1. Background

Sepsis is presently defined as a life-threatening organ dysfunction syndrome instigated by a dysregulated host response to infection, a condition that is garnering escalating interest in the field of emergency medicine [1]. Across the globe, sepsis is now estimated to result in more than 11 million deaths a year, and septic shock, the most severe form, leaves nearly 40% of patients dead at hospital discharge [2]. Over the past few decades, the mortality rate of sepsis has seen a gradual decrease due to the timely administration of antibiotics, fluid resuscitation, and multiple organ support therapies [3]. However, mortality rates persistently remain high among specific populations, such as immunosuppressed individuals. The Sequential Organ Failure Assessment (SOFA) score often falls short in identifying these patients, leading to a large segment of sepsis patients being erroneously classified as “immunocompetent” [4]. This misclassification results in prolonged intensive care unit (ICU) stays and increased financial burdens for these immunosuppressed patients.

To better identify these at-risk populations, numerous new technologies have been developed [5]. As an important immune cell, CD4⁺ T cells may play a significant role during sepsis. CD4⁺ T cells are a subgroup of lymphocytes that coordinates the cells of the immune system by releasing cytokines that are essential for their proliferation, differentiation, and bactericidal functions against pathogens [6]. It remains contentious whether CD4⁺ T cells are intimately involved in the early stages of sepsis. Some animal studies have shown that CD4⁺ T cells may directly mediate the host response to sepsis [6, 7], while others have shown that they had no impact on the inflammatory response [8]. Human patients and experimental mouse models of sepsis exhibit profound CD4⁺ T-cell apoptosis [9, 10]. However, the clinical relevance of these findings has not been sufficiently corroborated by research studies. Therefore, evaluating the relationship between sepsis and T lymphocyte subsets not only can it help to better understand the pathogenesis of sepsis-related immunity but it can also provide new insights as well as strategies for the treatment of sepsis. The purpose of this study was to investigate the predictive value of CD4⁺ T lymphocyte counts in peripheral blood using the Sepsis-3 criteria on 28-day mortality.

2. Methods

3. Results

3.1. Patient Characteristics

The study enrolled 104 patients, and 4 patients were withdrawn because 3 of them were eventually diagnosed with cancer or autoimmune diseases and 1 lost to follow-up (Figure 1). Thus, 100 patients were analyzed. There were 72 men (72%) and 28 women (28%), all of whom were Chinese, and the median age was 72 (60–78) years. A total of 29 patients died at or before 28 days. The demographic and clinical characteristics are shown in Table 1.

Table 1. Baseline characteristics of the patients.

Parameters	All patients, n = 100	Nonsurvivors, n = 29	Survivors, n = 71	p value
Demographic characteristics
Female, n (%)	28 (28)	8 (27.6)	20 (28.2)	0.953
Age (years)	72.00 (60.00–78.00)	72.00 (61.00–77.00)	72.00 (60.00–79.00)	0.918
Laboratory findings
WBC (×109/L)	14.97 (9.30–20.10)	11.86 (8.15–17.92)	15.94 (8.71–20.47)	0.118
Lac (mmol/L)	1.40 (1.10–2.00)	1.77 (1.23–2.56)	1.30 (1.10–1.80)	0.039
Bacteriology positive, n (%)	53 (53)	18 (62.1)	35 (49.3)	0.246
Vital signs
T (°C)	37.15 (36.70–38.2)	37.40 (36.65–38.10)	37.00 (36.70–38.40)	0.985
Heart rate (cpm)	100.00 (85.25–110.00)	105.00 (97.50–110.00)	96.00 (82.00–110.00)	0.047
MAP (mmHg)	89.00 (80.00–99.33)	85.00 (79.00–98.17)	89.00 (80.33–100.00)	0.569
Source of infection, n (%)
Bloodstream	4 (4)	1 (1.4)	3 (10.3)	0.072
Lungs	64 (64)	47 (66.2)	17 (58.6)	0.474
Abdomen	14 (14)	9 (12.7)	5 (17.2)	0.540
Urinary system	10 (10)	7 (9.9)	3 (10.3)	1.000
Central nervous system	4 (4)	4 (5.6)	0 (0)	0.320
Others	4 (4)	3 (4.2)	1 (3.4)	1.000
Severity of illness
GCS score	12.00 (7.13–15.00)	8.00 (3.50–13.50)	12.00 (9.00–15.00)	<0.01
APACHE II score	15.00 (10.00–20.00)	19.00 (15.00–24.50)	13.00 (9.00–17.00)	<0.01
SOFA score	5.00 (4.00–8.00)	9.00 (5.00–12.00)	4.00 (3.00–6.00)	<0.01
Shock, n (%)	24 (24)	12 (41.4)	12 (16.9)	<0.01

• Data are shown as median and interquartile range unless otherwise indicated. Pearson chi-square test was performed for sex, bacteriology positive, and shock while the nonparametric test of two independent samples (Mann–Whitney) were for the others. WBC: white blood cell, Lac: lactate, GCS: Glasgow Coma Scale, APACHE II: Acute Physiology and Chronic Health Evaluation, SOFA: Sequential Organ Failure Assessment, T: temperature, and MAP: mean arterial pressure.

