD4+ (%)	6.62 ± 1.06	14.43 ± 2.52	13.98 ± 1.39	0.867
PDL1+CD8+ (%)	7.75 ± 1.21	5.07 ± 3.22	8.59 ± 8.46	0.061
PDL1+CD4+ (%)	1.29 ± 0.23	7.95 ± 1.75	7.95 ± 1.40	0.812

^aP < 0.05.

Data are shown as mean ± SE. ALT: Alanine aminotransferase; HC: Healthy control; SR: Serologic response; NSR: Non-serologic response; n.d.: Not determined; HBV: Hepatitis B virus; PD-L1: Programmed death-ligand 1; IL-21: Interleukin-21; CXCR5: C-X-C chemokine receptor type 5.

Baseline comparison of CXCR5⁺, PD-L1, and IL-21 levels between CHB patients and HCs

The frequency of CXCR5⁺ T cells in PBMCs and on CD8⁺/CD4⁺ T cells in CHB patients before peg-IFN treatment was significantly lower than in HCs (P < 0.001, P < 0.001, P = 0.008; Figures 2 and 3). In contrast, PD-L1 expression on PBMCs and CD4⁺ T cells in CHB patients was substantially higher than in HCs (P < 0.001). There was no difference in the frequency of CD8⁺PD-L1⁺ T cells between CHB patients and HCs. Although the CHB group had relatively higher levels of IL-21 compared to the HC group, the difference did not reach statistical significance (P = 0.092).

Figure 2 Frequency of CD8⁺ and CD4⁺ T cells expressing C-X-C chemokine receptor type 5 and programmed death-ligand 1 in a single chronic hepatitis B patient at baseline. A: Frequency of C-X-C chemokine receptor type 5 (CXCR5)⁺CD8⁺ T cells; B: Frequency of CXCR5⁺CD4⁺ T cells; C: Frequency of programmed death-ligand 1 (PD-L1) ⁺CD8⁺ T cells; D: Frequency of PD-L1⁺CD4⁺ T cells. CXCR5: C-X-C chemokine receptor type 5; PD-L1: Programmed death-ligand 1.

Figure 3 Comparison of immune markers and interleukin-21 concentration between healthy controls and chronic hepatitis B patients at baseline. A: Frequency of programmed death-ligand 1 (PD-L1)⁺ cells; B: Frequency of C-X-C chemokine receptor type 5 (CXCR5)⁺ cells; C: Frequency of PD-L1⁺CD8⁺ T cells; D: Frequency of CXCR5⁺CD8⁺ T cells; E: Interleukin-21 concentration; F: Frequency of PD-L1⁺CD4⁺ T cells; G: Frequency of CXCR5⁺CD4⁺ T cells. CXCR5: C-X-C chemokine receptor type 5; PD-L1: Programmed death-ligand 1; IL-21: Interleukin-21.

Dynamic changes in markers during peg-IFN-α treatment

HBsAg, HBeAg, and HBV DNA levels gradually declined during peg-IFN-α treatment in both the SR and NSR groups. As shown in Figures 4 and 5.

Figure 4 Dynamic changes of various markers over time. A: Hepatitis B virus DNA levels; B: HBsAg levels; C: Hepatitis Be antigen; D: Frequency of C-X-C chemokine receptor type 5⁺CD8⁺ cells; E: Interleukin-21 concentration; F: Programmed death-ligand 1 expression. SR: Serologic response; NSR: Non-serologic response; HBeAg: Hepatitis Be antigen; IL-21: Interleukin-21; CXCR5: C-X-C chemokine receptor type 5; PD-L1: Programmed death-ligand 1.

Figure 5 Comparison of C-X-C chemokine receptor type 5 and programmed death-ligand 1 expression on peripheral blood mononuclear cells and CD8⁺ lymphocytes between the serologic response and non-serologic response groups at different time points. A: C-X-C chemokine receptor type 5 (CXCR5) expression on peripheral blood mononuclear cells (PBMCs); B: Programmed death-ligand 1 (PD-L1) expression on PBMCs; C: CXCR5 expression on CD8+ lymphocytes; D: PD-L1 expression on CD8⁺ lymphocytes. SR: Serologic response; NSR: Non-serologic response; CXCR5: C-X-C chemokine receptor type 5; PD-L1: Programmed death-ligand 1.

At baseline, the percentage of CXCR5 expression on CD8⁺ lymphocytes was significantly higher in the SR group than the NSR group (Figure 4C, P = 0.042). This pattern persisted during the 48-week peg-IFN treatment (P = 0.013, P < 0.001, and P < 0.001 from week 12 to week 48, respectively). Only at week 48 did CXCR5 expression in CD4⁺ cells show a significant difference in the SR group.

