T‐cell subsets in Pneumocystis pneumonia

Abstract

Pneumocystis pneumonia (PCP), caused by the opportunistic fungal pathogen Pneumocystis, remains a common fungal infection among immunosuppressed individuals. T cells are known to play a critical role in host defences against Pneumocystis. Two functional groups of T cells exist: CD4⁺ T and CD8⁺ T. Distinct subsets of CD4⁺ and CD8⁺ T cells have been shown to participate in PCP development through specific cytokines and interactions with other immune cells, significantly influencing the pulmonary fungal burden and disease severity. However, the host T‐cell responses required for an effective adaptive immune response to PCP remain incompletely defined. In this review, we explore how an in‐depth understanding of the integrated and well‐defined functions of different T‐cell subsets in the immune defence against Pneumocystis could provide insights into facilitating the development of anti‐Pneumocystis treatment.

In this article, we discuss the functional heterogeneity of T‐cell subsets in Pneumocystis pneumonia pathogenesis. Upon infection, naïve T lymphocytes undergo activation and differentiation into distinct effector populations, which orchestrate fungal clearance through polarised cytokine production.

Pneumocystis was thought to be a protozoan for approximately a century; however, it was reclassified as a fungus in 1988. ¹ Despite this reclassification, the species' obligate dependence on the mammalian lung environment makes it difficult to cultivate in vitro, hindering research into its biology and treatment strategies. ² As an opportunistic fungus, Pneumocystis can cause lethal pneumonia, particularly in individuals with compromised immune systems, ³ including those with congenital or acquired immunodeficiencies, such as human immunodeficiency virus (HIV), ⁴ and non‐HIV individuals undergoing immunosuppressive therapies. ⁵ , ⁶ , ⁷ According to global burden research, Pneumocystis pneumonia (PCP) causes approximately 505 000 illnesses and 214 000 deaths worldwide per year. ⁸ Importantly, Pneumocystis is now recognised as one of the most common invasive fungal infections in infants worldwide, ⁹ , ¹⁰ as highlighted by the landmark Pediatric Emergency Research Network study. ¹¹ The clinical symptoms of PCP are nonspecific and commonly include dry cough, low‐grade fever, progressive dyspnoea and chest pain. ¹² Pneumocystis jirovecii remains to be a prevalent opportunistic pathogen, especially among immunocompromised individuals.

In immunocompromised individuals with PCP, there is a substantial reduction in T cells and B cells in the lungs, contrasting with the more robust immune cell presence in immunocompetent individuals; however, the proportion of myeloid cells remains relatively stable in these compromised hosts. ¹³ Innate immune cells, such as antigen‐presenting cells, serve as the first line of defence and contribute to T‐cell priming in response to Pneumocystis. ¹⁴ Over the past two decades, T cells have emerged as central players in PCP pathogenesis, acting as both drivers and regulators of the immune response. ¹⁴ , ¹⁵ Peripheral CD4⁺ T cell counts are now routinely used to stratify infection risk in HIV‐positive patients. ¹⁶ , ¹⁷ , ¹⁸ It was observed that the percentage of T cells gradually increased in the lungs during Pneumocystis infection. ¹⁹ , ²⁰ We previously delineated the dynamic landscape of the host immune environment in Pneumocystis‐infected mice from 0 to 5 weeks, revealing that T cells consistently represent one of the most predominant immune cell populations in the lungs. ²¹ A subsequent study has confirmed that pre‐depletion of CD4⁺ and CD8⁺ T cells effectively prevents respiratory dysfunction and mortality in mice with PCP, even during progressive lung infection. ²² Our findings revealed that clonal CD4⁺ T cells, characterised by elevated T‐cell activation‐related gene expression, constitute the primary responders to Pneumocystis infection. Additionally, a reduction in T‐cell receptor (TCR) diversity among CD4⁺ T cells and an increase in CD8⁺ T‐cell diversity have been observed in Pneumocystis‐infected mice. ²³ These findings underscore the critical role of T cells in orchestrating the immune response to Pneumocystis infection and highlight their potential as therapeutic targets.