3.2. Comparison of Blood Cell Analysis, N/L, and Lymphocyte Subsets of Survivors and Nonsurvivors in Patients with Sepsis

Patients were divided into survivors and nonsurvivors at 28 days. We compared the two groups of the patients, and the results are shown in Table 2. In nonsurvivors, CD4⁺ T lymphocyte counts were significantly lower than those in survivors (Table 2, p < 0.05; Figures 2(a) and 2(b)). Similar results were also observed when compared with the lymphocyte counts, CD8⁺ T lymphocyte counts, and lactate (Table 2; p < 0.05). The representative flow dot plots are shown in Figure 2(a).

Table 2. Comparison of blood cell analysis, N/L, and lymphocyte subsets of survivor and nonsurvivors groups.

Parameters	All patients	Nonsurvivors	Survivors	p value (N/S)
Number	100	29	71
N (×109/L)	13.01 (8.00–17.67)	10.55 (6.88–16.77)	13.58 (8.62–18.24)	0.26
L (×109/L)	0.85 (0.48–1.30)	0.50 (0.37–1.07)	0.91 (0.62–1.40)	<0.01
N/L	13.04 (8.62–27.99)	14.01 (9.39–31.02)	11.82 (8.29–26.25)	0.248
CD4+ T cells counts (×109/L)	0.19 (0.09–0.42)	0.16 (0.03–0.24)	0.29 (0.11–0.47)	0.011
CD8+ T-cell counts (×109/L)	0.15 (0.06–0.30)	0.11 (0.02–0.25)	0.17 (0.06–0.30)	0.031
CD4/CD8	1.65 (0.96–2.49)	1.66 (0.78–2.24)	1.63 (1.02–2.67)	0.144

• Data are shown as median and interquartile range. Nonparametric test of two independent samples (Mann–Whitney) was performed for the comparison of nonsurvivors and survivors. N, neutrophils; L, lymphocyte; N/L, the ratio of neutrophil count to lymphocyte count.

3.3. Dynamic Changes in CD4⁺ T Lymphocyte Counts

CD4⁺ T lymphocyte counts of 45 patients were measured on days 1, 2, 4, and 7 after being enrolled. CD4⁺ T lymphocyte counts in nonsurvivors were significantly lower than those in the survivors on every time point (Figure 3). CD4⁺ T lymphocyte counts continued to increase in the survivors, notably in the early disease course (1-4d). However, CD4⁺ T lymphocyte counts began to decline at 4 days in the nonsurvivors.

3.5. ROC of CD4⁺ T Lymphocyte Counts for Predicting 28-Day Morality and Survival Analysis in Sepsis Patients

The ROC curve obtained with CD4⁺ T lymphocyte counts has an AUC of 0.662 (CI 0.551–0.774), and the ROC curve obtained with the SOFA score has an AUC of 0.752 (CI 0.641–0.864). The combination of these two parameters has an AUC of 0.778 (CI 0.679–0.876) for predicting 28-day mortality (Figure 4(a)). ROC curve analysis showed that 0.2810 (×10⁹/L) of CD4⁺ T lymphocyte counts was the optimal threshold for predicting 28-day mortality in patients with sepsis. The sensitivity, specificity, positive and negative predictive values, and its corresponding confidence interval of 95% for the three ROC curves were used to estimate the prognostic accuracy. The detailed results are shown in Figure 4(b).

Using cutoff values determined by the ROC curve, patients with the CD4⁺ T lymphocyte counts lower than 0.281 (×10⁹/L) had a lower probability of survival at day 28 than patients with higher CD4⁺ T lymphocyte counts (Figure 5).

4. Discussion

Previous studies found that the decrease in peripheral blood lymphocytes in sepsis patients is associated with the severity of the disease and the poor prognosis [11]. Martignoni found that CD4⁺ T cells may facilitate the early clearance of bacteria by regulating neutrophil function. The decrease of CD4⁺ T cells indicates the immunosuppression or immune paralysis of the organism [12]. Recently, the criteria for sepsis were updated [2] and the definition of sepsis was changed to a life-threatening organ dysfunction due to a dysregulated host response to infection (Sepsis-3). Changing the criteria may ultimately also affect the performance of prognostic biomarkers. As far as we know, the current study is one of the earliest studies correlating CD4⁺ T lymphocyte counts to a prognostic value of sepsis patients using the Sepsis-3 criterion.

In the present study, T-cell subgroups were selected as the target to explore their predictive performance on short-term mortality under the new sepsis criteria. We show here that CD4⁺ T lymphocyte counts and CD8⁺ T lymphocyte counts were significantly lower in nonsurvivors compared with nonsurvivors. Multivariate logistic regression analysis showed that only CD4⁺ T lymphocyte counts were independently associated with 28-day mortality in patients. Area under the curve of CD4⁺ T lymphocyte counts (0.662 and 0.551–0.774) indicated that it is an effective marker for predicting the short-term mortality of sepsis, although its predictive value was not too high.

