Article

The Role of PD-1/PD-L1 and IL-7 in Lymphocyte Dynamics and

Sepsis Progression: A Biomarker Study in Critically Ill Patients
Oana Coman 1 , Bianca-Liana Grigorescu 2, *, Adina Hut, anu 3,4 , Anca Bacârea 5 , Anca Meda Văsies, iu 6 ,
Raluca S, tefania Fodor 2 , Marius Petris, or 1 and Leonard Azamfirei 2

1 Department of Simulation Applied in Medicine, University of Medicine, Pharmacy, Science and Technology
“George Emil Palade”, 540142 Targu Mures, Romania; [email protected] (O.C.);
[email protected] (M.P.)
2 Department of Anaesthesiology and Intensive Care, University of Medicine, Pharmacy, Science and
Technology “George Emil Palade”, 540142 Targu Mures, Romania; [email protected] (R.S, .F.);
[email protected] (L.A.)
3 Department of Laboratory Medicine, University of Medicine, Pharmacy, Science and Technology
“George Emil Palade”, 540142 Targu Mures, Romania; [email protected]
4 Centre for Advanced Medical and Pharmaceutical Research, Immunology, University of Medicine, Pharmacy,
Science and Technology “George Emil Palade”, 540142 Targu Mures, Romania
5 Department of Pathophysiology, University of Medicine, Pharmacy, Science and Technology
“George Emil Palade”, 540142 Targu Mures, Romania; [email protected]
6 Department of Infectious Disease, University of Medicine, Pharmacy, Science and Technology
“George Emil Palade”, 540142 Targu Mures, Romania; [email protected]
* Correspondence: [email protected]; Tel.: +40-745-096-641

Abstract: Sepsis pathophysiology involves a dysregulated immune response to infection, excessive

inflammation, and immune paralysis. This study explores the relationships between cell death
biomarkers (serum-soluble levels of programmed cell death protein 1 (PD-1), programmed death
ligand 1 (PD-L1), and interleukin-7 (IL-7)) and the percentages of various lymphocyte subsets in
relation to the severity and progression of sepsis. This prospective, observational study included
Citation: Coman, O.; Grigorescu, 87 critically ill patients. We monitored parameters on days 1 (sepsis was diagnosed according to
B.-L.; Hut, anu, A.; Bacârea, A.; the Sepsis-3 Consensus) and 5. We established an IL-7 cutoff value of 1.94 pg/mL by comparing
Văsies, iu, A.M.; Fodor, R.S, .; Petris, or, levels between a healthy control group and patients with sepsis (p < 0.0001). Lymphopenia was
M.; Azamfirei, L. The Role of observed in all patients, with negative correlations between helper T lymphocytes and cytotoxic
PD-1/PD-L1 and IL-7 in Lymphocyte
and B lymphocytes, and positive correlations involving cytotoxic lymphocytes across all groups.
Dynamics and Sepsis Progression: A
We found correlations between PD-1/PD-L1 and lymphocyte subsets. IL-7 showed a statistical
Biomarker Study in Critically Ill
correlation with PD-1 in non-survivors. Assessing lymphocyte levels shows potential as a biomarker
Patients. Int. J. Mol. Sci. 2024, 25,
for evaluating the progression of sepsis. Monitoring IL-7 levels could help assess survival, as low
12612. https://doi.org/10.3390/
ijms252312612
levels are associated with higher mortality risk. Monitoring IL-7 levels could help assess survival,
as low levels are associated with higher mortality risk. Elevated PD-1/PD-L1 expression impairs
Academic Editor: Francisco Estevez
costimulatory signalling, reducing T cell responses and lymphopenia, which increases the risk of
Received: 8 October 2024 nosocomial infections.
Revised: 19 November 2024
Accepted: 21 November 2024 Keywords: sepsis; septic shock; apoptosis; lymphopenia; CD4+ lymphocytes; CD8+ lymphocytes;
Published: 24 November 2024 CD19+ lymphocytes; natural killer lymphocytes; PD-1/PD-L1 axis; IL-7

Copyright: © 2024 by the authors.

1. Introduction
Licensee MDPI, Basel, Switzerland.
This article is an open access article Sepsis is a critical condition resulting from an imbalanced inflammatory response of
distributed under the terms and the host to an infection, ultimately causing multiorgan failure. The inflammatory response
conditions of the Creative Commons contributes to the dysregulated reaction characteristic of sepsis, reflecting the host’s attempt
Attribution (CC BY) license (https:// to combat the progressing infection [1]. Despite advancements in medicine, sepsis remains
creativecommons.org/licenses/by/ the leading cause of mortality among critically ill patients, particularly elderly patients with
4.0/). multiple comorbidities [2,3]. In 2020, the World Health Organization (WHO) published its

Int. J. Mol. Sci. 2024, 25, 12612. https://doi.org/10.3390/ijms252312612 https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2024, 25, 12612 2 of 18

first global report on the epidemiology and burden of sepsis, highlighting that the estimated
in-hospital mortality rate for sepsis patients treated in the intensive care unit (ICU) was
over one-third, reaching 42% [4]. Timely assessment of the immune status in sepsis patients,
combined with enhanced supportive therapy, can potentially reduce mortality rates [5].
However, it continues to be a significant challenge.
Inflammatory processes play a crucial role in the pathophysiology of organ dysfunction
in critically ill patients. The susceptibility of these patients to secondary infections can
be attributed to both an overstimulated innate immune response and a failed immune
response, regardless of the initial insult [6].
The pathophysiology of sepsis is governed by the balance between proinflammatory
and anti-inflammatory mechanisms. This balance is reflected in the continuous interaction of
three pillars: lymphocyte subcategories, interleukin-7, and the modulatory component, the
programmed cell death protein 1/programmed death ligand 1 (PD-1/PD-L1) axis. The clinical
outcome of pathogen invasion hinges on the intricate interactions between the pathogen and
immune cells, prompting a variety of defence mechanisms. Among these, cytokine production
and cytotoxicity are the primary effector mechanisms in the acquired immune response [7,8].
During sepsis, both proinflammatory and anti-inflammatory cytokines are activated almost
simultaneously, with the clinical picture primarily dominated by the proinflammatory phase.
T helper cells (CD4+) play a significant role by secreting cytokines, while cytotoxic T lym-
phocytes (CD8+) and natural killer T CD3+ lymphocytes (NKT), the primary mediators of
cytotoxicity, are involved in the pathogenesis of septic shock [7].
Interleukin 7 (IL-7) is a molecule known for its antiapoptotic properties and ability to
induce significant proliferation of CD4+ and CD8+ T lymphocytes. IL-7 has been shown to
prevent lymphocyte apoptosis, restore the functionality of CD4+ and CD8+ T cells, and
improve survival rates in animal models of bacterial and fungal sepsis [9,10].
Immune checkpoints are immune-regulatory pathways indispensable for maintaining
self-tolerance, preventing autoimmunity, and reducing collateral tissue damage. They act
as brakes to modulate the adaptive immune response [11,12]. The interaction between
programmed cell death protein 1 (PD-1) and its ligand, programmed death ligand 1 (PD-
L1), is pivotal in dampening the activation signals triggered by the T cell receptor, also
suppressing the immune response across various cell types [13,14].
Despite years of research in this field, the information available on diagnostic biomark-
ers for sepsis that can evaluate the inflammatory response to pathogens and reflect immune
reactions is still limited.
This study aims to explore the relationships between cell death biomarkers (serum-
soluble levels of PD-1, PD-L1, and IL-7) and the percentages of various lymphocyte subsets
(CD4+ T helper lymphocytes, CD8+ T cytotoxic lymphocytes, natural killer T CD3+ lym-
phocytes, and CD19+ B lymphocytes) in relation to the severity and progression of sepsis.

