Lymphocyte subset analysis to evaluate the prognosis of HIV-negative patients with pneumocystis pneumonia

Pneumocystis pneumonia (PCP) is a life-threatening interstitial pneumonia caused by Pneumocystis jirovecii in immunocompromised patients [1, 2]. PCP has long been known for its high prevalence in acquired immunodeficiency deficiency syndrome (AIDS) patients [3], but the incidence of PCP in human immunodeficiency virus (HIV)-positive patients has been decreased largely due to the broader use of highly active antiretroviral therapy (HAART) and trimethoprim-sulfamethoxazole (TMP/SMZ) prophylaxis when the CD4+ T cell count is < 200/μL [4, 5]. Recently, PCP has been frequently diagnosed in HIV-negative patients, as immunosuppressive regimens are being increasingly used in a wide range of patients. The most common underlying conditions of PCP in non-HIV-infected patients are haematological malignancies, followed by solid tumours, autoimmune diseases, solid organ transplantation, etc. [6, 7]. The mortality rate among patients with PCP in this population is 30–60% [6]. HIV-negative PCP patients are more likely to have acute onset, more severe symptoms, and worse prognosis than HIV-positive patients [6]. Identifying potential prognostic factors in PCP patients could help clinicians choose appropriate treatment strategies and monitoring levels.

The major portion of the PCP process results from the host inflammatory response to apparent infection with P. jirovecii rather than direct tissue damage from P. jirovecii itself [8, 9]. Because PCP is a type of opportunistic infection (OI), lymphocyte subsets can be used as important indicators of the immune function of the body and can help with the clinical diagnoses of some infectious diseases, which is of great significance for analysing the pathogenesis, observing the curative effect, and predicting the prognosis [10]. T cells are known to be involved in the suppression of the autoimmune response and hyperinflammation mediated by CD4+ T cells [11]. B cells and NK cells are critical for an effective CD4+ T cell response that can control Pneumocystis infection [12]. In this article, we analysed the peripheral blood lymphocyte subsets of HIV-negative PCP patients to determine the influence of T lymphocyte, B lymphocyte and natural killer cell (NK cell) counts on prognosis.

In this study, all 88 HIV-negative patients with PCP had long-term and varying degrees of immunosuppression, mostly manifesting as diffuse lymphocyte depletion. Dichotomizing patients around the threshold calculated from ROC analysis, we identified a CD8+ T cell count < 300/μL as an independent risk factor for death in PCP patients. Additionally, the HR of CD8+ T cell count < 300/μL was 6.799, which was associated with 3-month mortality. Although low CD4+ T cell and NK cell counts were not independent risk factors for death, the mortality rates of PCP patients decreased sequentially with increasing CD4+ T cell and NK cell counts.

In the HIV epidemic of the 1980s, PCP was known as the most common OI among AIDS patients. In recent years, immunosuppressive therapy and biological agents, especially when used in combination, have increased the risk of infection by opportunistic pathogens such as P. jiroveci [15]. Many large retrospective cohort studies have shown that a low CD4+ T cell count is a risk factor for death in HIV-positive patients infected with PCP [16‐20], and lymphopenia is a poor prognostic factor in HIV-negative patients with PCP infection [21]. In animals without HIV infection, an inverse relationship between the P. carinii burden and the level of circulating CD4+ or CD8+ T lymphocytes has been found [22], which demonstrated the impact of immune system status on susceptibility to opportunistic pathogens. However, the prognostic value of lymphocyte subsets in HIV-negative PCP patients remains unclear. One small retrospective study revealed that CD4+ T cell count < 200/μL in kidney transplant recipients was a poor prognostic factor for PCP infection [23], and another showed that CD8+ T cell count < 160/μL was strongly associated with mortality in autoimmune disease patients with PCP [24]. However, we identified CD8+ T cell count < 300/μL as an independent risk factor for death in HIV-negative PCP patients. In addition, NK cell count was included in such an analysis for the first time.

As a threshold for predicting mortality, the sensitivity of CD8+ T cell count < 300/μL was low in our analysis. This means that other values below this threshold may also be correlated with poor prognosis. As shown in Fig. 2, the mortality rates of patients with CD8+ T cell counts ranging from 0 to 100/μL and 101–200/μL were very close to that of the 201–300/μL group. The mortality of the patients was significantly reduced when the CD8+ T cell count was > 300/μL, which demonstrates the high specificity of this threshold. We suggest paying more attention to patients with CD8+ T cell counts below 300/μL.

