B cell immune response to Talaromyces marneffei infection in HIV-negative patients and rat models

Abstract

Purpose

Talaromyces marneffei (T. marneffei), a thermally dimorphic fungus, causes systemic mycosis, with pathogenesis involving intracellular survival and dysregulation of host immunity. This study investigates the phenotypic and functional alterations in B lymphocytes induced by T. marneffei infection.

Methods

Retrospective analysis of peripheral blood B cell counts and IgG/IgM levels in 31 HIV-negative Talaromycosis (TSM) patients. Peripheral blood samples were collected to measure plasmablasts, plasma cells and related cytokine expression. The plasmablasts and plasma cells in spleen was determined using flow cytometry in T. marneffei infected rat models, and the levels of IgG/IgM, B-cell activating factor (BAFF), A Proliferation-Inducing Ligand (APRIL) and IL-21 were determined.

Results

TSM patients exhibited reduced CD19⁺B cell and plasmablasts proportions in peripheral blood, while plasma cell proportions increased significantly and IgG, IgM levels in serum were elevated. In T. marneffei infected rats, the proportion of plasmablasts and plasma cells in splenic lymphocytes increased, and levels of IgG, IgM, BAFF, APRIL and IL-21 were significantly elevated compared to normal control rats.

Conclusion

T. marneffei infection induces B-cell phenotypic and functional abnormalities, manifested as reduced B-cell numbers, increased proportions of plasma cells, accompanied by upregulation of BAFF, APRIL, and IL-21 expression. This suggests T. marneffei may participate in pathogenesis by regulating B-cell differentiation and humoral immune responses.

Clinical trial number

Not applicable.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12879-026-13277-8.

Introduction

Talaromycosis (TSM), a severe invasive disseminated fungal disease, is caused by Talaromyces marneffei (T. marneffei) and is prevalent in Southeast Asia. The infection rate is particularly high in China, especially in regions such as Guangxi, Guangdong and Hong Kong [1]. T. marneffei infections usually occur in immunocompromised individuals, including secondary immunodeficiency due to HIV infection, cancer and immunosuppressive therapy [2, 3]. However, T. marneffei infection can also occur in HIV-negative individuals without obvious immunosuppression, and the number of those patients is increasing now. The present study demonstrates that the extent of dissemination, duration of persistent infection, recurrence rate, and mortality rate are higher in HIV-positive subjects than in HIV-positive hosts. The clinical characteristics of TSM in HIV-negative hosts [4, 5], including treatment resistance and poor prognosis, present significant challenges in clinical diagnosis and management. The underlying immune mechanisms remain to be fully elucidated.

T. marneffei exists as a hyphal form (mold form) at room temperature, and transforms into a yeast form (pathogenic phase) at 37℃ or inside the host’s body. This unique morphological switching characteristic is known as dimorphism. After T. marneffei invades the host, it is first recognized and phagocytosed by antigen-presenting cells (APCs). These cells then present T. marneffei antigens to T cells and activate them. Recent studies have indicated that in cases where the host displays symptoms of immunodeficiency or a state of impaired function in their immune cells, as a result of T. marneffei mediated impairment, there is an observed tendency for impaired clearance of pathogens [6–9]. This impaired clearance has been identified as a pivotal factor in the progression of dissemination and the subsequent development of persistent infection [6]. The extant research has focused predominantly on the interactions between T. marneffei and the host’s innate and adaptive immune systems [7]. Macrophages, a pivotal element of the innate immune system, possess the capacity to phagocytose and attempt to eliminate invading T. marneffei spores. Nevertheless, the T. marneffei evade immune surveillance because they can survive in macrophages and transform into a yeast form (pathogenic phase) [8]. In addition, a decrease in CD4⁺T cell numbers has been demonstrated to result in a considerable elevation in the risk of infection and mortality [9]. However, the role of B cells and humoral immunity in T. marneffei infections remains to be elucidated. B cells have been identified as pivotal coordinators of both humoral and cellular immunity. In recent years, B cells have attracted considerable attention due to their established function as specialized APCs [10, 11], which has led to the validation of their pivotal roles in antigen presentation, T cell activation, and pathogen clearance. Therefore, the maintenance of the mature B cell pool depends on the survival of immature B cells during late development as well as the lifespan of the mature cells. Within the tumor necrosis factor family, BAFF and APRIL [12] have been demonstrated to enhance antibody expression. This is achieved by altering the proportion of cells transitioning through B-cell development and prolonging plasma cell lifespan [13, 14]. IL-21 has been demonstrated to induce the formation of germinal centers, promote the proliferation and differentiation of B cells into plasma cells, increase the secretion of immunoglobulin antibodies, and facilitate the development of memory B cells [15, 16]. Despite the lack of evidence on B cell responses post-T. marneffei infection, our groundbreaking study is the first to elucidate these immune mechanisms in both human and animal models.