2. Results
2.1. Population Analysis
A total of 87 patients were enrolled in this investigation: 31 female participants
(35.63%) and 56 male participants (64.34%). The average age was 68 years, ranging from
33 to 90 years. The majority of patients (30) were between 71 and 80 years old, followed
by 23 patients in the 61–70-year range. The smallest group consisted of just one patient in
the 30–40-year range. The mean BMI was 28.9 ± 5.6, with a range between 15.60 and 49.40.
Of the total, 63 patients (72.41%) had fatal outcomes, while 24 patients (27.59%) survived.
The typical duration of stay in the intensive care unit was 14 days. Sepsis was prevalent in
57 patients (65.52%), and septic shock in 30 patients (34.48%).
In the study group, the prevalent underlying conditions and the infectious site varied
as follows: cardiovascular disorders (83 patients, 83.91%), renal disease (58 patients, 69.88%),
and respiratory disease (55 patients, 63.22%). As “Other” we included pathologies such as
secondary anaemia, thrombocytopenia, chronic smoking, chronic alcohol intake, eschars,
Int. J. Mol. Sci. 2024, 25, 12612 3 of 18

acid-base disorders, multiple organ dysfunction syndrome, hypovolemia, malnutrition,

electrolyte disorders, etc., that can be found in Table S1.
Descriptive statistics for the entire study population and relevant demographic and
clinical variables are provided in Table S2.

2.2. Lymphopenia
We studied the variation in lymphocytes for each subcategory of patients. We found
that all patients presented lymphopenia on both studied days, with a statistically significant
variation, an increase in % between day 1 and day 5, for sepsis and for survivor patients
(Table 1).

Table 1. Comparison between median and IQR (interquartile range) values of studied groups with
normal lymphocyte range.

Median (IQR) Normal Range

p a Value
Day 1 Day 5 % ×103 /µL
0.067
% 0.085 (0.088) 0.0032
(0.0589)
Sepsis
0.910 1.02
×103 /µL 0.9323
(0.405) (0.77)
0.0502 0.064
% 0.2387
(0.0565) (0.0774)
Septic shock
0.86 1.020
×103 /µL 0.1089
(0.6605) (0.8) 0.205–0.45 1.2–3.4
0.0652
% 0.116 (0.113) 0.0014
(0.058)
Survivors
0.8985 1.035
×103 /µL 0.6322
(0.942) (1.718)
0.057 0.0635
% 0.1316
Non- (0.06) (0.0537)
survivors 0.91 1.015
×103 /µL 0.5014
(0.67) (0.7605)
Legend: a Wilcoxon test. Bold type indicates significance. Normal range measured by the laboratory of the County
Emergency Clinical Hospital in Târgu Mures, , Romania.

2.3. Cutoff Value for IL-7

The diagnostic accuracy of a biomarker is essential. Therefore, receiver operating charac-
teristic (ROC) curve analysis was used to assess the diagnostic accuracy and determine the
optimal cutpoint values for IL-7 levels between patients with sepsis/septic shock and control
groups (Figure 1). According to the ROC curve of IL-7 analysis, the cutoff value for IL-7 was
1.94 pg/mL, with a sensitivity of 74.23% and a specificity of 75.34% for sepsis/septic shock,
and the area under the curve (AUC) was 0.8547 (p < 0.0001), 95% CI 0.79–0.90.
Table 2 highlights the lowest and highest values of IL-7 in the four categories of
patients. We counted the number of patients with a value of IL-7 above the identified cutoff
value using the data found in Table S3.

Table 2. Comparative IL-7 values (median and IQR (interquartile range)) of the studied categories of
patients with the cutoff value.

Median (IQR)
IL-7 Cutoff Value
(pg/mL)
(pg/mL)
Day 1 Day 5
Sepsis 3.459 (4.204) 2.382 (2.189)
Septic shock 5.415 (5.35) 3.459 (4.052)
1.94
Survivors 3.921 (4.346) 3.227 (3.037)
Non-survivors 4.381 (5.834) 2.542 (2.686)
 Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 4 o
Int. J. Mol. Sci. 2024, 25, 12612 4 of 18

Figure
Figure 1.1.The
Thestandard
standard ROC
ROC curve
curve analysis
analysis of all patients
of all patients and thegroup.
and the control control group.
2.4. Comparison of Variables for the Sepsis and Septic Shock Groups
Table 2 highlights the lowest and highest values of IL-7 in the four categories of
We divided patients into sepsis and septic shock groups according to the Sepsis 3
tients. We counted
Consensus criteria [1]the
andnumber
analysed of the
patients with
studied a value on
parameters of IL-7
day 1above
and daythe5identified
after cu
value using the data found in Table S3.
meeting the inclusion criteria. Comprehensive descriptive statistics for the study cohort,
including demographic and clinical parameters, are presented in Table S4 for the sepsis
Table
group 2. Comparative
and Table S5 for IL-7 valuesshock
the septic (median and IQR (interquartile range)) of the studied catego
group.
of We observed
patients
Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW with thea statistically
cutoff value. significant variation for PD-L1 between days 1 and 5
(p = 0.0353) for septic shock patients (Figure 2). No other significant differences were
observed (Table S6). Median (IQR)
(pg/mL) IL-7 Cutoff Value (pg/m
Day 1 Day 5
Sepsis 3.459 (4.204) 2.382 (2.189)
Septic shock 5.415 (5.35) 3.459 (4.052)
1.94
Survivors 3.921 (4.346) 3.227 (3.037)
Non-survivors 4.381 (5.834) 2.542 (2.686)

2.4. Comparison of Variables for the Sepsis and Septic Shock Groups
We divided patients into sepsis and septic shock groups according to the Seps
Consensus criteria [1] and analysed the studied parameters on day 1 and day 5 after m
ing the inclusion criteria. Comprehensive descriptive statistics for the study cohort
cluding demographic and clinical parameters, are presented in Table S4 for the se
group and Table S5 for the septic shock group.
We observed a statistically significant variation for PD-L1 between days 1 and 5
0.0353) for septic shock patients (Figure 2). No other significant differences were obser
(Table S6).
Figure
Figure 2. 2. PD-L1
PD-L1 variation
variation forshock
for septic septic shock
patients patients
between daybetween
1 and day 5day 1 andtest).
(Wilcoxon day D1:
5 (Wilcoxon
day te
1,1,D5: day 5.
D5: day 5.

For the sepsis group, correlations between the subsets of lymphocytes p

statistically significant negative correlations between CD4+ and CD8+, and
NKT CD3+ on both day 1 and day 5, and between NKT and CD19+ on day
 Figure 2. PD-L1 variation for septic shock patients between day 1 and day 5 (Wilcoxon test). D1: day
Int. J. Mol. Sci. 2024, 25, 12612 5 of 18
1, D5: day 5.