Many biomarkers showed a relevant correlation with the clinical outcome of patients with sepsis, but their time courses may be more reliable than absolute levels [13]. Clinical studies had found that the immune status of sepsis was constantly changing, which is different from the early stage and the progression of diseases [14]. The present results have shown the dynamics change of CD4⁺ T lymphocytes on study days 1, 2, 4, and 7 of some patients with sepsis. Interestingly, the results showed that it is significantly associated with an increase in overall and sepsis-attributable mortality rates. As shown in time-serial measurements, CD4⁺ T lymphocyte counts in survivors were progressively increased. However, in the nonsurvivor group, the counts were increased at the beginning but decreased thereafter. This suggests that with the progression of the disease, the continued decline in CD4⁺ T lymphocyte counts is indicative of a poor prognosis of sepsis patients.

It is suggested that organ failure should be the core of sepsis. In this connection, SOFA is now widely accepted and is a relatively accurate measure of the severity of the disease [2]. As one of the scoring systems for sepsis patients, the SOFA score was proposed by scholars of ESICM (European Society of Intensive Care Medicine) in Paris in 1994, which has a history of 23 years [15]. Recently, Francesca’s study reported that the SOFA score shows a moderate prognostic stratification ability in sepsis patients [16]. Our study showed a similar result that the area under the curve of the SOFA score for predicting 28-day mortality is 0.752 (0.641–0.863). In comparative analysis of sensitivity and specificity of CD4⁺ T lymphocyte counts versus SOFA score, we found that the former is more sensitive at 82.76% for the prognosis, while the latter is more specific at 91.55%. More importantly, we have demonstrated that the SOFA score when combined with CD4⁺ T lymphocyte counts was a better factor for predicting 28-day mortality compared with the SOFA score and CD4⁺ T lymphocyte counts alone. There are six scoring systems (respiration, coagulation, liver, cardiovascular, central nervous system, and renal) in the assessment of SOFA [14], but the systemic nature and inflammatory response of sepsis involved large number of organs and cell systems [17], which is far more than the 6 items included in the SOFA score. It has been proposed that future iterations of the sepsis definitions should include an updated SOFA score with more optimal variable selection, cutoff values, and weighting, or more superior scoring systems [2]. More and more evidence showed that the function of the immune system played an important role in the occurrence and development of sepsis, and it is closely related to the prognosis of patients [9, 18]. Thus, it can be confidently assumed that the immune system could and should be a part of the improvement of the SOFA score, and CD4⁺ T lymphocyte counts can be used as an indicator of assessment.

We have further shown that besides CD4⁺ T lymphocyte counts, shock and heart rate were also the independent risks associated with 28-day mortality. Consistent with our findings, a retrospective study by Hayase et al. reported that tachycardia was a significant and independent predictor of a reduced survival rate in sepsis patients [6]. Recent studies have shown that controlling heart rate can reduce mortality in patients with septic shock and have a significant effect [18]. Furthermore, Jayaprakash et al. found that elevated modified shock index during early sepsis is associated with the development of the SOFA score and mortality [18]. This indicated that cardiac function plays an important role in sepsis, underscoring therefore the needs to obtain the necessary information.

Sepsis is a heterogeneous biological syndrome which is incompletely understood. Many of the established and emerging biomarkers are helpful for sepsis patients [18]. Notwithstanding, we have identified a biomarker that can reflect the immune function, predict the prognosis of sepsis patients, and increase the predictive accuracy of the SOFA score. Mounting data show that, in most ICU patients, sepsis-associated immunosuppression is associated with increased morbidity and mortality [18]. The immunomodulatory therapy of sepsis is one of the focuses of the current research study. Taken together with previous studies [7, 11, 18], we believed that CD4⁺ T lymphocyte counts can also play an important role in monitoring the therapeutic effect of immune regulation.

However, there were several limitations in this study. Firstly, it was a single-center study and the sample size was relatively small. Secondly, our study enrolled only ICU patients with the diagnosis of sepsis and who had higher acuity than the general population of infectious patients. Thirdly, we did not carry out further research on the cellular function of the remaining CD4⁺ T lymphocytes. Finally, this study was lacking a healthy control group.

5. Conclusions

In conclusion, our study strongly supports that CD4⁺ T lymphocyte counts are associated with mortality in sepsis patients. CD4⁺ T lymphocyte counts in combination with the SOFA score significantly increase the predictive accuracy for 28-day mortality. Furthermore, measurement of CD4⁺ T lymphocyte counts is a promising independent prognostic marker for sepsis patients.

Ethical Approval

The present prospective observational study was conducted with approval from Guangdong Provincial People’s Hospital Ethics Committee (no. GDREC2015374H(R1)).

Disclosure

The funding agents had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Authors’ Contributions

W.J and G.H designed the research. G.H, X.L, C.Z, H.L, Y.Z, C.H, L.X, and H.Z performed the research and collected the data. L.X carried out the flow cytometric analysis. H.Z, Y.X, and W.J analyzed the data. G.H, P.H, and W.J wrote the manuscript. All authors read and approved the final manuscript.