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\begin{document}

\title{CD28 monoclonal antibody improve sepsis mortality by amending Th17/Treg
balance}



\author[1]{yu wu}%
\author[1]{Dai Li}%
\author[1]{Jian Xie}%
\author[2]{Hang Wang}%
\author[3]{Yan Meng}%
\author[4]{Guosheng Wu}%
\author[5]{Xiaoming Deng}%
\author[1]{Xiaojian Wan}%
\affil[1]{Changhai Hospital, Naval medical university}%
\affil[2]{ Changhai Hospital, Naval medical university}%
\affil[3]{ Changhai Hospital}%
\affil[4]{Changhai Hospital, Navy Medical University}%
\affil[5]{Changhai Hospital}%


\vspace{-1em}



  
  \date{March 15, 2025}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Background: Sepsis is still developing exorbitantly high mortality. T
cell activation plays a crucial role in sepsis's initial innate immune
reactions. The imbalance between Th17 and Treg participates in sepsis
progression. CD28 signaling pathway was essential for the expression of
inflammatory cytokines related to Th17, and play a crucial role in the
maintenance of Treg. Methods: 60 sepsis patients' baseline conditions
were recorded, and the expressions of inflammatory factors in the
peripheral blood and levels of procalcitonin (PCT) were detected. PBMCs
were separated, subtypes of T cells and related biomarkers were measured
by FACS. Furthermore, the relationship between the above indicators and
patients' condition scoring (APACHEIIand SOFA) and ICU hospitalization
time were analyzed. To investigate effects of CD28 on the balance of
Th17 between Treg, anti-CD28 antibody was intraperitoneal administrated
to cecal ligation and puncture (CLP) mice. Results: Compared with septic
patients who stay in ICU more than 14 days, the Th17/Treg ratio of
patients fewer than 14 days was significantly lower. Moreover, the
expression of CD28 was significantly higher in sepsis patients than that
of healthy donors. After administration of CD28 monoclonal antibody,
7-day mortality and clinical score were significantly improved in septic
mice, with splenocyte Th17/Treg ratio decreased. CD28 antibody
alleviates the expression of pro-inflammatory factors and spleen injury
related to apoptosis. Conclusions: Th17/Treg ratio revealed septic
patient severity and as a predictor of ICU stay. CD28 monoclonal
antibody could improve 7-day mortality of septic mice by decreasing T
cell apoptosis and amending the ratio of Th17/Treg.%
\end{abstract}%



\sloppy


CD28 monoclonal antibody improve sepsis mortality by amending Th17/Treg
balance

Yu Wu\textsuperscript{1,2}, Dai Li\textsuperscript{1}, Jian
Xie\textsuperscript{1}, Han Wang\textsuperscript{1}, Yan
Meng\textsuperscript{1}, Guosheng Wu\textsuperscript{3}, Xiaoming
Deng\textsuperscript{1}, Xiaojian Wan\textsuperscript{1}

*Correspondence: deng\_x@yahoo.com, xiaojian.wan@yahoo.com

Yu Wu, Dai Li, Jian Xie these authors are co-first authors

1.Department of Anesthesiology and Critical Care Medicine, Changhai
Hospital, Naval medical university, Shanghai, China

2.Department of Anesthesiology and Critical Care Medicine, the 980th
Hospital (Bethune International Peace Hospital) of the Joint Logistics
Support Force, Shijiazhuang, China

Full list of author information is available at the end of the article

\textbf{Abstract}

\textbf{Background:} Sepsis is still developing exorbitantly high
mortality. In response to microbial molecules, T cell activation plays a
crucial role in sepsis's initial innate immune reactions. The imbalance
between Th17 and Treg participates in sepsis progression. CD28 signaling
pathway was essential for the expression of inflammatory cytokines
related to Th17, and play a crucial role in the maintenance of Treg. We
investigated the correlations of the balance between Th17 and Treg to
prognosis in sepsis patients and influence of anti-CD28 antibody on the
ratio of Th17 to Treg in sepsis mice.

\textbf{Methods:} 60 sepsis patients' baseline conditions were recorded,
and the expressions of inflammatory factors in the peripheral blood and
levels of procalcitonin (PCT) were detected. PBMCs were separated,
subtypes of T cells and related biomarkers were measured by
Fluorescence-activated cell sorting (FACS). Furthermore, the
relationship between the above indicators and patients' condition
scoring (APACHEIIand SOFA) and ICU hospitalization time were analyzed. To
investigate effects of CD28 on the balance of Th17 between Treg,
anti-CD28 antibody was intraperitoneal administrated to cecal ligation
and puncture (CLP) mice.

\textbf{Results:} Compared with septic patients who stay in ICU more
than 14 days, the Th17/Treg ratio of patients with ICU hospitalization
of fewer than 14 days was significantly lower. The sepsis patients with
higher expression of CD28 in peripheral blood lymphocytes have lower
APACHE II and SOFA scores. Moreover, the expression of CD28 was
significantly higher in sepsis patients than that of healthy donors.
After administration of CD28 monoclonal antibody, 7-day mortality and
clinical score were significantly improved in septic mice, with
splenocyte Th17/Treg ratio decreased. CD28 antibody alleviates the
expression of pro-inflammatory factors and spleen injury related to
apoptosis.

\textbf{Conclusions:} Th17/Treg ratio revealed septic patient severity
and can be used as a predictor of ICU stay. CD28 monoclonal antibody
could improve 7-day mortality of septic mice by decreasing T cell
apoptosis and amending the ratio of Th17/Treg.