For the sepsis group, correlations between the subsets of lymphocytes pointed out
For the sepsis group, correlations between the subsets of lymphocytes pointed out
statistically significant
statistically negative
significant correlations
negative between
correlations betweenCD4+
CD4+and CD8+,and
and CD8+, andCD4+
CD4+ andand
NKT
NKT CD3+ on both day 1 and day 5, and between NKT and CD19+ on day 1.
CD3+ on both day 1 and day 5, and between NKT and CD19+ on day 1. We also observed We also a
observed positive
a positive correlation
correlation between
between cytotoxic
cytotoxic T cells and
T cells (CD8+) (CD8+)
NKTand NKT
on both on days
study both (Figure
study 3
days (Figureand3Table
and S7).
Table S7).

Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW 6 of 19

Figure 3. Statistically significant correlation between lymphocyte subtypes Th CD4+, Tc CD8+, and
Figure 3. Statistically significant correlation between lymphocyte subtypes Th CD4+, Tc CD8+, and
NKT on day 1 (A–D) and day 5 (E–G) in the sepsis group;a a Spearman test,bb Pearson test.
NKT on day 1 (A–D) and day 5 (E–G) in the sepsis group; Spearman test, Pearson test.

By
Bycorrelating
correlating the lymphocytesubsets
the lymphocyte subsetsininthe
theseptic
septicshock
shockgroup,
group,
wewe observed
observed statis-
statisti-
tically significantnegative
cally significant negativecorrelations
correlationsonly
onlybetween
betweenCD4+
CD4+and
andCD8+
CD8+and
andbetween
betweenCD4+
CD4+
and NKT on both
and NKT on both dayday 1 and day 5 (Figure 4).
(Figure 4).
PD-1 was statistically positively correlated with PD-L1 on day 1 (r = 0.4566, p = 0.0190),
and with IL-7 on day 5 (r = 0.5750, p = 0.0274). The rest of the correlations can be found in
Table S8.
 Figure 3. Statistically significant correlation between lymphocyte subtypes Th CD4+, Tc CD8+, and
NKT on day 1 (A–D) and day 5 (E–G) in the sepsis group; a Spearman test, b Pearson test.

By correlating the lymphocyte subsets in the septic shock group, we observed statis
Int. J. Mol. Sci. 2024, 25, 12612 tically significant negative correlations only between CD4+ and CD8+ and between
6 of 18 CD4+
and NKT on both day 1 and day 5 (Figure 4).

Figure4.4.Statistically
Figure Statistically significant
significant correlation
correlation between
between lymphocyte
lymphocyte subtypes
subtypes Th CD4+, ThTcCD4+,
CD8+,Tc CD8+, and
and
NKTon
NKT onday
day1 1(A,B)
(A,B)
andanddayday 5 (C,D)
5 (C,D) inseptic
in the the septic
shockshock a Spearman
group;group; test, b Pearson
a Spearman test, b Pearson
test. test.

2.5. Comparison of Variables for the Survivor and Non-Survivor Groups

PD-1 was statistically positively correlated with PD-L1 on day 1 (r = 0.4566, p =
We divided
0.0190), and withtheIL-7
studied lot of
on day patients
5 (r intopsurvivor
= 0.5750, and
= 0.0274). non-survivor
The rest of the groups and can be
correlations
examined the studied parameters accordingly on day 1 and day 5 after meeting the inclusion
found in Table S8.
Int. J. Mol. Sci. 2024, 25, x FOR PEER REVIEW
criteria. Detailed descriptive statistics for the study population are available in Table S9
(survivor group) and Table S10 (non-survivor group).
2.5. Comparison
We studied the of Variables
variation for theparameters
in the Survivor and Non-Survivor
between Groups
the selected days. We observed a
We divided
statistically thevariation
significant studiedfor lotNKT
of patients
lymphocytesinto(psurvivor
= 0.076) inand non-survivor
the survivor groups and
group, and
and a statistically
aexamined
statistically significant variation for IL-7 (p = 0.069) in
the studied parameters accordingly on day 1 and day 5 after meeting5).
significant variation for IL-7 (p = 0.069) in the non-survivor the non-survivor
group (Figure gro
the inclu
ureother
No
sion 5). No other
variation
criteria. variation
was
Detailed was
significant
descriptive significant
(Table S11). for(Table
statistics S11).
the study population are available in Table
S9 (survivor group) and Table S10 (non-survivor group).
We studied the variation in the parameters between the selected days. We observed
a statistically significant variation for NKT lymphocytes (p = 0.076) in the survivor group

Figure5. 5.NKT
Figure NKT lymphocyte
lymphocyte variation
variation for the survivor
for the survivor lot between
lot of patients of patients
day 1between
and day 5day
(A).1 and d
IL-7 variation for the non-survivor lot of patients between day 1 and day 5 (B). D1:
IL-7 variation for the non-survivor lot of patients between day 1 and day 5 (B). D1: day 1, D5: day 5. day 1
5.

Correlating the subsets of lymphocytes, we observed statistically significant

tions between CD4+, CD8+, NKT, and CD19+ lymphocytes, as shown in Tables 3
For the survivor patients’ lot, statistically significant negative correlatio
found between CD4+ and CD8+ and between CD8+ and CD19+ lymphocytes on bo
Int. J. Mol. Sci. 2024, 25, 12612 7 of 18

Correlating the subsets of lymphocytes, we observed statistically significant correla-

tions between CD4+, CD8+, NKT, and CD19+ lymphocytes, as shown in Tables 3 and 4.

Table 3. Correlations between lymphocyte subsets on day 1 and day 5 for survivor patients.

Tc Cells (CD8+), % NKT (CD3+), % B Cells (CD19+), %

r = −0.9368 r = −0.4108 r = 0.3740
Day 1 (−0.9726 to −0.8575) (−0.7095 to 0.01305) (−0.04725 to 0.6823)
p b < 0.0001 p b = 0.0575 p a = 0.0718
Th cells (CD4+), %
r = −0.9788 r = −0.6998 r = 0.6929
Day 5 (−0.9922 to −0.9426) (−0.8833 to −0.3302) (0.3004 to 0.8847)
p b < 0.0001 p b = 0.0018 p b = 0.0029
r = 0.5205 r = −0.4517
Day 1 (0.1267 to 0.7726) (−0.7293 to −0.04645)
p b = 0.0130 p a = 0.0267
Tc cells (CD8+), %
r = 0.6379 r = −0.6794
Day 5 (0.2269 to 0.8561) (−0.8791 to −0.2770)
p b = 0.0059 p b = 0.0038
r = −0.4864
Day 1 (−0.7592 to −0.06832)
p a = 0.0217
NKT (CD3+), %
r = −0.4824
Day 5 (−0.7976 to 0.03964)
p b = 0.0686
Legend: a Spearman test, b Pearson test. Bold type indicates significance. B cells: B CD19+ lymphocytes, CD:
cluster of differentiation, NKT CD3+: natural killer T CD3+ lymphocytes, Tc cells: T cytotoxic CD8+ lymphocytes,
Th cells: T helper CD4+ lymphocytes.