The present study systematically investigates the effects of T. marneffei infection on B cell subsets, antibody production, and the expression of differentiation-related factors through clinical sample analysis, in vitro cell experiments, and animal models (rats). The objective of this study is to elucidate the immune response of B cells in HIV-negative patients and in rat models in response to T. marneffei infection.

Materials and methods

Data collection

A retrospective cohort of 31 HIV-negative TSM patients admitted to the First Affiliated Hospital of Guangxi Medical University and the Eighth Affiliated Hospital of Sun Yat-sen University between January 2019 and December 2023 was established for the purposes of this study. This study was approved by the Ethics Committee of The Eighth Affiliated Hospital of Sun Yat-Sen University (number: 2022-040-01). Written informed consent was obtained from all participants. A detailed flowchart illustrating the participant selection protocol and study steps is shown in Fig. 1.

Fig. 1.

Flowchart of participant selection

The inclusion criteria for the study are as follows: (1) Confirmed TSM diagnosis via pathogen culture, histopathology, or genetic testing; (2) Age ≥ 18 years; (3) Complete clinical records.

The following criteria were used to determine exclusion from the study: (1) Pregnant or lactating women; (2) HIV-positive status; (3) Patients in disease remission; (4) Lack of immunoglobulin and lymphocyte subset test results during hospitalization.

Fungal strains and experimental animals

The T. marneffei strain utilized in this study was provided by The First Affiliated Hospital of Guangxi Medical University. The strain was cultured on Sabouraud Dextrose Agar (SDS, Solarbio, China) and incubated at 25 °C for 10 days. Subsequently, colonies were washed with sterile phosphate-buffered saline (PBS), and yeast cells were collected by centrifugation. The harvested cells were resuspended in sterile saline to achieve a concentration of 1 × 10⁷ CFU/mL. The final suspension was stored at 4 °C for subsequent experiments.

32 healthy male SPF-grade SD rats (150–200 g) were procured from the Experimental Animal Center of Guangxi Medical University. All animal experiments comply with the ARRIVE guidelines and were carried out in accordance with the National Research Council’s Guide for the Care and Use of Laboratory Animals. Following 7 days of acclimatisation, the subjects were randomly divided into 2 groups: a control group and a T. marneffei infection group. Rats were appropriately restrained and administered general anesthesia via an intraperitoneal injection of 3% sodium pentobarbital (1.5 mL/kg). Following the induction of complete anesthesia, a rat model of T. marneffei infection was established by intravenous injection of 0.7 mL of T. marneffei suspension (1 × 10⁷ CFU/mL) via the tail vein. The control group was administered an equivalent volume of sterile saline. On days 7 and 14 post-modeling, the spleen was cut off and weighed after anesthesia. The spleen index was calculated as: spleen index=spleen weight (mg)/body weight (g), terminal procedures involved euthanasia via intraperitoneal pentobarbital overdose, followed by collecting blood through the tail vein.

Collection of blood samples

From 31 HIV-negative patients, 8 individuals who were admitted for the first time and had no concurrent infections were selected. Samples were collected from eight healthy controls and eight T. marneffei infected patients at the time of infection diagnosis, prior to the initiation of antifungal therapy. After centrifugation, divide the plasma or serum into multiple 1.5 mL centrifuge tubes and store them at -80℃. Then, isolate the human peripheral blood mononuclear cells (PBMCs) using a lymphocyte isolation medium. After washing twice with PBS, collect the cells following hemolysis.