\textbf{Keywords:} Sepsis, length of stay in ICU, Th17/Treg, CD28, Risk
stratification, Clinical outcomes

\textbf{Trial registration:} CHEC2019-133,
CTR20191855\textbf{.}Registered 27 August 2019

\textbf{Background}

Sepsis is a significant public health concern; the estimated number of
sepsis cases each year worldwide has doubled compared with previous
estimations and is almost 49 million now{[}1{]}. Sepsis refers to the
life-threatening organ dysfunction caused by the dysregulation of the
body's response to infection, especially immune dysregulation with
pro-inflammatory and anti-inflammatory imbalance{[}2{]}. The complicated
collapsed immune response to severe infection was considered the central
pathogenesis of sepsis{[}3-5{]}.

Meanwhile, immunosuppression also coincides with lymphopenia and loss of
immune function. T lymphocytes are one of the critical immune cells with
regulating antimicrobial phagocytic and cytotoxic activity. There are
many different subtypes T lymphocytes such as na\selectlanguage{ngerman}ïve T cells, helper T
(Th) cells, memory T cells, regular T (Treg) cells (CD4+CD25+ regulatory
T cells) and so on with different surface markers, transcriptional
regulators, effector molecules and functions. The subtypes of each cell
and the ratio between subtypes are different at different stages of T
cell immunity{[}6{]}. Among such subtypes, Treg and CD4+IL-17+ T helper
cells(Th17) share a common precursor cell (the naïve CD4 T cell) and
change appearance with the disease progressing{[}7, 8{]}. Mice that
knocked out the FoxP3 gene, which Treg cell-specific marker controls the
T cells development and their regulatory activities, developed fatal
inflammatory diseases{[}9{]}. Th17, secreting IL-17 and specific
transcriptional regulator ROR-\selectlanguage{greek}γ\selectlanguage{english}t, plays an essential physiological role
at mucosal barriers and is involved in inflammatory responses to
pathogens {[}10{]}. The balance between Th17 and Treg has emerged as a
prominent factor in regulating autoimmunity{[}11{]}. Accumulated
evidences suggest that the imbalance of Th17 and Treg is associated with
the development of many diseases{[}12{]}, such as primary Sj\selectlanguage{ngerman}ögren's
syndrome{[}13{]}, Experimental Autoimmune Encephalomyelitis{[}14{]},
human graft-versus-host disease{[}15{]} and asthma{[}16{]}.

The ratio of Th17/Treg might become predictors of immune status; the
identification and intervention of T cell immune response process in
sepsis may be the focus of future treatment. CD28 signal pathway, one of
the critical costimulatory molecules on T cells, can active and maintain
T cell functions, stimulate the body's immune response through combining
with CTLA-4 and B7(CD80/CD86) molecule on antigen-presenting cells (APC)
{[}17{]}. CD28 signaling is essential for effector T cells to overcome
Treg cell-mediated immunosuppression{[}17{]}. CD28~signaling pathway
plays a crucial role in maintaining Treg pool size and
each~Treg~subset's homeostasis by promoting the development and
proliferation of these cells {[}18, 19{]}. Then we observed the ratio of
Th17/Treg in predicting severity and outcomes of septic patients,
furthermore as the possible hint of the immunotherapy timing. Effects of
CD28 signaling pathway on the balance of Th17/Treg in septic mice need
to be identified, then providing a therapeutic reference for sepsis.

\textbf{Methods}

\textbf{Patient characteristic}

Patients with sepsis were recruited and the study was approved by the
human ethics committee of Changhai Hospital (CHEC2019-133), as
preliminary trial of sepsis treatment(CTR20191855), informed consent
from all of the subjects or their relatives was obtained before
enrollment. 65 patients suffering from sepsis were admitted to the
intensive care unit (ICU) in Shanghai Changhai Hospital from January to
October 2020, meanwhile 13 healthy volunteers, gender, age and ethnicity
was the same, as the control group were recruited. The sepsis diagnostic
criteria according to the definitions for sepsis and septic shock
established by The Third International Consensus (Sepsis-3){[}20{]}.
Eligibility criteria for enrollment in this study included the sepsis
patients of either sex by age 18--80 years with systolic blood pressure
\textless{}80 mmHg, presenting within 8 h of sepsis after taking proper
consent from the patient or relatives. The severity of the patients was
assessed by Acute Physiology and Chronic Health Evaluation score (APACHE
\selectlanguage{english}II) and the extent of development of organ dysfunction was assessed by
the Sequential Organ Failure Assessment score (SOFA; range, 0--24).
Exclusion criteria included: 1. age \textless{}18 years; 2. severe acute
head injury (GCS score \textless{}5); 3.irresuscitable terminal stage at
presentation; 4. autoimmune disease (such as rheumatoid arthritis,
systemic lupus erythematosus, asthma, or multiple sclerosis); 5. AIDS;
6. acute stroke, myocardial infarction or recent viral hepatitis;7.use
of hormones or immunosuppressors within the past 3 months before
hospitalization;8.transplant surgery;9.unexpected termination of CBP
treatment;10.Patients who resuscitated with anti-inflammatory drugs or
corticosteroids; 11. septic patients, of whom blood sampling was not
taken within the first 8 h after definition sepsis.

\textbf{Blood samples and preparation}

Venous blood samples were drawn into ethylenediaminetetraacetic acid
tubes within 24 hours after the patient is diagnosed with sepsis,
isovolumetric venous blood samples were collected from healthy
volunteers in the physical examination center of our hospital. Blood
samples were refrigerated at 4degC after EDTA anticoagulation. Density
gradient centrifugation was conducted at 2,000 rpm for 20 minutes to
isolate Peripheral blood mononuclear cells(PBMCs), which were used to
detect the expression of membrane markers, and plasma, which was stored
at -80degC for subsequent cytokine detection. The concentration of PBMCs
was adjusted to 1 x 10\textsuperscript{6}/mL in RPMI 1640 culture
solution supplemented with 100 U/mL penicillin, 100 \selectlanguage{greek}μ\selectlanguage{english}g/mL streptomycin,
2 mM/L-glutamine, and 10\% fetal calf serum (Gibco). The cell suspension
was seeded onto 12-well cell culture plates. Cells were treated with 1ul
Leukocyte Activation Cocktail, with BD GolgiPlug(BD), and incubated in
darkness at 37\selectlanguage{ngerman}°C under 5\%-CO\textsubscript{2} atmosphere for 6 hours.