Table 4. Correlations between lymphocyte subsets on day 1 and day 5 for non-survivor patients.

Tc Cells (CD8+), % NKT (CD3+), % B Cells (CD19+), %

r = −0.7114 r = −0.6415 r = 0.1206
Day 1 (−0.8155 to −0.5629) (−0.7722 to −0.4588) (−0.1451 to 0.3700)
p b < 0.0001 p a < 0.0001 p a = 0.3588
Th cells (CD4+), %
r = −0.9461 r = −0.4962 r = 0.1412
Day 5 (−0.9730 to −0.8939) (−0.7145 to −0.1899) (−0.2168 to 0.4657)
p b < 0.0001 p b = 0.0028 p a = 0.4257
r = 0.5488 r = −0.1183
Day 1 (0.3379 to 0.7072) (−0.3680 to 0.1474)
p a < 0.0001 p a = 0.3682
Tc cells (CD8+), %
r = 0.4641 r = −0.08319
Day 5 (0.1493 to 0.6934) (−0.4184 to 0.2720)
p b = 0.0057 p a = 0.6400
r = −0.2894
Day 1 (−0.5153 to −0.02578)
p a = 0.0276
NKT (CD3+), %
r = −0.2814
Day 5 (−0.5728 to 0.07306)
p a = 0.1069
Legend: a Spearman test, b Pearson test. Bold type indicates significance. B cells: B CD19+ lymphocytes, CD:
cluster of differentiation, NKT CD3+: natural killer T CD3+ lymphocytes, Tc cells: T cytotoxic CD8+ lymphocytes,
Th cells: T helper CD4+ lymphocytes.

For the survivor patients’ lot, statistically significant negative correlations were found
between CD4+ and CD8+ and between CD8+ and CD19+ lymphocytes on both studied
days, and between NKT and CD19+ on day 1, and between CD4+ and NKT on day 5.
Int. J. Mol. Sci. 2024, 25, 12612 8 of 18

Statistically significant positive correlations were found between CD8+ and NKT on both
studied days, and between CD4+ and CD19+ on day 5 (Table 3). The rest of the correlations
can be found in Table S12.
For non-survivor patients, correlating the subsets of lymphocytes showed statistically
significant negative correlations between CD4+ and CD8+, between CD4+ and NKT on
both day 1 and day 5, and between NKT and CD19+ on day 1. Statistically significant
positive correlations were found between CD8+ and NKT lymphocytes on day 1 and day 5
(Table 4). The rest of the correlations can be found in Table S13.
Correlating the lymphocyte subsets with the rest of the parameters, we observed a
statistically significant positive correlation between T helper CD4+ lymphocytes and PD-1
on day 1 of study inclusion, whilst on day 5 we observed a negative correlation. For T
cytotoxic CD8+ lymphocytes, we observed negative correlations on day 1 with PD-1 and
PD-L1, and NKT correlated negatively with PD-1 on day 1. IL-7 correlated positively with
the PD-1 component on day 5 (Table 5).

Table 5. Correlations between lymphocyte subsets and the studied parameters on day 1 and day 5 for
non-survivor patients.

Parameters (on Day 1) Th Cells (CD4+), % Tc Cells (CD8+), % NKT (CD3+), % B Cells (CD19+), %
r = 0.3463 r = −0.3873 r = −0.3663 r = 0.1884
PD-1, ng/mL (0.08886 to 0.5603) (−0.5920 to −0.1357) (−0.5775 to −0.1091) (−0.08889 to 0.4386)
p a = 0.0078 p a = 0.0027 p a = 0.0051 p a = 0.1683
r = 0.1843 r = −0.2790 r = 0.003428 r = −0.1443
PD-L1, ng/mL (−0.08264 to 0.4265) (−0.5049 to −0.01736) (−0.2696 to 0.2759) (−0.4035 to 0.1364)
p b = 0.1740 p b = 0.0373 p a = 0.9802 p a = 0.2979
r = 0.1172 r = −0.1029 r = −0.05624 r = 0.1398
IL-7, pg/mL (−0.1484 to 0.3671) (−0.3545 to 0.1625) (−0.3171 to 0.2125) (−0.1307 to 0.3908)
p a = 0.3723 p a = 0.4339 p a = 0.6750 p a = 0.2954
Parameter (on day 5) Th cells (CD4+), % Tc cells (CD8+), % NKT (CD3+), % B cells (CD19+), %
r = −0.4123
r = 0.3414 r = 0.05467 r = −0.1472
(−0.6749 to
PD-1, ng/mL (−0.02566 to 0.6273) (−0.3155 to 0.4104) (−0.4851 to 0.2289)
−0.05702)
p a = 0.0602 p a = 0.7702 p a = 0.4294
p a = 0.0212
r = −0.2116 r = 0.2440 r = −0.1061 r = −0.02838
PD-L1, ng/mL (−0.5536 to 0.1915) (−0.1584 to 0.5768) (−0.4739 to 0.2934) (−0.4212 to 0.3734)
p b = 0.2995 p b = 0.2297 p b = 0.6061 p a = 0.8906
r = −0.1133 r = 0.1375 r = 0.1150 r = −0.1754
IL-7, pg/mL (−0.4757 to 0.2819) (−0.2592 to 0.4944) (−0.2803 to 0.4770) (−0.5233 to 0.2225)
p a = 0.5659 p a = 0.4855 p a = 0.5600 p a = 0.3719
PD-1, ng/mL on day 1 PD-1, ng/mL on day 5
r = 0.2317 r = 0.3774
IL-7, pg/mL (−0.04387 to 0.4744) (0.001264 to 0.6600)
p a = 0.0888 p a = 0.0436
Legend: a Spearman test, b Pearson test. Bold type indicates significance. B cells: B CD19+ lymphocytes, CD:
cluster of differentiation, IL-7: interleukin-7, NKT CD3+: natural killer T CD3+ lymphocytes, PD-1: programmed
cell death protein 1, PD-L1: programmed death ligand 1, Tc cells: T cytotoxic CD8+ lymphocytes, Th cells: T
helper CD4+ lymphocytes.