T‐cell heterogeneity has been shown to significantly influence disease progression and severity. ²⁴ , ²⁵ CD4⁺ T cells have been extensively studied in the context of T‐cell‐mediated adaptive immunity to PCP, whereas fewer investigations have been conducted on the role of CD8⁺ T cells. ²⁶ , ²⁷ , ²⁸ , ²⁹ , ³⁰ This review focuses on the role of αβ T‐cell subsets as evidenced in recent studies and summarises their central functions in adaptive immunity against Pneumocystis infection (Figure 1 and Table 1).

Figure 1.

Graphical representation of T cells in PCP. Upon arrival in the lungs, Pneumocystis is recognised by APCs, which subsequently induce the differentiation of naïve T cells. Naïve CD4⁺ T cells differentiate into more specialised functional subsets and exert immune responses by through the secretion of specific cytokines. Naïve CD8⁺ T cells contribute to host defence a Tc1‐type response. AEC, alveolar epithelia cell; APC, antigen presenting cell; Tc1, cytotoxic CD8⁺ T cell; Tfh, follicular helper T cell; Th, T helper cell; Treg, regulatory T cell.

Table 1.

T ‐ cell subsets and key cytokines that participate in PCP progression

T‐cell subsets	Animal model of immunosuppression	Treatment	Final fungal burden	Lung inflammation	References
CD4+ T cell	Reconstituted SCID mice	Anti‐CD4 mAb	↑	n.d.	28
	Balb/c mice	Anti‐CD4 mAb	↑	↑	31
	Reconstituted SCID mice	Anti‐CD4 mAb	↑	↑	32
Th1	Stat4‐deficient mice	—	↓	n.d.	33
	Ifn‐γ‐deficient mice	—	†	n.d.	34
	Ifn‐γ receptor‐deficient mice	—	†	↑	35
	γδ‐TCR+ T‐cell‐deficient mice	IFN‐γ neutralisation	†	n.d.	36
	Reconstituted SCID mice	IFN‐γ neutralisation	†	↑	37
Th2	Stat6‐deficient mice	—	↓	n.d.	33
	Il‐4‐deficient mice	—	†	†	38
Th9	Il‐9‐deficient mice	—	†	n.d.	39
Th17	Il‐17‐deficient mice	‐	†	↑	40
	C57BL/6 mice	IL‐17 neutralisation	↑	n.d.	41
	Stat3 fl/fl Cd4 Cre transgenic mice	—	↑	n.d.	33
Tfh	Il‐21‐deficient mice/Il‐21 receptor‐deficient mice	—	↑	n.d.	33
Treg	Il‐10‐deficient mice	—	†	↓	42 , 43
	C57BL/6 mice	Anti‐CD25 mAb	†	↑	44
CD8+ T	Balb/c mice	Anti‐CD8 mAb	†	†	31

†, not significant; mAb, monoclonal antibody; n.d., not done; SCID, severe combined immunodeficiency; Stat, signal transducer and activator of transcription; TCR, T‐cell receptor; Tfh, follicular helper T cell; Th, T helper cell; Treg, regulatory T cell.

CD4 ⁺ T cells

CD4⁺ T cells serve as central regulators in the immune response by assisting other immune cells, such as B cells, macrophages, neutrophils, eosinophils, and basophils, through the secretion of various cytokines and chemokines. ⁴⁵ CD4⁺ T cells differentiate into distinct functional subsets that orchestrate immune responses, exerting effects that either enhance or suppress the activity of other immune cells while directly mediating pro‐inflammatory or anti‐inflammatory influences on resident cells. ⁴⁵ , ⁴⁶ This functional diversity underscores the vital role of CD4⁺ T cells in PCP.

Clinical studies have highlighted the importance of CD4⁺ T cells in PCP. ¹⁶ , ⁴⁷ HIV‐infected patients with CD4⁺ T‐cell counts below 200 per cubic millimetre are at a significantly higher risk of developing PCP. ²⁷ Additionally, the Pneumocystis burden is negatively correlated with the level of circulating CD4⁺ T cells. ⁴⁸ Furthermore, the depletion of CD4⁺ T cells, whether genetically or via antibody treatment, suppresses Pneumocystis infection progression. ²⁸ , ³¹ , ³² This is most definitively demonstrated in humans by genetic conditions such as MHC class II deficiency (Bare Lymphocyte Syndrome), where the absence of antigen‐specific CD4⁺ T‐cell responses invariably leads to PCP, as illustrated by clinical case reports of affected children. ⁴⁹ , ⁵⁰ , ⁵¹ Notably, anti‐CD4 monoclonal antibodies significantly diminish the ability to control the fungal burden in severe combined immunodeficiency (SCID) mice with naturally acquired PCP that were previously transferred spleen cells to enable infection clearance, emphasising the critical role of CD4⁺ T cells in immune defences. ²⁸ In summary, the aforementioned studies highlight the pivotal function of CD4⁺ T cells in PCP, demonstrating that identifying key mechanisms is crucial for further understanding the immunopathogenesis of this disease.