\textbf{Animals}

Male C57BL/6 mice aged 6--8 weeks and weighing 20-25g were used and bred
carefully in the Changhai Hospital Animal Laboratory Centre under the
ethical guidelines of the National Institutes of Health on Animal Care.
Mice were raised in specific pathogen-free cages and maintained at
temperatures between 20 and 25 °C and relative humidity of 50--70\%. The
experimental protocols were approved by the Animal Care and Use
Committee of Navy Medical University, Shanghai.

\textbf{Reagents}

Mouse TNF-a, IL-17A, IL-10, IL-6 enzyme-linked immunosorbent assay
(ELISA) kits, CD28 functional antibody, fluorescent-labeled monoclonal
antibodies anti-mouse CD4-FITC, anti-mouse IgG1-FITC, anti-mouse
CD25-PE, anti-mouse IgG1-PE, anti-mouse FoxP3-APC, anti-mouse
IFN-gamma-PE, anti-mouse IgG1-APC, and anti-mouse IL-17-APC were bought
from eBiosciences (San Jose, CA, USA). FIX \& PERM medium from
Invitrogen, USA. Leukocyte Activation Cocktail from BD, Catalog
No.550583, contain PMA(Phorbol 12-Myristate 13-Acetate), a calcium
ionophore (Ionomycin) and the protein transport inhibitor BD GolgiPlug\selectlanguage{english}
(Brefeldin A).

\textbf{Animal Treatment and cecal ligation and puncture (CLP)}

Mice were anesthetized with isofluorane and a midline abdominal incision
was made. The cecum was mobilized, ligated below the ileocecal valve,
and punctured twice with a 22 gauge needle to induce polymicrobial
peritonitis. Sham-operated mice underwent the same procedure, including
opening the peritoneum and exposing the bowel, but without ligation and
needle perforation of the cecum. After surgery, the mice were injected
with 1 mL physiologic saline solution for fluid resuscitation and 20ug
butorphanol for post-operative analgesia. All mice had unlimited access
to food and water both pre- and post-operatively, allowed to die
spontaneously from sepsis. According to the literature and our previous
research, 5 mg/kg CD28 functional antibody was administered
intraperitoneally at the same time{[}21{]}. Mice were scored with murine
sepsis severity scale and survival rate by two independent, blinded
researchers.

\textbf{Mouse clinical score after treatment.}

After CLP-induced polymicrobial sepsis successfully in mice, all of them
moved free and none needed analgesia for pain immediately. The clinical
scores of mice at 0h, 2h, 6h, 12h, 24h, 48h, 72h, 4d, 5d and 7d by
incorporating to the Murine Sepsis Score (MSS) were evaluated. The
scoring criteria are as follows: spontaneous activity, response to touch
and auditory stimuli, posture, respiration rate and quality (labored
breathing or gasping), and appearance (i.e., degree of piloerection),
each of these scores variables between 0-4{[}22{]}.

\textbf{Spleen index}

After the mice were completely anesthetized by sevoflurane on the third
day after CLP, mice were weighed and mice's spleen was harvested then
measure the length. Spleen index can be expressed as the weight of the
spleen divided by the weight of the mouse.

\textbf{Histology}

At 3 days after CLP challenge{[}21{]}, mice were sacrificed by
CO\textsubscript{2} inhalation. Then, the spleens were collected and
fixed in 4\% paraformaldehyde solution for more than 48 hours before
being embedded in paraffin and sectioned. After deparaffinization and
rehydration, the sections were stained with hematoxylin and eosin
(H\&E). The congestion and necrosis of the spleen tissue and the
aggregation of inflammatory cells were evaluated under an optical
microscope. Then the spleen injury scores of mice based on the results
of H\&E{[}23{]} was evaluated by two independent, blinded researchers.

\textbf{Culture splenocytes}

After the mice were completely anesthetized by sevoflurane, the mice
were killed by cervical dislocation. Then place the mouse in a bottle
filled with alcohol for 5 minutes to sterilize, and operate in a sterile
biological operation table: use a sterile instrument to perform
laparotomy to remove the spleen tissue of the mouse and place it into a
sterile mill previously added with sterile PBS Grind gently in the
grinding dish to completely separate the spleen cells, then add
erythrocyte lysate and let stand for 10 minutes, then stop with sterile
PBS, centrifuge to remove the lower layer of cells. After filtering out
the non-cellular components with a filter, count
1x10\textsuperscript{6}cells into a sterile culture dish, add Leukocyte
Activation Cocktail 1ul and incubate for 6 hours at 37 deg C with 5\%
CO\textsubscript{2}.

\textbf{Western blot analysis}

Proteins from spleen tissues were separated by SDS-PAGE and transferred
to PVDF membranes. The membranes were then incubated overnight at 4 degC
with antibodies against the protein of interest, including BCL-2 (D17C4;
3498; Cell signaling), Bax (D2E11; 5023; Cell signaling), GAPDH (D16H11;
5174T; Cell signaling) over-night at 4 degC. Protein quantification was
measured in optical density units using Image Lab software (Bio-Rad, CA,
USA) and was normalized to the corresponding sample expression of GAPDH.