3. Discussion
Three consensus definitions of sepsis have guided physicians in understanding and
managing this insidious disease. However, in 2016, these definitions were revised to ad-
dress the significant limitations of previous versions, which included a simplistic model
of the illness. The earlier model suggested that sepsis progresses from a systemic inflam-
matory response to a compensatory anti-inflammatory response syndrome. The Sepsis-3
Int. J. Mol. Sci. 2024, 25, 12612 9 of 18

definition has redefined the understanding of sepsis, encompassing the essence of the
pathophysiology behind this disease. Nevertheless, much is still to understand [1,15].
In 2020, the WHO offered its first global report on sepsis, urging healthcare pro-
fessionals to step up efforts to improve data about sepsis. The condition is linked to
suboptimal quality of care, inadequate health infrastructure, poor infection prevention
measures, delayed diagnosis, and inappropriate clinical management. Additionally, an-
timicrobial resistance complicates sepsis management in all settings, especially among
high-risk populations, such as neonates and intensive care unit patients [4,16].
In 2017, a comprehensive global study identified 49 million sepsis cases and 11 million
sepsis-related deaths; around 20% of worldwide annual deaths were related to sepsis.
There were notable regional differences, with lower-middle-income countries experiencing
the highest incidence and mortality rates. The estimated average hospital cost of treating
sepsis was over USD 32,000 per patient; however, these figures primarily reflect data from
high-income countries [2,17]. In the 2020 WHO report, the in-hospital mortality rate for
sepsis patients receiving ICU treatment was estimated at 41.9%; for the European region,
mortality reached 42.7%, with an incidence of 139/100,000 adults/year [4,18].
The development of organ dysfunction is the most critical clinical event during sepsis,
as it is directly linked to mortality and morbidity. Despite the new definition by the
Sepsis-3 Consensus, which emphasises sepsis as a “life-threatening organ dysfunction
caused by a dysregulated host response to infection” [1], the mechanisms through which
sepsis induces organ dysfunction remain incomplete. This knowledge gap is significant
because mortality from sepsis remains very high, therapeutic options are limited, and
nonspecific, and morbidity continues to be a substantial burden for patients after hospital
discharge [19]. Nonetheless, the understanding of immunomodulatory mechanisms opens
up opportunities for personalised immune therapies.
In our study, the three leading infectious sites were pulmonary, abdominal, and urinary
tract, while the prevalent underlying conditions were cardiovascular, renal, respiratory,
neurological, and diabetes. In a 2022 study, Prest et al. studied sepsis-related mortality
trends based on the site of infection and found that pulmonary, abdominal, and geni-
tourinary sepsis were the leading causes of mortality in the United States of America;
demographical factors, such as age, sex, and race, played an important role [20]. In a 2022
study, Chen et al. examined the relationship between infection site and mortality in patients
with cancer and sepsis or septic shock. They found that lung, urinary tract, unspecified site,
and gastrointestinal infections were the most common, with gastrointestinal infections and
pneumonia showing the highest mortality rates [21].
To develop new immunotherapy approaches for patients with sepsis and septic shock,
it is essential to have a thorough understanding of the extensive changes occurring in the
immune system. In recent years, there has been increasing emphasis on strategies aimed at
effectively mitigating the immunosuppressive state in sepsis patients. A timely and accurate
assessment of immune status is crucial for promptly identifying immune dysfunction in
these patients and determining the optimal timing for administering immunomodulatory
therapy [3,22].
Lymphocytes play a crucial role in both initiating and sustaining the body’s response to
sepsis. Their significance lies in their ability to interact with the innate and adaptive immune
systems, as well as their capacity to coordinate, amplify, and regulate the inflammatory
response. Lymphocyte anergy or loss of their numbers can significantly impair the immune
system [23].
Our study observed lymphopenia in all the studied categories of patients from day
1 of study inclusion compared to the standard laboratory values. On day 5, we observed
an increase in the medians of the lymphocyte count, especially in the sepsis and survivor
groups, without statistical significance. A statistically significant variation was observed in
the percentage of total lymphocytes in the sepsis and survivor groups. In a study by Drewry
et al. involving 335 patients hospitalised with bacterial or fungal sepsis, lymphopenia
was observed in both survivors and non-survivors on the first day. However, by day
Int. J. Mol. Sci. 2024, 25, 12612 10 of 18

4, a notable difference emerged: Survivors had higher lymphocyte counts compared

to non-survivors [24]. Another study, published by Hohlstein et al., assessed 105 ICU
patients (52 of whom had sepsis) and found that survivors had significantly higher baseline
counts of leukocytes, lymphocytes, and T cells. Notably, patients with a T cell count below
0.36 × 109 /L at admission had a significantly higher 100-day mortality rate (60%) compared
to those with a count above the same value (20%) [25]. Two studies observed that a low
lymphocyte count sustained for more than 3 days is a predictor of 28-day mortality [24,26],
and one study found that lymphocyte percentage is a significant predictor of prognosis
in patients with lung cancer [27], supporting our findings of persistent lymphopenia in
the non-survivor group on day 5, together with the increased mortality rate of 72.41%.
However, lymphocyte percentage has not yet been proven to be a reliable tool in sepsis
evolution and prognosis.
A hallmark of sepsis is the significant depletion of lymphocytes due to apoptosis.
Postmortem examinations of patients who succumbed to sepsis revealed a substantial loss
of lymphocytes in the spleen, intestines, and other organs [28,29]. Lymphopenia involves
the loss of various lymphocyte classes, including CD4+ and CD8+ T cells, B cells, and
natural killer cells [28]. The T lymphocyte response becomes highly dysfunctional, and a
state of T cell exhaustion rapidly develops following sepsis [30,31].
A reduction in total lymphocyte count and lymphocyte infiltration in pathological
specimens is linked to poor overall survival [32]. IL-7 is an essential cytokine for the
immune system, and deficiencies in IL-7 or its receptor can result in severely impaired
development of immune cells. Studies have shown that IL-7 is essential for the devel-
opment and maintenance of various immune cells. It aids lymphocyte development in
the thymus, supports lymph node formation, and sustains activated T cells in secondary
lymphoid organs. Increased IL-7 production enhances the survival of both naïve and
memory T cells. IL-7 is also implicated in multiple stages of B-cell progenitor development,
including commitment, survival, differentiation, and proliferation. Additionally, IL-7 is
a critical cytokine that regulates the recruitment of leukocytes, such as neutrophils and
monocytes [28,33–35].
Recent literature regarding the IL-7 cutoff value is scarce, especially concerning sep-
sis and septic shock. The value of IL-7 has been studied thoroughly in cancer patients,
radiation-induced lymphopenia patients, and HIV patients [32,34,36]. Analysing the sep-
sis/septic shock groups, on day 1 of study inclusion, with the healthy control group, we
found the cutoff value of IL-7 to be 1.94 pg/mL with an AUC of 0.8547 (p < 0.0001). Patho-
logic values in the aforementioned studied groups were considered those above the cutoff
value of 1.94 pg/mL. We observed that on day 1, the median IL-7 levels in both the sepsis
and septic shock groups were elevated, surpassing the identified cutoff value. Although
a decrease by day 5 was not statistically significant, the median value remained elevated
above the cutoff threshold.
In the survivor and non-survivor groups, we observed a similar result: an increased
median value of IL-7, above the cutoff value, and a gradual decrease towards day 5, with a
statistically significant variation for the non-survivor group. This is explained by the fact
that IL-7 is a powerful antiapoptotic cytokine produced in various organs, including the
thymus gland, liver, peripheral lymphoid organs, and in small amounts by dendritic cells.
It plays an essential role in lymphocyte survival, development, and expansion [23]. The
increased values of IL-7 above the cutoff threshold and maintained throughout the studied
period, observed in our studied groups, serve as a means to restore depleted lymphocyte
populations [37]. The statistically significant decrease in IL-7 in the non-survivor group
is translated into lymphocyte fatigue and functional exhaustion, predisposing the patient
to secondary infections and subsequent immunosuppression in individuals who survive
the initial septic episode [1,23]. A study from 2023 by Leśnik et al. found a cutoff value for
IL-7 on admission day for the septic shock group of 15.8 ng/l (15.8 pg/mL) and an AUC
of 0.556 (p = 0.81), the values gradually decreasing on days 3 and 5 [9]. The cutoff value
Int. J. Mol. Sci. 2024, 25, 12612 11 of 18