Preparation of spleen mononuclear cell suspensions

The rat spleens were placed in a 200-mesh sieve. The spleen fragments should then be gently crushed and ground using the piston head of a syringe. The application of PBS is then required to facilitate the filtration of cells through the sieve. The liquid should then be collected into a centrifuge tube, following which the process of centrifugation should be carried out in order to remove the plasma. The cells should then be resuspended in PBS in order to adjust the density of rat splenic lymphocytes to 1 × 10⁶ cells/mL.

Flow cytometry

Transfer 100 µL of the prepared PBMCs or Spleen Mononuclear Cell suspension to a flow cytometry tube. Add an equal volume of each of the following monoclonal antibodies to the tube: CD20-anti-human-FITC, CD38-anti-human-APC and CD138-anti-human-PE or CD138-anti-mouse-APC, CD20-anti-mouse-FITC and CD38-anti-mouse-PE. Incubate at room temperature in the dark for 30 min. After incubation, add PBS to the tube to dilute and remove the antibodies. Centrifuge at 300×g for 5 min, remove the supernatant and repeat the washing step twice. Fix the cells with paraformaldehyde, then analyse them using a flow cytometer after 30 min. The results were analysed using FlowJo for gating and calculating cell subset proportions.

ELISA

Serum samples from healthy controls, TSM patients and rats were tested in accordance with the instructions for the ELISA kit. Absorbance was measured at 450 nm using a microplate reader. Sample concentrations were calculated by substituting the OD values into the equation provided.

RT-qPCR

Total RNA was isolated from PBMCs using TRIzol. The quality and quantity of the RNA were analysed using a spectrophotometer. The RNA samples were then reverse transcribed into cDNA using a reverse transcription kit. SYBR Green I and RT-PCR were then used to detect the levels of IL-21, BAFF and APRIL mRNA relative to the control gene GAPDH. The forward and reverse primer sequences are listed in the Table 1.

Table 1.

Human PCR Primer Sequences

Gene	Forward primer	Reverse primer
BAFF	5’-GGGTCCAGAAGAAACAGGATCT-3’	5’-CTTCTAGGGCACTTCCCCTTT-3’
APRIL	5’-GCAGCAGCACTCTGTCCT-3’	5’-CTCTGTCACATCGGAGTCATCCTT-3’
IL-21	5’-CCAAGGTCAAGATCGCCACA-3’	5’-GGCAGAAATTCAGGGACCAAG-3’
GAPDH	5’-GATTCCACCCATGGCAAATTC-3’	5’-CTGGAAGATGGTGATGGGATT-3’

Statistical analysis

Normally distributed quantitative data were expressed as mean ± SEM in the figures. Comparisons between 2 groups were performed using t-tests, qualitative data were expressed as number and percentage. P < 0.05 indicated statistical significance. Data management and analyses were performed with GraphPad Prism 9.0 software.

Results

Patient baseline characteristics

This study enrolled 48 TSM patients, of whom 31 HIV-negative patients were ultimately included. Of these, 18 were male and 13 were female, with an average age of 51.27 ± 11.3 years. 28 cases presented with disseminated infection, while three cases had localised pulmonary infection. The organs most commonly affected were the lungs (100%), lymph nodes (65%), and bones (58%). The clinical baseline data for the 31 TSM patients are shown in Table 2 and Table S1. Reduced CD19⁺B cells were observed in 58.06% of patients, while elevated IgG and IgM were observed in 74.19% and 54.84% respectively (Fig. 2).

Table 2.