Th1

Th1 cells mediated inflammation and pathogen clearance. ⁵² The hallmark features of Th1 cells include high expression of T‐box expressed in T cells and the presence of chemokine receptors CXC‐chemokine receptor 3 and CC‐chemokine receptor 5. ⁵³ Cytokines produced by Th1 cells, such as IFN‐γ and IL‐2, have been found to play a significant role in influencing the course of PCP. ⁵⁴ , ⁵⁵ , ⁵⁶ The proportion of pulmonary Th1 cells is significantly higher in infected lungs than in uninfected controls, ⁵⁷ and a low Th1 cell frequency is associated with a poor prognosis. ⁵⁷ , ⁵⁸ Numerous studies have demonstrated that the proportion of Th1 cells among CD4⁺ T cells increased following Pneumocystis infection. ²³ , ⁵⁷ , ⁵⁹ Research focussing on intrinsic signal transducer and activator of transcription (STAT) family members that control T‐cell differentiation has indicated that Stat4‐deficient (Th1) cells show only a modest impairment in their ability to clear Pneumocystis infection. ³³

IFN‐γ, a key cytokine produced by Th1 cells, has been implicated in conferring protection against immunopathogenesis while delaying fungal clearance during PCP. ⁶⁰ , ⁶¹ Studies in rodent models have demonstrated that IFN‐γ reduces the fungal burden. ⁶² , ⁶³ , ⁶⁴ IFN‐γ acts directly by stimulating alveolar macrophages, which triggers the L‐arginine‐dependent cytocidal pathway, ⁶⁵ and by priming CD8⁺ T cells. ⁶² However, IFN‐γ is not essential for Pneumocystis clearance, as mice deficient in IFN‐γ or its receptor exhibit unimpaired fungal clearance. ³⁴ , ³⁵ Furthermore, IFN‐γ neutralisation does not affect Pneumocystis elimination. ³⁶ , ³⁷ In contrast, IFN‐γ plays an important regulatory role in controlling inflammation. ³⁷ In a mouse model of graft‐versus‐host disease, anti‐IFN‐γ treatment exacerbated Pneumocystis‐driven interstitial pneumonia. ³⁷ Consistent with this observation, SCID mice inoculated with Pneumocystis and reconstituted with splenocytes from IFN‐γ^−/− mice exhibited prolonged and exacerbated inflammatory responses compared with those reconstituted with splenocytes from wild‐type (WT) mice. ³⁷ Although IFN‐γ is the most extensively studied Th1 cytokine in PCP, the involvement of other Th1‐associated cytokines in PCP pathogenesis remains plausible. Notably, dedicated studies examining these alternative cytokines are currently lacking.

Th2

Th2 cells, regulated by GATA binding protein 3, play an important role in coordinating immune responses through the synthesis of IL‐4, IL‐5 and IL‐13. ⁶⁶ , ⁶⁷ Pneumocystis acts as a respiratory allergen that can initiate a Th2‐type immune response. ⁶⁸ , ⁶⁹ In immunocompetent mice, Th2 cells are dominant both in the lung and lymph nodes, with increases in Th2 cells in lung tissues persisting for 3 weeks before declining. ⁷⁰ However, their role is not indispensable, as STAT6‐deficient (Th2) mice exhibit only a modest decrease in their ability to clear Pneumocystis infection. ³³ In further confirmation of the absence of complementary roles between Th1 cells and Th2 cells, infection experiments with STAT4/STAT6‐deficient mice demonstrated no additional predisposition to Pneumocystis murina. ³³ Interestingly, Th2‐dominant immunity is associated with alleviated immunopathological outcomes. PCP mice treated with both anti‐IFN‐γ and Th2‐promoting cytokines maintained enhanced fungal clearance capacity, demonstrating less severe pulmonary immunopathology and faster recovery rates compared to those receiving anti‐IFN‐γ alone. ⁶¹ Intriguingly, Th2 cells are involved in the development of inducible bronchus‐associated lymphoid tissue, which may enhance host defences in PCP. ⁷¹