of 15.8 pg/mL is superimposed on the maximum value of Il-7 that we found in the septic
shock group on day 5 (15.3 pg/mL).
In sepsis, immunosuppression involves various cell types and mechanisms, most
notably the increased apoptosis of T and B cells, T cell exhaustion, and reduced expression
of activating surface molecules. The apoptosis of immune cells is particularly pronounced
in CD4+ T cells, CD8+ T cells, B cells, natural killer cells, and follicular dendritic cells [7,38].
For the sepsis group, we observed a statistically significant negative correlation be-
tween CD4+ and CD8+ and between CD4+ and NKT on both studied days, suggesting a
decrease in the value of both helper and cytotoxic lymphocytes. We observed the same
statistically significant negative correlation between CD4+ and CD8+ and between CD4+
and NKT in the septic shock group. Similar negative correlations were found in the survivor
and non-survivor groups. The negative correlation between helper T lymphocytes and
cytotoxic lymphocytes, irrespective of sepsis or septic shock, can be attributed to profound
immunosuppression and heightened inflammation. The fluctuation in T lymphocytes is
indispensable for the immune response against sepsis. Nevertheless, even if lymphocyte
counts remain within the normal range, immunoparalysis can severely impair their func-
tion [23,39]. These findings are consistent with recent studies that have also reported a
significant reduction in CD4+ cells among sepsis patients [5].
Known for their role in adaptive immunity, helper T lymphocytes activate B cells
and cytotoxic T lymphocytes [39]. Our study observed a statistically significant negative
correlation between NKT lymphocytes and CD19+ B lymphocytes, in the sepsis group,
survivor and non-survivor groups, on day 1. This is sustained by how sepsis affects
these lymphocyte subpopulations. B lymphocytes are central to the adaptive immune
response, producing antigen-specific antibodies targeting pathogens. In sepsis, they become
dysregulated and exhausted, rendering the cells of the adaptive immune system incapable
of mounting an effective defence against infection. A recent study by Wang et al. found that
the frequency and absolute number of regulatory B lymphocytes (Bregs) were significantly
decreased in septic patients compared with healthy controls, and that low levels of Bregs
were associated with an increased risk of non-survival in septic patients [40].
NKT lymphocytes are a unique subset of T cells, primarily acting in the body’s defence
against infections. NKT cells are abundant in liver, spleen, and peripheral blood tissues.
They respond rapidly to various pathogens and perform multiple roles, including cytotoxic
activity, supporting antibody production, influencing Th1/Th2 differentiation, and linking
innate and adaptive immune responses. In sepsis, NKT cells are important initiators of
immune responses, where they activate and regulate downstream effector immune cells
and trigger an inflammatory cytokine cascade, supporting pathogen clearance and immune
response coordination. [41,42]. The role of NKT cells in regulating sepsis is complex.
While they trigger an inflammatory cytokine cascade necessary for clearing pathogens, an
excessive inflammatory response can negatively impact patient outcomes. The heightened
inflammatory activity can lead to NKT cell apoptosis and immunosuppression, significantly
contributing to sepsis mortality [41].
An interesting finding of our study is the statistically significant variation between
day 1 and day 5 for NKT lymphocytes, an increase in the median value towards day 5 for
the survivor group. Although not statistically significant, we observed a reversal of the
medians in the septic shock and non-survivor groups, with a decrease towards day 5. In
a 2015 study, Young et al. observed that a significant decrease in NKT lymphocytes due
to apoptosis reduces IFN-gamma secretion, thus contributing to immunosuppression in
sepsis non-survivors [43]. However, the mechanisms behind NKT cell apoptosis triggered
by sepsis remain poorly understood. In 2023, Wu et al. found that NKT cell subsets
were significantly reduced by up to 60% within the first two days following sepsis onset,
primarily due to increased apoptosis [41]. In our study, the increased median value of NKT
on the fifth day compared to the first day in the sepsis group places the subjects in the
initial hyperinflammatory phase of sepsis; meanwhile, the reversed median values for the
Int. J. Mol. Sci. 2024, 25, 12612 12 of 18

septic shock and non-survivor patients represent the immunosuppression manifested in

the late stages of sepsis, together with functional exhaustion and anergy.
In our study, the cytotoxic answer can be observed by the statistically significant
positive correlation between CD8+ T cytotoxic lymphocytes and NKT lymphocytes on
day 1 and day 5 in the sepsis group, a correlation found in the survivor and non-survivor
groups as well. Doganyigit et al. published a study stating that cytotoxic T lymphocytes are
key components of the cytotoxic cell axis, playing an important role in defending the host
by lysing cells infected with intracellular pathogens [8]. Another study published by Chen
et al., investigating the immune status of CD8+ lymphocytes by examining the expression
of granzyme A, granzyme B, and perforin, found that in septic shock non-survivors the
activated CD8+ T lymphocytes were consumed, followed by a suppression of their immune
function, but in septic shock survivors, there was a compensatory increase [7], suggesting
that a rebalancing surge of cytotoxic players occurs during sepsis. These studies endorse
our findings that CD8+ and NKT correlate positively in the sepsis group and in both
survivors and non-survivors groups.
Immune checkpoint inhibitory receptors on immune cells trigger signalling pathways
promoting immunosuppression. These molecules function as regulatory mechanisms,
similar to brakes, moderating the adaptive immune response. PD-1, an immune checkpoint
receptor, plays a pivotal role in regulating both the quantity and functional capacity of
T cells [44]. PD-1 is expressed on T cells, B cells, antigen-presenting cells, and some non-
lymphoid tissues. When ligands bind to PD-1 receptors on T cells, it leads to immune
suppression by inhibiting T-cell activation and encouraging apoptosis. Additionally, PD-L1
is found in cardiac endothelial cells, placental tissues, and pancreatic islets, indicating its
role in promoting immunological tolerance [45–47]. Soluble forms of PD-1 and PD-L1 can
be produced by shedding the membrane-bound forms or as alternative splice variants.
PD-1 has both a membrane-bound form and a soluble form, known as soluble PD-1 (sPD-1),
which enters the bloodstream and acts similarly to a cytokine, helping to regulate the
immune response. Recently, sPD-1 has gained attention as a crucial natural inhibitor that
helps maintain the balance of the PD-1/PD-L1 signalling pathway. It has been linked
to risk stratification and prognosis assessment [48,49]. Zhao et al. studied the soluble
form of PD-L1, which regulates the PD-1/PD-L1 axis. During sepsis, serum levels of
sPD-L1 are elevated, which may indicate the severity of the condition and predict clinical
outcomes [50].
In non-survivors, we observed strong evidence of immunosuppression, indicated by a
statistically significant positive correlation between CD4+ helper cells and PD-1 on day 1,
which shifted to a statistically significant negative correlation by day 5 of the study. Similar
correlations were found between CD4+ and PD-L1, although not statistically significant.
Also, statistically significant negative correlations on day 1 between CD8+ cytotoxic T
lymphocytes, PD-1, and PD-L1, together with a negative correlation between NKT and
PD-1 on day 1, suggest an increased inflammatory response leading to immune suppression.
These align with findings from Shao et al., who reported increased PD-L1 expression on
immune cells in septic shock patients, which has been associated with higher mortality rates
within the first 28 days of illness. The study also noted the activation of inhibitory immune
checkpoints, metabolic disruptions, impaired effector cytokine production, reduced cell
proliferation, and increased susceptibility to apoptotic cell death [13,51].
To enhance the profound immunosuppression brought by the expression of the PD-
1/PD-L1 axis, we found a statistically significant positive correlation in the septic shock
group on the first day of study between PD-1 and PD-L1. Despite not being statistically
significant, a positive correlation between the two components was found on day 5 as well.
In a previously published study [52], we found positive correlations between PD-L1 and
the sequential organ failure assessment score, reflecting the presence of multiple organ
dysfunction and an immunosuppressed status and increasing the risk of mortality in these
patients. In a separate study, Zhao et al. discovered that non-survivors of sepsis had higher
peripheral blood levels of soluble PD-1 and PD-L1, as well as increased PD-1 expression
Int. J. Mol. Sci. 2024, 25, 12612 13 of 18