\textbf{Analysis of apoptosis}

According to the manufacturer's instructions, the induction of apoptosis
in the spleen was detected using terminal deoxynucleotidyl transferase
dUTP nick end labeling (TUNEL){[}24{]}. The apoptotic index was
quantified by the proportion of TUNEL-positive cells{[}25{]}, three
parallel experiments were done for each group.

\textbf{Flow cytometry}

All anti-bodies listed were obtained from eBioscience, San Diego, CA,
USA, 5ul labeled antibody was added in ench procedure. The expression of
CD28, CTLA-4, CD80 and CD86 in CD4\textsuperscript{+} T Lymphocytes was
assayed in PBMCs and splenocytes. Add anti-human (mouse)CD4-FITC
monoclonal antibodies, anti-human(mouse) CD28-PE, anti-human(mouse)
CTLA-4-APC or anti-human(mouse) CD80-PE, anti-human(mouse) CD86-APC at
room temperature away from light for 30 minutes. A parallel control
group was treated with isotype controls according to the manufacturer's
recommendations.

Stimulated and cultured mononuclear cells were collected, pre-incubated
for 15 minutes with unlabeled isotype control Abs (IgG1 or IgG2b). Then
incubated with anti-human CD3-FITC, anti-human CD8-PE, or anti-human
CD4-FITC and anti-human CD25-PE, a parallel control group was treated.
splenocytes incubated with anti-mouse CD4-FITC, with or without
anti-mouse CD25-PE at room temperature away from light for 30 minutes,
then incubated at room temperature away from light for 15 minutes after
treating with 100 \selectlanguage{greek}μ\selectlanguage{english}L FIX \& PERM medium A and B (Invitrogen, USA). Then,
anti-human IL-17-APC, anti-human IFN-g-APC, anti-human IL-4-APC or
anti-human FoxP3-APC was added in PBMCs and anti-mouse IL-17-APC,
anti-mouse IFN-g-APC, anti-mouse IL-4-APC or anti-mouse FoxP3-APC was
added in splenocytes. A parallel control group was treated d with
isotype controls. Both suspensions were incubated at 4 temperature away
from light for one night.

\textbf{Plasma cytokine}

The patients' serum was collected as shown above, and mice blood samples
were collected by heart puncture when anesthetized by sevoflurane 3 days
after CLP and CD28 challenge. Then, the mice's blood was sent into
ethylenediaminetetraacetic acid tube and placed in a centrifuge tube,
centrifuge at 12,000 rpm for 5 minutes, take the supernatant, measure
the content of TNF-a, IL-17, IL-10, and IL-6 in patients or mice serum
according to Elisa manufacturer's recommendations (Thermo Fisher).

\textbf{Statistics}

All analyses were done using SPSS, version 22.0 (IBM Corp. Armonk, NY,
USA), MedCalc Statistical Software, version 19.5 (MedCalc Software Ltd,
Ostend, Belgium) and GraphPad Prism 8.0(GraphPad Software, San Diego,
CA, USA). Continuous variables were reported as mean \selectlanguage{ngerman}± SD or as median
(IQR) after testing their normal distribution using the
Kolmogorov-Smirnov test. For two-group comparisons, the independent
samples t-test was used for normally distributed data, and the
Mann-Whitney test was used for non-normally distributed data. For the
comparison of multi-group, one-way analysis of variance and the
Kruskal-Wallis test was used to analyze normally and non-normally
distributed data. Categorical data are summarized using number
(percentage), and were compared using the chi-square or Fisher's exact
test. Spearman's rank correlation was applied to determine the
correlation between variables. The area under the receiver operating
characteristic (ROC) curve was calculated to evaluate the tested
parameters' diagnostic and prognostic value and a value of P
\textless{}0.05 was considered statistically significant.

\textbf{Results}

\textbf{Sepsis induced Th17 and ratio of Th17/Treg increasing}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

Th17 and Treg expression on CD4\textsuperscript{+}T lymphocytes and
their ratio were compared between sepsis patients and volunteers.
Although there was no difference in health volunteers and septic
patients {[}4.998(3.474-6.523) vs 4.625(4.117-5.133), P=0.5573{]},
sepsis caused Th17 increased obviously {[}0.6977
(0.4852-0.9102)\emph{vs} 3.580(3.209-3.951), P\textless{}0.01{]}. And
the ratio of Th17 to Treg higher in sepsis patients {[}0.2131
(0.1400-0.2600) vs 0.7907 (0.5575-1.038), P \textless{}0.01{]}. This
suggest in initial sepsis Th17 cells increase significantly, while Treg
which acted as an inflammatory brake remained relatively stable.

Figure 1: \textbf{A,} Th17 expression in healthy volunteers and sepsis
patients, take CD3 as abscissa and CD8 as ordinate, find out the
CD3+CD8-cell group, and then mark IL-17 (ordinate). \textbf{B,} Treg
expression in healthy volunteers and sepsis patients, take CD4 as the
abscissa, find out CD4+cell group, and then mark FoxP3
(ordinate).\textbf{C,} Expression of Th17 between health volunteers and
septic patients. \textbf{D} , Expression of Treg between health
volunteers and septic patients. E, Expression of Th17/Treg between
health volunteers and septic patients, ***, p\textless{}0.001, ****,
p\textless{}0.0001.

\textbf{Patient characteristics and Comparison}

Sixty-five adult patients with sepsis were admitted, including 41 males
and 24 females, and 5 cases died in the ICU. According to the length of
ICU stay more or fewer than 14 days, 60 patients of survival were
divided into two groups. There were no significantly difference in
demographic data of two groups (Table 1), except that the median age of
patients whose ICU stay more than 14 days was older than that of group
of ICU stay fewer than 14 days (63.33\selectlanguage{ngerman}±10.41 vs 72.63±7.45, P =0.015),
which was in parallel with SOFA score {[}5.17 (3.00-6.00) vs 8.00
(5.00-11.00), P =0.01{]} and APACHE II score {[}11.79 (7.00-14.00) vs
16.62 (13.00-21.00), P =0.01{]}.