Within the SR group, the percentage of CXCR5⁺CD8⁺ lymphocytes was significantly higher at weeks 12, 24, 48, and 72 compared to the baseline (Figure 5). After treatment, the expression of CXCR5 in CD8⁺ lymphocytes decreased to a significantly lower level at week 72 compared to the other time points. Concurrently, HBeAg levels gradually declined during treatment in the SR group. However, there was no significant difference in HBeAg levels between week 48 and 24 weeks post-treatment, although HBeAg continued to decrease after treatment. In contrast, in the NSR group, at week 12, the mean expression of CXCR5 in CD8⁺ lymphocytes increased with less than a 0.5 Log10 difference from baseline. However, CXCR5 expression in CD8⁺ lymphocytes decreased rapidly and remained at a low level at all time points after week 12. In addition, HBeAg levels fell at week 12 but exhibited a stalled decline at other time points.

Correlations between CXCR5, PD-L1 expression, serum IL-21 concentration, and peg-IFN-α response

At week 12, univariate regression analysis revealed that CXCR5⁺CD8⁺, PD-L1⁺CD4⁺ lymphocytes, and HBV DNA, HBsAg, and HBeAg were significantly associated with HBeAg seroconversion (P < 0.01). Multivariate regression analyses revealed a close relationship between HBeAg and CXCR5⁺CD8⁺ lymphocytes at week 12 with SR achievement (P < 0.05) (Table 2).

Table 2 Logistic regression analyses for sustained Hepatitis Be antigen seroconversion at week 12.

Characteristics	Univariate analysis			Multivariate analysis
	OR	95%CI	P value	OR	95%CI	P value
ALT	1.002	0.998-1.002	0.383	1.008	0.999-1.014	0.063
IL-21	0.999	0.994-1.004	0.645
PDL1	0.957	0.891-1.029	0.236
CXCR5	1.038	0.953-1.131	0.395	0.652	0.387-1.098	0.107
PDL1+CD8+	1.070	0.959-1.193	0.225
PDL1+CD4+ (%)	0.85	0.733-0.984	0.018a
CXCR5+CD8+	1.459	1.125-1.892	0.004b	4.820	1.081-21.487	0.039a
CXCR5+CD4+	0.952	0.853-1.062	0.375
log10 HBV DNA	0.412	0.241-0.705	0.001b
log10 HBsAg	0.126	0.032-0.507	0.004b
log10 HBeAg	0.300	0.144-0.626	0.001b	0.009	0.001-0.490	0.021a

^aP < 0.05.

^bP < 0.01.

ALT: Alanine aminotransferase; HBV: Hepatitis B virus; HBeAg: Hepatitis Be antigen; IL-21: Interleukin-21; CXCR5: C-X-C chemokine receptor type 5; PD-L1: Programmed death-ligand 1.

DISCUSSION

This study highlights the significant role of CXCR5⁺CD8⁺ T cells in chronic HBV infection, particularly in the context of peg-IFN-α treatment. Our findings suggest that these cells may contribute to immune responses associated with HBeAg seroconversion, indicating potential therapeutic implications for chronic HBV management.

Chronic HBV infection affects approximately 2 billion people worldwide, particularly in Southeast Asia and China, where over 50% of hepatocellular carcinoma cases are linked to HBV infection[7]. Dysfunction of effector CD8⁺ T cells is often attributed to the immune system's failure to control viral replication. However, recent clinical and experimental studies on HIV and lymphocytic choriomeningitis virus (LCMV) infections challenge this notion, demonstrating that a subset of exhausted CD8⁺ T cells expressing CXCR5 plays a critical role in regulating viral replication[8,9].

CXCR5⁺CD8⁺ T cells are a unique subset with significant antitumor and antiviral activities, found in human tonsils and follicular lymphoma. They inhibit T (FH)-mediated B cell differentiation and show enhanced cytotoxicity, with adoptive transfer resulting in greater antitumor efficacy compared to CXCR5- cells. Research by Chu et al[5] identified 77 differentially expressed genes, with 33 linked to improved survival in follicular lymphoma. Additionally, these cells can be expanded using IL-23 and TGF-beta, highlighting their potential for therapeutic applications. Overall, CXCR5⁺CD8⁺ T cells play a critical role in suppressing B cell differentiation and enhancing antitumor responses.

Li et al[4] found that CXCR5⁺CD8⁺ T cells, particularly the intrahepatic CXCR5⁺ subset, retain effector functions characterized by enhanced production of HBV-specific IFN-γ and IL-21. Notably, PD-1 blockade and exogenous IL-21 enhance the production of IFN-γ from these cells. Furthermore, this subpopulation exhibits more potent antiviral activity than their CXCR5- counterparts, inhibiting HBsAg expression and supporting antibody production by B cells. This aligns with our findings that patients with higher CXCR5⁺CD8⁺ T cell expression during peg-IFN treatment exhibit stronger antiviral capabilities and a greater likelihood of achieving HBeAg seroconversion.