Peripheral blood mononuclear cells obtained from HIV‐infected patients with a history of PCP exhibited significantly elevated IL‐4 secretion upon major surface glycoprotein stimulation. ⁷² Additionally, the effective fungal clearance observed in IL‐4^−/− mice indicates that Pneumocystis resistance mechanisms can operate independently of IL‐4, suggesting that IL‐4 may have a limited effect on PCP pathogenesis. ³⁸ The levels of IL‐13 in bronchoalveolar lavage fluid (BALF) were notably lower in infected versus uninfected individuals, whereas the levels of IL‐5 showed no significant differences. ⁷³ The use of plasmid‐mediated IL‐5 expression resulted in elevated eosinophil counts and reduced fungal infection in mice lacking CD4⁺ T cells. ⁷⁴ These findings suggest that Th2 cells may not be essential for controlling fungal load but contribute to alleviating pulmonary inflammation. ³⁸ , ⁷⁴

Th9

Th9 cells are characterised by the transcriptional factor PU.1 ⁷⁵ ; however, their immunological function in combating PCP is less reported. ¹⁴ IL‐9, the signature cytokine of Th9 cells, exerts multiple biological effects, including promoting the migration and activation of mast cells and eosinophils, and inducing mucus secretion from epithelial cells. ⁷⁶ The role of the Th9/IL‐9 axis in fungal immunity is complex and context‐dependent. On the one hand, its proper function is crucial for host defence, as evidenced by patients with chronic mucocutaneous candidiasis who exhibit a broad T helper defect encompassing Th9 cells and significantly reduced IL‐9 production. ⁷⁷ On the other hand, its dysregulation can be pathogenic, as prominently displayed in allergic bronchopulmonary aspergillosis, where Th9 cells drive a deleterious allergic response to Aspergillus fumigatus, fuelling eosinophilic inflammation and mucus hypersecretion. ⁷⁸

Given these divergent roles, the function of Th9/IL‐9 in PCP has been an open question. Addressing this gap, we have previously demonstrated that Il‐9^−/− mice exhibit no significant difference in the final clearance of Pneumocystis organisms from lung tissues; these gene‐depleted mice demonstrate a markedly reduced pulmonary fungal burden 3 weeks after Pneumocystis infection compared with that observed in WT mice. This reduction was accompanied by an increased absolute number of Th17 cells in both BALF and lung tissues, along with an augmentation of IL‐17A in BALF, suggesting an enhanced pulmonary Th17 response in Il‐9^−/− mice with PCP. In vitro differentiation experiments revealed that splenocytes from Il‐9^−/− mice are more prone to differentiate into Th17 cells, with higher concentrations of IL‐17A in the cell culture supernatant. ³⁹ Thus, our study provides valuable insights into the immunoregulatory role of IL‐9 in PCP. It suggests that, unlike its protective role in candidiasis, the IL‐9 pathway may act as a negative regulator of protective immunity in PCP, potentially by constraining the Th17 response. This positions IL‐9 signalling not merely as an understudied topic, but as a promising and novel immunotherapeutic target for modulating host defence in PCP.