on CD4+ and CD8+ T cells and PD-L1 expression on monocytes, compared to survivors.
Additionally, the levels of soluble PD-1 and PD-L1 were found to correlate with the severity
of the disease [50]. Enhanced PD-1/PD-L1 axis expression increases mortality and the
development of secondary infections [53,54].
The battle between cell survival and cell death mechanisms can be seen in the statisti-
cally significant positive correlation we found on day 5 in the septic shock and non-survivor
groups between PD-1 and IL-7. Recent studies have shown that PD-1 expression levels
on responding T lymphocytes decrease when the activated antigen is eliminated. If the
antigen is not cleared, as seen in persistent infections and malignancies, PD-1 expression re-
mains elevated [13], rendering T cells fatigued, dysfunctional, vulnerable to apoptosis, and
unable to participate in an effective immune response [44,55]. Conversely, IL-7 enhances
cell survival by stimulating the strong proliferation of CD4+ and CD8+ T lymphocytes,
preventing lymphocyte apoptosis, restoring CD4+ and CD8+ T cell function, and ultimately
improving survival [9].
This study has several limitations. First, we measured only the serum-soluble levels
of PD-1 and PD-L1 without assessing the expression of membrane-bound PD-1/PD-L1.
It is essential to highlight that detecting serum-soluble PD-1/PD-L1 is easier and more
cost-effective than detecting the membrane-bound forms, which also speeds up disease
diagnosis in clinical settings. Second, our study cannot affirm that there is a direct relation-
ship between serum-soluble PD-1/PD-L1 and the variations in certain lymphocyte subsets.
This can only be achieved by measuring the expression of membrane-bound PD-1/PD-L1
on lymphocytes. Nevertheless, the clinical use of serum-soluble PD-1/PD-L1 testing in
sepsis shows valuable predictive capacity, aiding the physician in reaching the outcome.
Third, the relatively small sample size made it difficult to draw definitive conclusions.
Furthermore, as a single-centre study, there was potential bias in evaluating the pathology.
In the future, the authors plan to expand the study group and further evaluate the examined
parameters in a larger patient cohort.

4. Materials and Methods

4.1. Ethical Approval
This prospective observational single-centre study enrolled 87 sepsis patients admitted
to the ICU of the County Emergency Clinical Hospital in Târgu Mures, , Romania, between
July 2021 and March 2023.
The study was conducted in accordance with the Helsinki Declaration of 1975. Ethical
approval for this study was obtained from the Ethics Committee of the University of
Medicine and Pharmacy, Science, and Technology ‘George Emil Palade’ in Târgu Mures,
(Mures, , Romania; approval no 1425/01.07.2021). Informed consent for study participation
and data publication was obtained from each patient or their legal guardian.
We enrolled 82 healthy volunteers as the control group. Informed consent for study
participation and data publication was obtained from each volunteer, and the General Data
Protection Regulation was adhered to.

4.2. Study Cohort

Participants included individuals aged 18 and above diagnosed with sepsis or septic
shock according to the Sepsis-3 Consensus criteria [1]. The exclusion criteria were as
follows: current cancer with chemotherapy or radiation therapy, ongoing treatment with
corticosteroids or immunosuppressive medication, and evidence of autoimmune disorders.
We divided the patients into four subcategories according to diagnostic (sepsis and
septic shock) and survival status (survivors and non-survivors).
Eighty-two healthy volunteers were studied alongside the patients as a control group
to assess the cutoff values of IL-7. The control group comprised unrelated individuals
(24 males, 58 females) with an average age of 32 years and without known infections in the
previous 18 months.
Int. J. Mol. Sci. 2024, 25, 12612 14 of 18

4.3. Evaluated Parameters

The parameters under examination included age, gender, complete blood count (CBC),
T-helper lymphocytes (CD4+), T-cytotoxic lymphocytes (CD8+), natural killer T CD3+
lymphocytes (NKT), programmed cell death-1 (PD-1), programmed cell death-1 ligand
(PD-L1), and interleukin-7 (IL-7). We assessed the parameters on the first and fifth days
after confirmation of either sepsis or septic shock in the intensive care unit (ICU), aligning
with the findings of the IRIS-7 trial [33].
Venous blood samples were collected from each study participant via peripheral
puncture using 10 mL syringes. Blood was collected in K2 EDTA tubes for leukocyte subset
identification and sent for processing. For PD-1/PD-L1 expression quantification, blood
was collected in clot activator tubes, centrifuged for 15 min at 750 × g, aliquoted, and
stored at −80 ◦ C in 1.5 mL Eppendorf tubes until all patients were recruited.

4.4. CBC and Lymphocyte Subsets

For CBC and immunophenotyping, venous blood samples were collected in K2 EDTA
tubes. The identification of leukocyte subsets was performed using the BD FACSCalibur™
flow cytometer analyser (Becton, Dickinson and Company, Franklin Lakes, NJ, USA), and
the interpretation of data was performed with BD CellQuest™ Pro (Becton, Dickinson and
Company, Franklin Lakes, NJ, USA). The subsets of leukocytes were expressed as % of total
peripheral blood mononuclear cells. We gated on lymphocytes using the Side Scatter/CD45
density plot.
The following combination of monoclonal antibodies conjugated to specific fluo-
rochromes from BD® Biosciences reagents (Becton, Dickinson and Company, Franklin
Lakes, NJ, USA) was used:
• CD4/CD8/CD3 (BDTritest, Cat. No. 342414) to identify and enumerate the following
T-lymphocyte subset populations: CD3+ T lymphocytes, CD3+CD4+ helper/inducer
T lymphocytes, and CD3+CD8+ suppressor/cytotoxic T lymphocytes.
• CD3/CD16+CD56/CD45/CD19 (BD Multitest, Cat. No. 342416) to identify and
enumerate the following T-, B-, and NK-lymphocyte subset populations: CD3+ T
lymphocytes, CD19+ B lymphocytes, and CD3-CD16+CD56+ NK lymphocytes.
Lymphocytes were identified using CD45 and side scatter (SSC) gating strategy as
CD45bright with low SSC. The relative proportion of B (CD3-CD19+) and T (CD3+CD19-)
cells was then quantified as the plot was split into four quadrants, allowing the identifi-
cation of the cells’ single positive for each marker. Then, T cells were identified by CD3+
expression, and CD8+ cytotoxic and CD4+ helper T cells were identified by a CD8 versus
CD4 dot plot.