B-lymphocyte-related immune levels in 31 TSM patients

Item	Number of Cases (n)	Proportion (%)
Gender
Male	18	58.06
Female	13	41.94
CD19+ Absolute B-cell count
Increased (> 324 cells/µL)	5	16.12
Normal (180–324 cells/µL)	8	25.81
Decreased (< 180 cells/µL)	18	58.06
IgG
Increased (> 18 g/L)	23	74.19
Normal (8–18 g/L)	6	19.35
Reduced (< 8 g/L)	2	6.45
IgM
Increased (> 1.32 g/L)	17	54.84
Normal (0.84–1.32 g/L)	7	22.58
Decreased (< 0.84 g/L)	7	22.58

Fig. 2.

Proportion of CD19 ⁺ B cells and IgG/IgM in peripheral blood of HIV-negative TSM patients

Decreased plasmablasts and increased plasma cells in TSM patients

Flow cytometry analysis of PBMCs from 8 TSM patients and 8 healthy volunteers revealed that the plasmablast proportion in healthy controls was significantly higher than that in TSM patients (P = 0.0004; Fig. 3A-B), whereas the plasma cell proportion in TSM patients was significantly higher than that in healthy controls (P < 0.0001; Fig. 3C-D). This suggests that T. marneffei infection may influence B cell differentiation pathways in patients, potentially promoting the transformation of plasmablasts into plasma cells.

Fig. 3.

Flow cytometric analysis of B cell subsets in PBMCs from HC and TSM patients. (A, B) Flow cytometry analysis of plasmablast in PBMCs from HC and TSM patients. (C, D) Flow cytometry analysis of plasma cell in PBMCs from HC and TSM patients (n = 8). All data are presented as the mean ± SEM

Elevated IgG, IgM and B-cell differentiation-related factor expression in TSM patients

ELISA analysis of peripheral blood serum revealed significantly higher IgG, IgM antibody concentrations in TSM patients than in healthy controls (P < 0.01; Fig. 4A, B). Furthermore, BAFF and APRIL, which are factors related to B cell differentiation, were found to be significantly higher in TSM patients than in healthy controls (P < 0.01, Fig. 4C-E). qRT-PCR analysis showed that the relative mRNA expression of the BAFF, APRIL and IL-21 genes was higher in TSM patients than in healthy individuals (P < 0.01, Fig. 4F-H). Elevated BAFF and APRIL levels suggest B cell activation and the promotion of pathogen-specific plasma cell expansion, which we hypothesize could lead to increased circulating IgG and IgM antibody levels. Elevated IL-21 indicates enhanced B cell proliferation and plasma cell differentiation capacity.

Fig. 4.

Expression levels of IgG, IgM and B cell differentiation-related factors in serum from HC and TSM patients (n = 8). (A-B) IgG and IgM protein levels of HC and TSM patients assessed by ELISA. (C-E) BAFF, APRIL and IL-21 protein levels of HC and TSM patients assessed by ELISA. (F-H) BAFF, APRIL and IL-21 mRNA levels in PBMCs determined by real-time quantitative RT-PCR. All data are presented as the mean ± SEM

T. marneffei induces splenic inflammation in rats

Compared with the normal control group, the T. marneffei infected groups exhibited significantly increased spleen weight and spleen index (P < 0.001). Furthermore, spleen weight and spleen index were significantly higher in the 14-day T. marneffei infected group than in the 7-day T. marneffei infected group (P < 0.01, Fig. 5A-B). Spleen histopathology revealed: In the normal control group, hematoxylin and eosin (H&E) staining showed clearly demarcated splenic corpuscles of uniform size, with no significant inflammatory cell infiltration in the splenic septa. HE-stained pathological sections of spleens from rats infected with T. marneffei for 7 days showed enlarged splenic lymphoid nodules, a disorganised structure between splenic lobules and abundant inflammatory cell infiltration. Spleen lymphoid nodules in rats infected with T. marneffei for 14 days were significantly more numerous than in those infected for 7 days (Fig. 5C).

Fig. 5.