Th17

The subset of CD4⁺ T cells that generate IL‐17A, known as Th17 cells, exhibits remarkable adaptability in the immunopathogenesis of various autoimmune disorders and is regulated by retinoid‐related orphan receptor gamma t. ⁷⁹ Pneumocystis colonisation in COPD patients is associated with elevated IL‐17 levels. ⁸⁰ Single‐cell TCR sequencing of Pneumocystis‐infected mice revealed that Th17 cells were predominantly clonal CD4⁺ T‐cell subsets, displaying characteristics similar to those of tissue‐resident memory Th17 cells. ²³ IL‐17 depletion leads to delayed elimination of Pneumocystis and worsened pulmonary damage in comparison with observations in WT PCP mice, ⁴⁰ emphasising the critical role of Th17 cells in controlling Pneumocystis infection. Notably, WT mice treated with anti‐IL‐17 antibodies showed a 36‐fold increase in fungal burden 3 weeks after Pneumocystis inoculum. ⁴¹ Anti‐IFN‐γ treatment enhances fungal clearance, which can be blocked by anti‐IL‐17 antibodies. ⁶¹ Our previous study further demonstrated that IL‐17 neutralisation exaggerates the fungal burden in an Il‐9^−/− mouse model of PCP. ³⁹ Additionally, Th17 cells, together with Th2 cells, contribute to the formation of the inducible bronchus ‐ associated lymphoid tissue structure. ⁷¹ Th17 cells not only influence the progression of Pneumocystis infection through the secretion of IL‐17, but also exert their functions via complex interactions with other T‐cell subsets. ³⁹ , ⁴⁰ , ⁴¹ , ⁷¹

Tfh

Follicular helper T (Tfh) cells, which are characterised by the expression of the transcription factor B‐cell lymphoma 6, reside in B‐cell follicles and predominantly produce IL‐21. ⁸¹ , ⁸² Despite the importance of Tfh cells in humoral immunity, few studies have been conducted on their role in Pneumocystis infection. Both Il‐21^−/− mice and patients carrying IL‐21 receptor mutations show increased susceptibility to PCP, indicating a potential protective role for Tfh cells in combating Pneumocystis infection. ³³ , ⁸³ Moreover, our previous study provided further insights into the functions of Tfh cells, demonstrating that they might regulate cell proliferation, cell–cell adhesion and the positive regulation of B‐cell differentiation, thereby highlighting their potential contribution to immune defence mechanisms during Pneumocystis infection. ²¹ The critical importance of Tfh is underscored by the susceptibility of patients with CD40L deficiency to PCP. As this gene is critical for Tfh function and germinal centre formation, its failure disrupts antibody affinity maturation. However, the fact that pure B‐cell deficiencies (e.g. BTK deficiency) confer a much lower risk of PCP suggests that the essential role of Tfh extends beyond providing B cell help, likely encompassing direct effector mechanisms such as macrophage activation. ⁸⁴

Treg

Regulatory T cells (Tregs), a specialised subset of CD4⁺ T cells, play an important role in suppressing immune responses and are characterised by the expression of the key transcription factor forkhead box protein 3 (FOXP3). ⁸⁵ , ⁸⁶ Treg cells suppress the function of other immune cells through IL‐10, TGF‐β, CTLA‐4 and CD25. ⁸⁷

Patients with IPEX syndrome resulting from FOXP3 mutations are at risk for developing PCP. ⁸⁸ , ⁸⁹ However, it remains challenging to definitively attribute this risk solely to the FOXP3 mutation itself, as these patients frequently receive potent immunosuppressive therapies to manage their severe autoimmunity, which independently predisposes them to opportunistic infections. ⁹⁰ Therefore, while FOXP3 deficiency is clinically associated with PCP, iatrogenic immunosuppression is likely a significant contributing factor. ⁸⁸ In rodent models, faster fungal clearance and heightened inflammation occur when IL‐10 or Tregs are depleted, ⁴² , ⁴³ , ⁴⁴ indicating a complex role for Treg cells in balancing immune responses during PCP. The adoptive transfer of CD25⁺ CD4⁺ T cells to infected SCID mice alleviates inflammation without affecting the fungal load. ⁹¹ Similarly, intratracheal administration of IL‐10 in CD4⁺ T‐cell‐depleted mice has been found to reduce inflammation without affecting Pneumocystis clearance. ⁹² These findings suggest that Tregs primarily modulate inflammation rather than directly influencing fungal clearance.

CD8 ⁺ T cells

CD8⁺ T cells play a critical role in identifying and eliminating pathogens and abnormal cells through cytotoxic mechanisms. ⁹³ , ⁹⁴ Clinical studies have delineated the importance of CD8⁺ T cells in PCP outcomes. ⁹⁵ , ⁹⁶ In non‐HIV patients diagnosed with PCP, a low CD8⁺ T‐cell count is identified as an independent risk factor for poor prognoses. ⁹⁷ Furthermore, in another study, low levels of CD8⁺ T cells were found to be strongly related to PCP and its mortality, ⁹⁸ indicating their potential role in disease progression.