4.5. PD-1/PD-L1
The cytokine/target proteins PD-1 and PD-L1 levels were assessed using an ELISA
protocol. Commercial kits designed for detecting recombinant and natural human pro-
grammed cell death-1 and programmed cell death-1 ligand were employed following
the manufacturer’s instructions (EIAab Science Inc., Wuhan, China) and processed using
automated ELISA equipment (Dynex DSX, Dynex Technology USA, Chantilly, Virginia,
VA, USA). Blood samples were collected via venous puncture using clot activator tubes,
then centrifuged for 15 min at 750× g. The serum was subsequently aliquoted and stored
at −80 ◦ C in 1.5 mL Eppendorf tubes until all patients were recruited.
The ELISA plates used for PD-1/PD-L1 quantification were precoated with anti-
bodies with specificity to PD-1 or PD-L1 protein, respectively. Samples and standards
with known concentrations were incubated with a biotin-conjugated antibody for 2 h at
37 ◦ C. During this step, the antigen–antibody complex was formed. After the subsequent
wash, avidin conjugated with horseradish peroxidase was added to all wells, followed
by 1 h incubation at 37 ◦ C. The second washing step, followed by the addition of the 3,3′ ,
5,5′ -tetramethylbenzidine substrate, allowed the formation of immune complexes to be
revealed.
Int. J. Mol. Sci. 2024, 25, 12612 15 of 18

The concentration of the target protein, either PD-1 or PD-L1, was proportional to the
intensity of the colour developed in each reaction well; the change in the colour intensity
was measured at 450 nm. The software generated a 4-parameter logistic curve, allowing
the quantification of the target proteins in the neat serum samples. The detection range for
PD-1 was 0.156–10 ng/mL, sensitivity was <0.067 ng/mL, and the coefficients of variations
were ≤4.2% for intra-assay and ≤8.7% for inter-assay precision. For PD-L1, the detection
range was 0.312–20 ng/mL, sensitivity was <0.098 ng/mL, and the coefficients of variations
were ≤6% for intra-assay and ≤7.9% for inter-assay precision.

4.6. IL-7 Quantification

For IL-7 detection in serum samples, the ELISA sandwich immunoassay from Ab-
clonal (Abclonal Technology. Co., Ltd., Cummings Park, Woburn, MA, USA) followed
the manufacturer’s instructions. Briefly, neat serum samples and calibrators with known
concentrations were incubated for 2 h in precoated wells with anti-IL-7 antibodies. The
captured antibodies have specificity for both natural and recombinant human IL-7.
After a washing step, a second 1 h incubation with a secondary biotin-conjugated
antibody was performed. The addition of streptavidin–horseradish peroxidase and subse-
quent 3,3′ , 5,5′ -tetramethylbenzidine substrate revealed the antigen–antibody complexes
by changing the colours in the wells. The concentration was directly related to the intensity
of the colour, which was read spectrophotometrically at 450 nm. The sample concentration
was evaluated using the 4PL calibration curve performed by the Dynex DSX instrument.
The measuring range for IL-7 was 7.8–500 pg/mL, and the minimum detectable concentra-
tion was 3.9 pg/mL. The precision was <10% for intra-assay and <15% for inter-assay.

4.7. Statistical Analysis

The information was inputted into MS Excel (Microsoft® Excel® for Microsoft 365
MSO Version 2406 Build 16.0.17726.20078, Microsoft Corporation, Redmond, WA, USA).
Subsequent statistical analyses, encompassing descriptive and inferential processing, were
conducted using GraphPad Prism version 8.4.3 (686), released on 10 June 2020 (GraphPad
Software, San Diego, CA, USA). Descriptive statistics included the computation of means
or medians with corresponding confidence intervals. The Kolmogorov–Smirnov test was
performed to verify the normality of the data distribution. The mean was determined for
data exhibiting a normal distribution, while for non-Gaussian distributions, the median and
the interquartile range were calculated. We used either the Student’s t-test or the Wilcoxon
test for paired data, depending on whether the distribution was Gaussian or non-Gaussian.
For receiver operating characteristic (ROC) analysis, GraphPad Prism version 8.4.3 (686)
was used. p ≤ 0.05 was considered to indicate a statistically significant difference.

5. Conclusions
Monitoring lymphocyte levels shows potential as a biomarker for tracking sepsis
progression. In our study, we noted a mild increase in lymphocyte percentages among both
septic and surviving patients. However, lymphocyte percentages alone have not yet been
validated as a consistent predictor of sepsis outcomes.
The study emphasises the significance of monitoring IL-7 levels in patients with sepsis
and septic shock; low levels were linked to a higher risk of mortality, as seen in the non-
survivor group. IL-7 plays a dual role in sepsis; it can also be used to guide exogen therapy
with IL-7.
PD-1/PD-L1 axis is a biomarker of the immune system; its upregulated expression
weakens costimulatory signalling, resulting in reduced T cell response capacity, lym-
phopenia, higher mortality rates, and greater vulnerability to nosocomial infections. The
immunosuppressive state was particularly evident in the non-survivor group of patients,
as indicated by the correlations with both helper and cytotoxic lymphocytes.
Int. J. Mol. Sci. 2024, 25, 12612 16 of 18

Supplementary Materials: The supporting information can be downloaded at: https://www.mdpi.

com/article/10.3390/ijms252312612/s1.
Author Contributions: Conceptualization, O.C. and B.-L.G.; data curation, O.C. and B.-L.G.; formal
analysis, O.C. and M.P.; investigation, O.C., A.H. and A.B.; methodology, O.C., B.-L.G., and R.S, .F.;
project administration, B.-L.G. and R.S, .F.; resources, O.C. and B.-L.G.; software, O.C. and M.P.;
supervision, L.A. and B.-L.G.; validation, A.B. and A.M.V.; writing—original draft, O.C. and B.-L.G.;
writing—review and editing, R.S, .F., A.M.V., and L.A.; funding acquisition, B.-L.G. All authors have
read and agreed to the published version of the manuscript.
Funding: This research was funded by the University of Medicine, Pharmacy, Science and Technology
‘George Emil Palade’ of Târgu Mures, (grant no. 10126/17.12.2020).
Institutional Review Board Statement: This study was conducted according to the guidelines in the
Declaration of Helsinki, and ethical approval for this study was obtained from the Ethics Committee
at the University of Medicine, Pharmacy, Science, and Technology ‘G.E. Palade’ in Târgu Mures, ,
Mures, , Romania; approval no. 1425/01.07.2021.
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.
Written informed consent was obtained from the patients to publish this paper.
Data Availability Statement: The data generated in the present study may be requested from the
corresponding author.
Conflicts of Interest: The authors declare that there are no conflicts of interest.

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