Alterations in spleen in SD rats during the 14 days following T. marneffei infection. (A-B) Spleen weight and spleen index(mg/g) in SD rats during the 14 days following T. marneffei Infection (n = 8). (C) Histopathological examination of spleen by Hematoxylin and Eosin (H&E) staining in the control group, the 7- and 14-days post-T. marneffei Infection Group. All data are presented as the mean ± SEM. Scale bar 20 μm

Increased proportions of plasmablasts and plasma cells in T. marneffei infected rats

Flow cytometry analysis revealed a statistically significant increase in the proportion of plasma cell precursors in rat spleens at 7- and 14-days post-T. marneffei infection compared to normal controls (P < 0.001, Fig. 6A-B). However, no significant difference in the proportion of plasma cell precursors was observed between the group infected with T. marneffei for 7 days and the normal control group. Nevertheless, the proportion of plasma cells in the spleen of rats infected with T. marneffei for 14 days was significantly higher than in the normal control group (P < 0.0001, Fig. 6C-D).

Fig. 6.

Changes in plasmablasts and plasma cells in rat spleens by flow cytometry. (A-B) Flow cytometry analysis of Plasmablast cell in rat spleens from control group, 7- and 14-days post-T. marneffei infection group and the percentage analysis (n = 8). (C-D) Flow cytometry analysis of Plasma cell in rat spleens from control group, 7- and 14-days post-T. marneffei infection group and the percentage analysis (n = 8). All data are presented as the mean ± SEM

Elevated IgG, IgM and B cell differentiation-related factor levels in T. marneffei infected rats

ELISA assays revealed that serum IgG and IgM antibody concentrations were significantly higher in T. marneffei infected rats than in normal controls at both 7- and 14-days post-infection (Fig. 7A-B). However, no significant differences in IgG or IgM concentrations were detected between the 7-and 14-days T. marneffei infected groups. Secondly, the expression levels of the B-cell differentiation factors BAFF, APRIL and IL-21 in the peripheral blood were higher in the 14-days group than in the normal control group. BAFF and IL-21 expression levels were higher in the 7-days T. marneffei infected group than in the normal control group, while APRIL expression levels were lower in the 7-days T. marneffei infected group than in the normal control group (Fig. 6C-E).

Fig. 7.

Expression levels of IgG, IgM, and cytokine (BAFF, APRIL, IL-21) in rats across different groups (n = 8). (A-B) IgG and IgM of control group, 7- and 14-days post-T. marneffei infection group assessed by ELISA. (C-E) BAFF, APRIL and IL-21 protein levels of control group, 7- and 14-days post-T. marneffei infection group assessed by ELISA. All data are presented as the mean ± SEM

Discussion

This study systematically elucidates the abnormal immunophenotype of B lymphocytes and the underlying regulatory mechanisms in T. marneffei infection, integrating clinical observations, in vitro experiments and animal models. Our key finding is that T. marneffei infection induces B-cell dysregulation, characterised by a reduction in total B-cell numbers and a significant increase in the proportion of plasma cells. This is accompanied by elevated serum IgG and IgM levels and marked upregulation of the BAFF, APRIL and IL-21 signaling pathways. Further animal studies revealed that this process evolves dynamically: early infection is characterised by plasmacytosis, while later stages are characterised by plasma cell accumulation and changes in the immune function and histopathology of the spleen. These findings suggest that T. marneffei infection may alter the intrinsic differentiation trajectory of B cells.