In contrast to CD4⁺ T cells, the role of CD8⁺ T cells in combating fungal infection during PCP is less clear and remains controversial. It has been suggested that CD8⁺ T‐cell deficiency alone does not impair the clearance of Pneumocystis; however, depletion of both CD4⁺ and CD8⁺ T cells exacerbates pulmonary infection, ³¹ implying an important interplay between CD8⁺ T cells and CD4⁺ T cells in PCP. However, CD4⁺ cell‐depleted mice presented opposite results. ⁶² , ⁹⁹ It has been found that IFN‐γ induces an increase in IFN‐γ‐positive CD8⁺ T cells, resulting in stronger clearance of Pneumocystis infection. ⁶² Furthermore, CD8⁺ T cells demonstrating a cytotoxic CD8⁺ T (Tc1) response to Pneumocystis in BALB/c mice effectively killed the pathogen in vitro and facilitated its clearance in adoptive transfer models. ¹⁰⁰ Subsequent evidence suggested that recombinant human IL‐7 can increase the number of CD8⁺ T cells in CD4‐depleted mice, facilitating the clearance of Pneumocystis. ¹⁰¹ Additionally, it has been found that the fungal burden remains consistent across all time points, regardless of the presence of CD8⁺ T cells, implying that the role of CD8⁺ T cells in Pneumocystis clearance is not well‐supported. ⁹⁹ The conflicting findings regarding CD8⁺ T cells in PCP may result from the heterogeneity within the CD8⁺ T‐cell population. Some subsets, such as Tc1 cells, exhibit protective functions; however, others may contribute to lung injury without significantly impacting fungal clearance.

Existing studies have documented that CD8⁺ T cells play a causative role in lung injury during PCP. Pneumocystis infection triggers a strong infiltration of CD8⁺ T cells into the alveolar and interstitial areas of the lung, especially when CD4⁺ T cells are absent. This infiltration causes lung injury, evidenced by decreased pO₂ and albumin leakage into the BALF, similar to observations in other interstitial lung diseases mediated by CD8⁺ T cells. ³⁰ , ¹⁰² , ¹⁰³ In one study, non‐T cytotoxic‐1 CD8⁺ T cells showed no in vitro activity and were linked to lung damage when transferred. ¹⁰⁰ Aligning with this conclusion, Tc1 CD8⁺ T cells have also been identified as a good prognostic marker among autoimmune patients. ⁵⁷ This diversity highlights the need for an in‐depth investigation to clarify the specific roles of different CD8⁺ T‐cell subsets in PCP.

Immune reconstitution inflammatory syndrome and PCP

Immune reconstitution inflammatory syndrome (IRIS) is characterised by the clinical deterioration or new onset of an infectious disease following the reversal of immune deficiency. ¹⁰⁴ This reversal can be triggered by multiple events, including antiretroviral therapy (ART) in HIV patients, neutrophil recovery after chemotherapy or stem cell transplantation, suboptimal immunosuppression in solid organ transplant recipients, and postpartum immune reconstitution. ¹⁰⁵ , ¹⁰⁶ IRIS complicates 4–5% of cases of PCP in patients with HIV, occurring following the initiation of both ART and antimicrobial therapy for PCP. ¹⁰⁷ , ¹⁰⁸ The withdrawal of glucocorticoids or other immunosuppressants, which has been associated with clinical deterioration in a considerable proportion of patients, contributes to increasing PCP mortality rates in non‐HIV populations. ¹⁰⁹ According to one case series, elevated pre‐ART HIV viral burden and reduced CD4⁺ T cell counts are predictive of PCP‐IRIS after ART initiation. ¹¹⁰

The first experimental model of IRIS associated with PCP was established using Rag1 ^−/− and SCID mice. ³⁰ , ⁴⁴ In this model, immunodeficient mice were infected with Pneumocystis murina for 2 weeks and subsequently reconstituted with whole splenocytes or CD4⁺ T cells. ³⁰ , ⁴⁴ Studies using this system have demonstrated that CD4⁺ T cells contribute to the pathological immune response in PCP‐IRIS. Depletion of Tregs was found to exacerbate IRIS in this model. ⁴⁴ Furthermore, IFN‐γ is a critical mediator in the exacerbated inflammatory response, decreasing the CD8⁺ Foxp3⁺ regulatory T cell subset in PCP‐IRIS. ³⁵ Moreover, CD8⁺ T cells are the primary inflammatory mediators in the absence of CD4⁺ T cells. ³²

Omics approaches in PCP

The increasing accessibility of omics platforms has enabled research using high‐throughput sequencing strategies to map dynamic alterations in the host pulmonary immune response and the pathogenic landscape of PCP.