\textbf{Table 1} Baseline levels of sepsis patients with different
length of stay in ICU\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
\begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
Parameter\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
Total\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
ICU stay\textless{}14d (n=36)\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
ICU stay\textgreater{}14d (n=24)\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
p\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Age, years\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
67.05\selectlanguage{ngerman}±10.35\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
63.33\selectlanguage{ngerman}±10.41\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
72.63\selectlanguage{ngerman}±7.45\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.015\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
gender(M/F)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
39/21\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
24/12\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
15/9\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.740\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
height, cm\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
166.73\selectlanguage{ngerman}±9.46\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
166.78\selectlanguage{ngerman}±9.21\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
166.67\selectlanguage{ngerman}±10.03\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.862\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
body weight, kg\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
67.65\selectlanguage{ngerman}±13.13\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
65.64\selectlanguage{ngerman}±13.18\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
70.67\selectlanguage{ngerman}±12.73\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.516\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
White blood cells ,103/\selectlanguage{greek}μ\selectlanguage{english}L\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
18.12(11.09-23.68)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
19.00(12.38-24.27)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
16.79(8.26-20.95)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.165\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Lymphocytes, 103/\selectlanguage{greek}μ\selectlanguage{english}L\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.79(0.38-0.97)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.70(0.39-0.91)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.91(0.34-1.13)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.868\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
SOFA score\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
6.30(4.00-9.00)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
5.17(3.00-6.00)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
8.00(5.00-11.00)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.001\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
APACHE II score\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
13.72(8.00-17.00)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
11.79(7.00-14.00)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
16.62(13.00-21.00)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.001\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

As showed in Table 2, despite the severer condition of septic patients
that ICU stay more than 14 days, neither Th1, Th2, Treg cells subtypes
nor their remarkable cytokines were changed obviously when compared with
patients that stay fewer than 14 days. However, both the ratio of Th17
and the IL-17 level of septic patients that ICU stay more than 14 days
were increased significantly {[}compared with patients that stay fewer
than 14 days, 3.20 (2.21-3.92) vs 4.32 (3.33-5.47), P=0.002 and 139.68
(98.53-162.81) vs 169.32 (131.86-206.94), P =0.02{]}. As a result, the
ratio of Th17/Treg of septic patients that ICU stay more than 14 days
was also increased significantly (compared with patients that stay fewer
than 14 days, 0.76\selectlanguage{ngerman}±0.31 vs 0.98±0.18, P =0.001, Fig.2a). The ratio of
Th17/Treg of septic patients was positively correlated with the length
of stay in ICU (Fig.2b) and SOFA score (r\textsuperscript{2}=0.2061,
Fig. 2c) and APACHE II score (r\textsuperscript{2}=0.2583, Fig. 2d) when
the patients were admitted to ICU.

\textbf{Table 2} Cytokines in sepsis patients with different length of
stay in ICU\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
Parameter & Total & ICU stay\textless{}14d(n=36) & ICU
stay\textgreater{}14d(n=24) & p\tabularnewline
\midrule
\endhead
IL-6(pg/ml) & 258.07(44.46-374.59) & 202.90(43.65-301.07) &
340.83(65.60-376.26) & 0.154\tabularnewline
TNF-a (pg/ml) & 32.47(13.55-34.00) & 35.80(13.10-34.28) &
27.48(18.05-34.00) & 0.415\tabularnewline
IL-10(pg/ml) & 47.52(8.56-50.80) & 52.40(8.38-35.33) &
40.19(10.61-57.09) & 0.327\tabularnewline
IL-8(pg/ml) & 1024.47(74.08-1024.47) & 1066.58(63.40-1181.35) &
961.30(175.50-976.77) & 0.284\tabularnewline
IL-17(pg/ml) & 151.54(107.60-188.90) & 139.68(98.53-162.81) &
169.32(131.86-206.94) & 0.020\tabularnewline
PCT & 13.37(1.41-16.48) & 10.21(1.13-9.99) & 18.10(3.115-23.30) &
0.097\tabularnewline
Th1 & 14.02(6.91-19.47) & 13.43(7.82-15.12) & 14.90(5.87-21.70) &
0.350\tabularnewline
Th2 & 2.61(1.19-2.96) & 1.98(1.08-2.81) & 3.55(1.35-3.60) &
0.182\tabularnewline
Th1/Th2 & 9.22(3.65-12.78) & 9.61(3.48-10.63) & 8.63(3.65-14.93) &
0.461\tabularnewline
Th17 & 3.65(2.42-4.73) & 3.20(2.21-3.92) & 4.32(3.33-5.47) &
0.002\tabularnewline
Treg & 4.63(3.31-6.20) & 4.62(2.78-6.48) & 4.64(3.33-5.85) &
0.946\tabularnewline
Th17/Treg & 0.85\selectlanguage{ngerman}±0.28 & 0.76\selectlanguage{ngerman}±0.31 & 0.98\selectlanguage{ngerman}±0.18 & 0.001\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

Figure 2. \textbf{A:} Comparison of Th17/Treg ratio among patients with
different length of hospital stay; \textbf{B:} Correlation between
Th17/Treg and length of ICU stay; \textbf{C:} Correlation between SOFA
score and Th17/Treg ratio; \textbf{D:} Correlation between APACHE II
score and Th17/Treg ratio. SOFA, Sequential Organ Failure Assessment;
APACHE II, Acute Physiology and Chronic Health Assessment II. ** p
\textless{} 0.01.