This discovery provides a new perspective on understanding T. marneffei pathogenesis. Previous research has largely focused on T. marneffei ‘s interactions with host non-specific and cellular immunity. There is substantial evidence that macrophages polarise from pro-inflammatory M1 to anti-inflammatory M2 types during the early stages of T. marneffei infection [17, 18], producing anti-inflammatory factors such as IL-10 and TGF-β. This creates conditions that facilitate T. marneffei immune evasion and dissemination. Furthermore, certain antigenic components of T. marneffei pathogens can upregulate the autophagic function of dendritic cells [19–21], facilitating the clearance of intracellular T. marneffei pathogens [22, 23]. Pathogen-associated molecular patterns (PAMPs) can activate the ERK pathway in dendritic cells, thereby upregulating co-stimulatory molecules (CD40, CD80, CCR7 and CXCR4) and chemokine receptors. This promotes the secretion of pro-inflammatory cytokines (IL-12, IFN-γ and TNF-α), which participate in pathogen clearance [22, 24]. In cellular immunity, the cytotoxic role of Th1 cells is well established; however, an imbalance in the Treg-to-Th17 ratio has been shown to play a crucial part in persistent, disseminated infection [7, 25, 26]. For example, Treg cells can suppress effective Th1 and Th17 immune responses by releasing inhibitory factors such as IL-10 and TGF-β, thereby enabling the persistence of pathogens [7, 27]. While recent studies suggest that IgM provides broad-spectrum antifungal immunity through high-avidity binding and complement activation, yet its therapeutic potential against life-threatening systemic mycoses remains underexplored [28]. Although antibodies may play a role in T. marneffei infections as a novel immunosuppressive mechanism, the specific immune characteristics, dynamic changes and upstream regulatory mechanisms of B lymphocytes, which are the source of antibody production, remain an unexplored area in this field.

This study addresses this gap by conducting an in-depth investigation. We demonstrate that the synergistic upregulation of BAFF, APRIL and IL-21 likely constitutes the core molecular basis of the aforementioned B-cell phenotypic and functional abnormalities. Specifically, BAFF and APRIL, which are key factors in B-cell survival and differentiation, may directly promote abnormal plasma cell proliferation and antibody secretion. Meanwhile, IL-21 drives B-cell differentiation towards plasma cells, offering highly promising new targets for future immune intervention strategies against T. marneffei infections. However, the results obtained from the T. marneffei rat model seem to contradict the clinical observations of human patients. This is likely due to the fact that rats are the natural hosts of T. marneffei, and the host’s adaptive immune response to T. marneffei infection is enhanced, with a more rapid and stronger immune response, manifested as continuous activation of B cells. This might be the result of self-immune regulation of B lymphocytes, compensating by increasing the proportion of Plasmablasts. However, the proportion of plasma cells in both the rat model and TSM patients increased, indicating that humoral immunity was effectively activated and produced a large number of antibodies.

What is more, this study has certain limitations. For example, the animal model used does not fully mimic the complex immune characteristics of humans, and there is a lack of long-term clinical follow-up data. Future research could use cutting-edge technologies such as single-cell sequencing to refine the analysis of how different B-cell subsets evolve during T. marneffei infection and future in vitro research will be required to elucidate the precise molecular mechanisms involved. This would allow us to explore their precise association with patient prognosis and lay a solid theoretical foundation for precision immunotherapy regimens.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

T. marneffei

Talaromyces marneffei

TSM

Talaromycosis

BAFF

B-cell activating factor

APRIL

A Proliferation-Inducing Ligand

APCs

Antigen-presenting cells

PBS

Phosphate-buffered saline

PBMCs

Peripheral blood mononuclear cells

Author contributions

Z. Mai and Y. Wei designed the study and analyzed data. Z. Mai wrote a draft of the manuscript. Y. Wei, M. Pan and G. Fang contributed to data collection and investigation. J. Deng, Y. Huang, H. Lin and W. Ruan drafted or revised the article and gave final approval of the version to be published. J. Zhang was responsible for critical revision of the manuscript. All authors contributed to manuscript revision. All authors read and approved the final manuscript.

Funding

This work was supported by grants from the National Natural Science Foundation of China (No. NSFC82570010), Futian Healthcare Research Project (grant numbers: FTWS2025099), the Natural Science Foundation of Guangdong Province (grant numbers: 2024A1515011073), and the Shenzhen Science Technology Program (grant numbers: JCYJ20230807110914029), the Science and Technology Department of Guangxi Zhuang Autonomous Foundation of Guangxi Key Research and Development Program (grant numbers: 2024AB17061).

Data availability

All the relevant raw data and materials are freely available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

This study and animal experiments were approved by the Ethics Committee of The Eighth Affiliated Hospital of Sun Yat-Sen University (number: 2022-040-01, 2022-040-02). Written informed consent was obtained from all participants. This study was conducted in accordance with the Declaration of Helsinki.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.