Notably, studies have shown that CXCR5⁺CD8⁺ T cells exhibit a phenotype similar to T follicular helper cells and reside within B cell follicles, suggesting they play a crucial role in mediating humoral responses. This resemblance indicates that in the context of HBV infection, CXCR5⁺CD8⁺ T cells may facilitate interactions with B cells, potentially enhancing antibody production against HBV[10]. Given their association with successful peg-IFN-α treatment and HBeAg seroconversion, enhancing the functionality or frequency of these cells could improve treatment outcomes.

In pediatric HBV infection, recent literature has identified a potential antiviral role for CCR5⁺CD8⁺ T cells. A study by Tan et al[6] highlighted the diverse phenotypes and functions of CD8⁺ T cells in children with HBV, noting that the frequency of CCR5⁺CD8⁺ T cells correlated positively with patient age and CCR5⁺CD4⁺ T cells, while negatively correlating with liver function and viral markers. These findings underscore the complexity of CD8⁺ T cell responses in children and suggest that CCR5⁺CD8⁺ T cells may serve as promising immunotherapeutic targets. Our research findings align with these conclusions, demonstrating the dynamic changes of CCR5⁺CD8⁺ T cells during interferon treatment and their role in distinguishing treatment responses, further validating the complexity of pediatric CD8⁺ T cell responses and the therapeutic potential of CCR5⁺CD8⁺ T cells.

Successful IFN treatment in CHB patients exhibiting HBeAg seroconversion was associated with higher levels of CXCR5⁺CD8⁺ T cells and a sustained increase in these lymphocytes during peg-IFN-α therapy[11,12]. The CXCR5 expression level in CD8⁺ cells negatively correlated with HBV DNA, HBsAg, and HBeAg but did not correlate with serum IL-21 or ALT. This suggests that CXCR5⁺CD8⁺ lymphocytes may contribute to CHB immunotherapies, indicating their role in the immune response against HBV rather than liver inflammation severity. Recent findings by Khanam et al[13] provide valuable insights into the role of CXCR5⁺CD8⁺ T cells in CHB infection. They demonstrated that while the overall frequencies of CD8⁺CXCR5⁺ cells remain stable during CHB, a higher prevalence of these cells is observed in patients with low HBsAg and HBV-DNA levels, HBeAg negativity, and elevated fibrosis scores. This suggests that CXCR5+CD8+ T cells may play a protective role in viral control, correlating with reductions in HBsAg and HBV-DNA levels.

While previous studies reported significant differences in serum IL-21 concentrations between patients with sustained and non-SR[14,15], our study did not replicate these findings. Similar to our results, those studies indicated that IL-21 concentrations decreased over time during peg-IFN therapy in both groups. This aligns with evidence suggesting that IL-21 primarily participates in liver cirrhosis inflammation and fibrogenesis. We observed a positive association between IL-21 and CXCR5⁺CD4⁺ T cells, consistent with IL-21 being predominantly produced by CD4⁺ T cells.

PD-1 expression plays a unique role in CXCR5⁺CD8⁺ T cells. Although in a chronic LCMV infection model[16], PD-1 expression on CXCR5⁺CD8⁺ T cells was lower than on CXCR5−CD8⁺ T cells, other studies have shown that both cell types express PD-1, with CXCR5⁺ T cells displaying slightly higher levels[17,18]. In HIV infection, PD-1 may help localize CXCR5⁺CD8⁺ T cells to follicles rather than solely marking exhaustion[19]. Our findings show that PD-L1 expression on PBMCs significantly decreased at weeks 24 and 72 and on CD8⁺ lymphocytes at week 24, with no differences observed at other time points. Furthermore, we identified a negative correlation between PD-L1 and CXCR5 lymphocytes in peg-IFN-α-treated patients, indicating that PD-L1 may influence circulating CXCR5 expression in CD8⁺ lymphocytes, thereby impacting viral kinetics during treatment.

In summary, this study demonstrated that circulating CXCR5⁺CD8⁺ T cells may contribute to the immunotherapy mechanism of peg-IFN-α-2b in chronic HBV patients. The relationship between PD-L1 and CXCR5 was found to be inverse. Although CXCR5⁺CD8⁺ T cells did not produce most IL-21 in CHB patients, they interacted with CXCR5⁺CD4⁺ T cells. Future research could analyze how IFN, PD-L1, and CXCR5⁺CD8⁺ T cells interact to influence signaling pathways and immune responses.

Limitations

This study has several limitations. The small sample size, especially when comparing the SR and NSR groups, may reduce statistical power and affect the reliability of the findings. Furthermore, discrepancies in demographic variables between the HC and patient groups could have introduced biases. Additionally, the study lacked transcriptional marker analysis and focused primarily on peripheral rather than liver-resident CCR5⁺CD8⁺ T cells.

CONCLUSION

The HBeAg seroconversion patients treated with IFN exhibited higher levels of CXCR5⁺CD8⁺ cell expression. The CXCR5 expression on CD8⁺ T cells is most likely related to IFN-induced antiviral immune responses against hepatitis B infections.