Hu et al. ¹¹¹ used transcriptome analysis to examine the immune response in the lungs of corticosteroid‐treated PCP mice, revealing downregulated expression of genes associated with innate immunity, including antigen processing and presentation, inflammatory response and phagocytosis, as well as those involved in adaptive B‐ and T‐cell‐mediated immunity. These findings partly explain the susceptibility of patients using glucocorticoids to Pneumocystis and provide potential targets for clinical intervention. Unlike bulk RNA sequencing, single‐cell RNA sequencing (scRNA‐seq) enables the identification of specific groups of immune cell subtypes and the analysis of immune cell profiles during infection processes. ¹³ , ²¹ TCRs and B‐cell receptors (BCRs) are key components of the adaptive immune system and play a crucial role in responding to pathogen infections. ¹¹² , ¹¹³ , ¹¹⁴ Integrated scRNA‐seq and BCR sequencing of murine lungs revealed that Pneumocystis infection induces persistent plasma cell expansion, reduced BCR diversity, biased V(D)J gene usage, and an expanded naïve B cell subset marked by high ATF3 expression, providing insights for biomarker and immunotherapy development. ¹¹⁵ In a complementary study, integrated scRNA‐seq and TCR‐seq analysis showed that clonally expanded CD4⁺ T cells dominate the immune response in Pneumocystis‐infected mice, accompanied by reduced TCR diversity and biased VDJ gene usage. A distinct population of clonal Th17 cells exhibiting a tissue‐resident memory‐like phenotype was also identified. ²³ Qiao et al. collected TCR data from plasma of 10 patients with PCP and HIV‐1 infection, eight asymptomatic HIV‐infected patients, and eight healthy subjects, revealing that patients with PCP exhibited different immune cell proportions and reduced TCR pool diversity compared with healthy individuals. Furthermore, anti‐PCP treatment could restore cytokine dynamics and TCR diversity, suggesting that the immune response plays a key role in host infection control. ¹¹⁶

While Th1, Th2 and Th17 cell responses are all elicited during murine infection, studies using gene‐deficient mouse models have demonstrated that these subsets are individually insufficient to mediate pathogen clearance. ³³ , ³⁸ , ⁴⁰ These findings suggest that intercellular crosstalk among immune subsets, rather than isolated effector functions, may play a pivotal role in PCP immunopathogenesis. Current high‐throughput sequencing studies predominantly focus on delineating alterations within discrete cell populations, as previously discussed. ¹³ , ²¹ , ¹¹⁵ However, the inability of single lineage‐deficient models to recapitulate PCP susceptibility phenotypes implies that coordinated multicellular networks, mediated by cell–cell communication, are critical for host defences. This gap is underscored by the limited exploration of immune synapse dynamics, cytokine chemokine circuits, or receptor ligand interactions specific to the Pneumocystis‐infected lung microenvironment.

Future directions

Despite progress in defining the contributions of specific cell populations to the pathogenesis of PCP, an in‐depth exploration of the crosstalk and cooperative networks among these cells remains lacking. Future studies should therefore prioritise elucidating these complex cellular interactions, both spatially and temporally, across the spectrum of infection. Key directions include applying high‐dimensional single‐cell technologies, such as spatial transcriptomics and multiplexed imaging, to map intercellular communication within the lung microenvironment. Unravelling this intricate cellular crosstalk will not only advance our fundamental understanding of PCP immunopathogenesis but also reveal novel therapeutic targets for modulating host immunity to improve disease outcomes.