\textbf{The predictive of Th17/Treg ratio, SOFA score, APACHE II score
on length of ICU stay}

The limited data suggested the ratio of Th17/Treg predicted the length
of septic patient ICU stay. The area under the ROC curve(AUC)of the
Th17/Treg ratio, SOFA score and APACHE II score was 0.747(95\%CI:
0.618-0.850), 0.764(95\%CI: 0.637-0.864) and 0.760(95\%CI: 0.6333-0.861)
respectively for ICU stay more than 14 days. Combined either SOFA score
or APACHE II score with the ratio of Th17 / Treg, although there is no
statistically difference each other, but its AUC of predictive value on
the length of sepsis patients' ICU stay is higher, with AUC value of
0.782 (95\% CI: 0.656-0.878) or 0.800 (95\% CI: 0.676-0.892). The
sensitivity and specificity for predicting ICU stay based on cut-off
values are shown in Figure 3.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

Fig.3- Receiver operating characteristic (ROC) curves for the Th17/Treg
ratio, acute physiology and chronic health evaluation (APACHE) II,
sequential organ failure assessment (SOFA) and Th17/Treg ratio in
combination with APACHE II score, Th17/Treg ratio in combination with
SOFA score for predicting sepsis patients more than 14-day ICU stay
time. The area under the curve (AUC) demonstrates that the Th17/Treg
ratio measures 0.747 (95\% CI 0.618 to 0.850), the APACHE II score was
0.760 (95\% CI 0.633 to 0.861), the SOFA score measures 0.764 (95\% CI
0.637 to 0.864), PCT measures 0.627 (95\% CI 0.493 to 0.749), the
Th17/Treg ratio in combination with the SOFA score measures 0.782 (95\%
CI 0.656 to 0.878), and the Th17/Treg ratio in combination with the
APACHE II score measures 0.800 (95\% CI 0.676 to 0.892).

\textbf{CD28 and ligands expression levels in sepsis patients}

The expression level of CD28 of peripheral blood CD4+T lymphocytes was
obviously increased in sepsis patients {[}compared with healthy
volunteers, 84.21 (73.24-95.05) vs 17.05 (12.40-22.10),
p\textless{}0.001, Figure 4{]}. Meanwhile neither CD28, CTLA-4 nor their
ligands CD80, CD86 expression levels was without significant in all
sepsis patients. The Th17/Treg ratio was positively correlated with the
expression level of CD28 in CD4+ T lymphocytes (r2 =0.2615,
P\textless{}0.01, Figure 4c).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

Fig 4. \textbf{A,} the expression of Treg in healthy volunteers and
sepsis patients. \textbf{B} , CD28 molecules expression on CD4+
lymphocyte cells between sepsis and healthy. Compared with
healthy.\textbf{C} , Th17/Treg ratio correlate with the expression of
CD28 in CD4+ lymphocytes. ****p\textless{}0.001.

\textbf{Table 3} Comparison of CD28 expression in sepsis patients with
different length of stay in ICU\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
\begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
Parameter\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
Total\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
ICU stay\textless{}14d(n=36)\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
ICU stay\textgreater{}14d (n=24)\strut
\end{minipage} & \begin{minipage}[b]{0.19\columnwidth}\raggedright\strut
p\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
CD28\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
84.21(73.24-95.05)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
85.79(79.50-95.39)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
81.84(69.92-92.30)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.152\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
CD80\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1.39(1.02-1.51)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1.45(1.13-1.61)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1.29(0.80-1.34)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.145\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
CD86\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
52.54(38.48-66.13)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
55.93(43.38-67.09)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
47.45(34.22-62.89)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.145\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
CTLA-4\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1.79(0.79-1.70)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1.94(0.81-1.70)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
1.56(0.75-1.74)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.442\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textbf{Anti-CD28 improved mortality of CLP-induced sepsis mice}

Therefore, animal models were designed to verify effects of CD28 on
ratio of Th17 to Treg in septic mice. With administrated anti-CD28
functional antibody (5 mg/kg) after CLP, both 7-days survival rate
(Fig5.a) and clinical scores (Fig5.b) of mice were improved obviously.
The white pulp nodule in septic mice's spleen was disordered, and the
boundary between white pulp and red pulp was blurred (Fig5. c). After
detur CD28 antibody, the disorder of white pulp nodule and
histopathological score (Fig5. d) were alleviated while spleen index
(Fig5. e) were aggrandized.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

Fig.5 \textbf{A} , the Kaplan-Meier survival analysis 7 d after CLP in
isotype treat and CD28 functional antibody mice. The Kaplan-Meier
survival curves were compared by the log-rank test. Line-graphs show
mean and standard error(n=10 mice per group). \textbf{B} , Mice's Murine
Sepsis Score (MSS) over time that were administered 5 mg/kg CD28
functional antibody (n = 5). \textbf{C} , Histology of spleen (Original
magnification: 200\selectlanguage{ngerman}×), Scale bar, 100 mm. \textbf{D} , Spleen
histopathological score(n=5), the values presented are mean ±SEM,
n=6.\textbf{E} , spleen index(n=5). The values presented are mean ±SEM,
*p\textless{}0.05, ** p\textless{}0.01, ***p\textless{}0.001, The data
are representative of 3 independent experiments.