The pathogenesis and prognosis of opportunistic infection are closely related to immune status, and the importance of lymphocytes in combatting P. jirovecii infection has been recognized. Lymphocytes participate in host defence by regulating other immune cells, producing antibodies against pathogens (B cells) and killing organisms (cytotoxic CD8+ T cells and NK cells) [25]. CD4+ T cells can promote the proliferation and differentiation of B cells, T cells, and other immune cells; coordinate the interaction between immune cells; and play a central role in coordinating host defence mechanisms. CD8+ T cells include inhibitory T cells and killer T cells [26]. If the inflammatory response is driven by CD4+ T cells without sufficient inhibitory activity from CD8+ T cells, it will cause an effective but excessive inflammatory response similar to immune restoration disease (IRD), and the elimination of pathogens will incur severe lung injury as a cost [27]. Dysregulation of CD4+ T cells can lead to a decrease in their ability to assist CD8+ T cells, which in turn leads to a reduction in the killing and dissolving function of CD8+ T cells; that is, the regulation of the immune response becomes imbalanced in the negative direction. In our study, a CD8+ T cell count < 300/μL was an independent risk factor for death in PCP patients. Although CD4+ T cells play a central role in coordinating host defences against P. jirovecii, the inhibition and cytotoxic effects of CD8+ T cells seem to be critical to control and terminate the infection.

The NK cell count was also associated with the prognosis of PCP patients. Most striking of our findings was the progressive increase in mortality as NK cell count decreased from > 100/μL (26% mortality) to < 25/μL (65% mortality). NK cells play an important role in the immune defence against P. jirovecii infection by releasing IFN-γ, granzyme, and perforin and by their direct microbial activity [28, 29]. Recent studies have demonstrated that NK cells directly regulate adaptive immune responses through their interaction with CD4+ T cells [30‐32]. Therefore, we believe that the NK cell count has certain prognostic value for PCP patients. Being aware of the importance of NK cells in combatting P. jirovecii infection, it is necessary for us to further clarify the immune process in response to P. jirovecii infection in humans.

We usually believe that the longer the exposure time and the higher the dose of immunosuppressive therapy, the more severe the degree of immunosuppression is. However, in our study, no association was found between the prognosis of PCP and the type of immunosuppressive regimen, the dosage, or the exposure time. The reason for this lack of correlation may be that the number of patients taking the different immunosuppressive regimens was very small, so the fact that almost all patients received high-dose immunosuppressive therapy eliminated the distinction between low and high levels of immunosuppression. The findings also imply that the prognosis of PCP patients cannot be simply reflected by their immunosuppressive therapy. Our study revealed that low CD4+ T cell, CD8+ T cell, and NK cell counts were correlated with a poor prognosis of PCP, especially CD8+ T cell counts. Therefore, for patients receiving high-dose immunosuppressive therapy, lymphocyte subset analysis can greatly help clinicians assess the degree of immunosuppression. In some studies, CD4+ T cell count < 200/mm³ was taken as the indicator of a prevention strategy of PCP in HIV-negative patients, following the practice in HIV-positive patients [33]. Our study demonstrates that several patients have CD4+ T > 200/mm³, and CD8+ T and NK cells are also associated with the prognosis of these patients. Because there are a variety of changes in the immune system in HIV-negative patients, they cannot be described by the CD4+ T cell count alone. Therefore, the CD4+ T cell count should not be the only indication of prophylaxis in HIV-negative patients, and the levels of CD8+ T and NK cell counts should also be considered.

For HIV-positive patients with severe PCP, a large amount of medical-based evidence has shown that CSs as an adjuvant treatment can reduce mortality [34]. However, for HIV-negative PCP patients, due to the non-specific anti-inflammatory effects and serious side effects of CSs, the application of CSs in immunocompromised individuals is still controversial [35]. Therefore, alternative treatments for HIV-negative patients with PCP need to be more specific. Recent studies have suggested that CD4 + CD25+ T cells [36] and CD40 ligand [30] seem to be helpful in the regulation of immunity to P. jirovecii infection. CD4 + CD25+ T cells seem to suppress proliferation and cytokine production to limit the occurrence of immune reconstitution disease [37]. CD40L+ effector memory CD4+ T cells can control the reactivation and maintain the response of NK cells through IL-2 production and can promote the secretion of chemokine ligand-10 (CXCL10) from dendritic cells [38]. In a murine experimental model, CD40 ligand [39‐41] and anti-CD3 monoclonal antibody [42] have been confirmed to improve the prognosis of PCP infection. For HIV-negative patients with PCP, in addition to extensive antifungal therapy, adjusting immune disorders and attenuating excessive inflammatory responses may be a new treatment strategy.

There were several limitations to this study. First, it was a single-site study. Second, autoimmune diseases were the predominant underlying conditions among our study patients. Studies of HIV-negative PCP patients with other underlying disorders, such as organ transplantation, may yield different findings. Third, we only analysed the lymphocyte subsets of the patients after onset of the infection, but baseline lymphocyte subsets, which may also have an influence on mortality prediction, were unknown. A prospective study may be meaningful for further analysis. Fourth, follow-up results beyond 3 months were not available, so we do not know the long-term outcome of this patient cohort. Our study provides evidence of the correlation between lymphocyte subsets and short-term prognosis among HIV-negative PCP patients. Our findings suggest that a lower CD8+ T cell count is an independent risk factor for mortality, and in addition to effective anti-fungal therapy, treatments aimed at regulating immune function should be investigated.