The current standard therapeutic regimen for PCP remains trimethoprim‐sulfamethoxazole (TMP‐SMX), which serves as the first‐line treatment. ⁴ , ¹¹⁷ Toxicities associated with TMP‐SMX, such as rash, fever, nephrotoxicity, bone marrow suppression, electrolyte disorders and hepatotoxicity, necessitate a treatment change in up to 40% of patients. ¹¹⁸ , ¹¹⁹ , ¹²⁰ Other alternative agents include dapsone, pentamidine, atovaquone, clindamycin and primaquine, ¹²¹ which are less efficacious than TMP‐SMX and possess their own profiles of serious toxicities. ¹²² Although T‐cell therapies have not yet been explored for PCP, pioneering work in invasive fungal infection highlights their transformative potential. ¹²³ Given the limitations of current pharmacotherapies, the development of T‐cell‐based immunotherapies for PCP represents a compelling and innovative direction. Ultimately, harnessing T‐cell immunity may pave the way for targeted, toxin‐sparing therapeutic modalities that overcome the drawbacks of existing antifungal regimens.

Conclusion

Host defence against Pneumocystis is uniquely dependent on functional T‐cell immunity, a principle unequivocally demonstrated by the specific spectrum of human inborn errors of immunity. Profound susceptibility to PCP is observed in patients with severe T cell deficiencies, such as SCID (e.g. because of mutations in in IL2RG, ¹²⁴ IL7R, ¹²⁵ RAG1 ¹²⁶ and STAT3 ¹²⁷ ) and in those with impaired function (e.g. CD40L ⁸⁴ ). A highly informative dichotomy, however, reveals the distinct nature of this defence: mutations affecting the IL‐23/IL‐17/Th17 axes (e.g. IL12B ¹²⁸ and RORC ¹²⁹ ) or the IFN‐γ signalling pathway ¹³⁰ confer susceptibility to intramacrophage infections such as mycobacterial disease; however, they do not typically predispose otherwise healthy individuals to PCP. This key distinction indicates that the protective host response to Pneumocystis is less dependent on the classical Th1 and Th17 pathways required for intracellular pathogen control; instead, it is more reliant on other T‐cell functions, potentially including CD4⁺ T‐cell‐mediated macrophage activation through alternative pathways and B‐cell help for antibody production. Furthermore, the occurrence of PCP in patients with certain forms of humoral immunodeficiency (e.g. agammaglobulinemia because of BTK mutations ¹³¹ ) suggests that while antibodies may play a contributory role, they are individually insufficient to confer protection in the absence of adequate T‐cell immunity.

While murine models have implicated numerous CD4⁺ T helper subsets (Th1, Th2, Th9, Th17, Tfh) in fungal control, the findings are often inconsistent, and the roles of CD8⁺ T cells and the precise mechanisms of inter‐cellular crosstalk remain poorly defined. This heterogeneity underscores the complexity of the immune response to Pneumocystis and highlights a critical gap between phenomenological observations and mechanistic understanding.

Bridging this gap necessitates a paradigm shift in research strategies. Future efforts must prioritise the application of high‐resolution tools, such as single‐cell and spatial transcriptomics on human PCP samples, to move beyond murine models and directly map the landscape of protective versus pathological T‐cell responses in the human lung. This refined understanding will be the foundation for rational therapeutic design, shifting the focus from broad‐spectrum antimicrobials towards targeted immunotherapies. Promising avenues include engineering adoptive T cell transfers or modulating specific cytokine pathways (e.g. IL‐9, IL‐17) to augment defective immunity, particularly in immunocompromised hosts.

In conclusion, unravelling the intricate functional network of T‐cell subsets in PCP is no longer just a biological question but a translational imperative. By defining the precise rules of T‐cell‐mediated host defence, we can pioneer a new class of immune‐complementary therapies that overcome the limitations of current antifungals and finally improve outcomes for this challenging infection.

Author contributions

Yuxi Chen: Writing – original draft; visualization. Hengmo Rong: Writing – original draft; visualization; funding acquisition. Ting Li: Writing – original draft. Chao Zhang: Writing – original draft. Huqin Yang: Writing – original draft. Han Sun: Writing – original draft. Dong Wang: Writing – original draft. Xiaoxia Zhou: Writing – original draft. Kan Zhai: Conceptualization; writing – review and editing. Zhaohui Tong: Conceptualization; writing – review and editing; funding acquisition.

Conflict of interest

None.

Ethics approval statement

The authors have nothing to report.

Data availability statement

Data sharing not applicable to this article as no datasets were generated or analysed during the current study.