\textbf{Anti-CD28 Ab amended Th17/Treg imbalance in sepsis mice}

As Th17 and Treg variation in sepsis patients, Th17 and Treg variation
in CLP induced sepsis mice had similar trends. Treg and Th17 in spleen
of CLP mice were decreased obviously (Figure 6a, b). After treated with
CD28 antibody, the extent of Treg and Th17' increase amplitude was
lower, as well as the proportion of Th17/Treg (Figure 6c, d, e). Serum
levels of IL-6\selectlanguage{english},TNF-\selectlanguage{greek}α \selectlanguage{english}and IL-17A were increased significantly in CLP
mice, while the level of IL-10 was increased slightly. Meanwhile, such
trends were reversed by administrated with CD28 functional antibody
(Fig6. F, G, H and I).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

Fig.6 Flow cytometry of Treg cell(\textbf{A} ), Th17 cell(\textbf{B} )
in spleen after 3 days of CLP. The percentage of Treg(\textbf{C} ) and
the Th17 (\textbf{D} ) in different groups by flow cytometry. \textbf{E}
, the ratio of Th17 to Treg is calculated. Expression of serum related
inflammatory factors in mice 3 days after CLP, IL-6(\textbf{F} ),
TNF-a(\textbf{G} ), IL-17A(\textbf{H} ) and IL-10(\textbf{I} ). The
values presented are mean \selectlanguage{ngerman}±SEM (n=5). *p\textless{}0.05,
**p\textless{}0.01, ***p\textless{}0.001. The data are representative of
3 independent experiments.

\textbf{Anti-CD28 reduced CLP-induced splenocytes apoptosis}

T lymphocyte subsets differentiation and lymphocytes apoptosis played an
important role in septic rats{[}26, 27{]}. The proportion of spleen cell
apoptosis was significantly increased in CLP mice, with reduced nucleus
number, irregular shape of the nucleus, state of condensation or nuclear
fragments appear, while anti-CD28 relieved such spleen apoptosis
significantly (Fig.7a, b). The BCL-2 and Bax signaling pathways are
required in spleen homogenate for apoptosis in CLP-induced septic
mice{[}28{]}. As illustrated in Figure.7c, expression of the BCL-2 was
significantly increased after anti-CD28 challenge (Fig.7d,
p\textless{}0.01), while expression of the Bax was remarkably decreased
(Fig.7e, p\textless{}0.01).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image7/image7}

\end{center}
\end{figure}

Fig.7 \textbf{A,} apoptosis by TUNEL analysis (Original magnification:
200X Scale:100um). Representative confocal fluorescence microscopy
images of spleen cells stained with antibodies against Tunnel (green)
and DAPI(blue). DAPI ,4'6-diamidino-2-phenylindole. Scale bar, 20
mm.\textbf{B,} the percentage of apoptosis were determined average gray
value by Image J. \textbf{C,} western-blot of apoptosis BCL-2 and Bax in
spleen, \textbf{D} and \textbf{E} , Densitometric analysis of the
relevant bands was performed by Image J. The values presented are mean
\selectlanguage{ngerman}±SEM (n=5). *p\textless{}0.05, **p\textless{}0.01, ***p\textless{}0.001,
n=5, The data are representative of 3 independent experiments.

\textbf{Discussion}

The body immune system plays an important role in sepsis like
double-edged sword, which protects against invading pathogens, while its
overreaction can cause body organ injury. During sepsis, the invasion of
a large number of pathogens not only causes the body's immune
overreaction, but also causes a large number of lymphocytes to consume
and cause immune paralysis. As Hotchkiss RS described that the T cells
were the band conductor of the orchestra, and all immune components
worked in harmony to play the best role{[}30{]}. Due to the intertwined
complex relationship, Th17, Treg and their balance proportion has been
attracted more and more attention, which is essential for immune
homeostasis{[}31{]}. Although Treg represent less than 10\% of
circulating CD4+T cells, it can limit the overreaction of anti-infective
effector cells, protect surrounding normal tissues from damage, and
maintain the body's immune balance {[}32, 33{]}. Th17 terminally
differentiated cells fulfill opposite Treg's functions; IL-17 can
promote the occurrence of inflammation in sepsis{[}34{]}. Studies have
found that Th17 can appear in lung tissue to help eliminate bacteria in
the bacterial infection model, including Klebsiella pneumoniae that is
resistant to carbapenem{[}35{]}, which is very important to resistance
in sepsis bacterial infection. Th17 cause autoimmunity and inflammation,
whereas Treg inhibit these phenomena and maintain immune homeostasis.
The two are functionally antagonistic to each other, but also inhibit
each other in differentiation, even switch between each other{[}36{]}.
Thus, unraveling the mechanisms that affect the Th17/Treg balance is
critical to understand autoimmunity and tolerance better. Limited data
had shown the ratio of Th17/Treg might be related to the clinical
severity and prognosis of sepsis {[}37{]}, {[}38{]}, {[}39{]}.

The length of stay in intensive care units (LSICU) is an essential
factor for quality assessment and a strong determinant for the total
cost of ICU admission, of which septic patients represent a major
part{[}40{]}. Age was an independent risk factor of many diseases, and
some researches showed that the odds for death in ICU patients with
sepsis increased with age with the maximal rate of increase {[}46{]}.
Age is a factor affecting the length of stay in ICU in patients with
sepsis, and there is a positive correlation between age and length of
stay in ICU. Clinical scores except age, such as the APACHE II{[}41{]},
SOFA{[}42{]} have been widely used in clinical practice to predict
outcome in critically ill patients; the early risk-stratification of
these patients and their prognosis, as well as accurate monitoring of
clinical treatment effects. Th17/Treg ratio can predict 28-day mortality
in sepsis patients with ARDS {[}43{]}. There is currently an apparent
lack of an accurate biomarker for predicting sepsis patients the length
of stay in ICU. In the present study, positive correlations were
observed between the Th17/Treg ratio and the length of stay in ICU,
indicating that the higher the Th17/Treg ratio, the longer the length of
stay in ICU in sepsis patients. The Th17/Treg ratio, APACHE II score,
and SOFA score were independent predictors of the length of stay in ICU
in sepsis patients, the Th17/Treg ratio in combination with the APACHE
II score increased the AUC for predicting the length of stay in ICU.
Taken together, our findings strongly suggest that the Th17/Treg ratio
can reflect the intensity of the inflammatory response in sepsis, and
might be a potential indicator for the length of stay in ICU in sepsis
patients.

CD28 is the primary costimulatory molecule for naive CD4+ conventional T
cell activation{[}44{]}, binding to B7 ligands leads to increased
duration and magnitude of T cell responses, enhanced survival and
glucose metabolism, and acquisition of migratory properties{[}45{]}.
CD28 activates integrin-mediated adhesion of T cells and promotes actin
polymerization. Selective blockade of CD28-mediated T cell costimulation
protects rhesus monkeys against acute fatal experimental autoimmune
encephalomyelitis(EAE){[}46{]}. In septic patients, there is a strong
positive correlation between CD28 expression on CD4+ T cells and
Th17/Treg ratio; we speculate that CD28 may be able to change Th17/Treg
ratio then short the length of stay in ICU.

The mice sepsis model showed that the clinical score and Th17/Treg ratio
of septic mice were the highest on the third day after CLP. After
administration of CD28 antibody, the 7-day survival rate of mice
increased, the Th17/Treg ratio of spleen cells decreased, and the
expression of inflammatory factors in peripheral serum also decreased.
Simultaneously, the spleen's length, weight and index were improved, and
the histopathological damage were alleviated. These suggested the
decrease of apoptosis of spleen cells in mice. It is speculated that
CD28 antibody may affect the balance between Th17 and Treg through
ROR-\selectlanguage{greek}γ\selectlanguage{english}t and FoxP3, thereby improving the survival rate of sepsis mice.

The function of T cells is related to cell death, which can be divided
into accidental cell death (ACD) and regulatory cell death (RCD). The
signal cascade reactions involved in regulating cell-related functions
are mainly RCD, RCD involving effector molecules, which have unique
biochemical characteristics, morphological characteristics and
immunological consequences {[}47{]}. RCD mode includes apoptosis,
necroptosis, pyroptosis, ferroptosis, entotic cell death, and so
on.{[}48{]}. T cell dysfunction is manifested specifically in increased
T cell apoptosis, decreased proliferative capacity and decreased
reactivity or unresponsive state{[}49{]}. Our data showed that the
administration of CD28 antibody to CLP processed mouse could reduce the
apoptosis of splenocytes. Western blot in tissues confirmed that the
expression of pro-apoptotic protein BCL-2 decreased and the expression
of anti-apoptotic protein BAX increased in mouse spleen under the action
of CD28 antibody.

\textbf{Limitations}

There were some limitations to this study. First, this is a
single-center study and the sample size was not large, thus restricting
generalizability. Our data were used to observe the circulating Th17 and
Treg changes and the correlation between Th17/Treg ratio and length of
ICU stay. In addition, patients were included for sepsis as a variety of
risk factors, and our findings need to be confirmed by further studies
and more patients. Second, the change in functional immunocompetent
cells was only measured at one time point, because we aimed to
understand the early functional immune cells influence on clinical
outcomes. Many factors affect the length of hospital stay of patients
with sepsis, not all of them were observed. Therefore, further research
is necessary to include more factors that influence hospital stay length
of sepsis patients. Finally, Due to the cost and experimental
conditions, we did not purify CD4+T lymphocytes from human PBMC, nor did
we conduct further mechanism verification. CD28 knockout mice were not
used in our in vivo study for verification, and the intervention only
used CD28 antibody, and CD28 agonist was not used. As for the mechanism
of the effect of CD28 antibody on T cell function, we only focused on
the splenocytes' apoptosis, but did not further explore other possible
mechanisms.

\textbf{CONCLUSIONS}

Th17/Treg ratio can be used as a predictor of ICU stay in sepsis. CD28
functional antibody may have therapeutic significance for sepsis
patients and treat septic mice by affecting the imbalance of Th17/Treg
ratio and inhibiting T cell apoptosis.

\textbf{Abbreviations}

APACHE II: Acute Physiology and Chronic Health Evaluation II; SOFA:
Sepsis-related organ failure assessment; AUC: Area under the receiver
operating characteristic curve; EDTA: Ethylenediaminetetraacetic acid;
GCS: Glasgow Coma Scale; ICU: Intensive care unit; IFN-\selectlanguage{greek}γ: \selectlanguage{english}Interferon-\selectlanguage{greek}γ;
\selectlanguage{english}IL-4: Interleukin-4; IL-8: Interleukin-8; IL-10: Interleukin-10; IL-17A:
Interleukin-17A; OR: Odds ratio; PCT: Procalcitonin; ROC: Receiver
operating characteristic; WBC: White blood cell; CD3: Cluster
of~Differentiation 3; CD4: Cluster of~Differentiation 4; CD8: Cluster
of~Differentiation 8; CD28: Cluster of~Differentiation 28; CD80: Cluster
of~Differentiation 80; CD86: Cluster of~Differentiation 86;
CTLA-4:cytotoxic T-lymphocyte-associated protein 4;

\textbf{Acknowledgements}

Not applicable.

\textbf{Authors' contributions}

Yu Wu performed the mostly experiments, analyzed the data, drafted the
manuscript. Dai Li and Jian Xie analyzed the data, and critically
revised the manuscript. Han Wang, Yan Meng and Guosheng Wu contributed
to the data analysis and interpretation. Xiaoming Deng conceived and
designed the study. Xiaojian Wan conceived and designed the experiments,
analyzed the data, edited the manuscript. All authors substantially
contributed to editing, revising, and finalizing the manuscript. All
authors read and approved the final manuscript.

\textbf{Funding}

This work was supported by Shanghai Natural Science Foundation
(19ZR1456600),National Natural Science Foundation of China (81600955),
Shanghai Pujiang Program (2020PJD059) and Hebei Medical Science Research
Key Project Plan (ZD20170953)

\textbf{Availability of data and materials}

The datasets generated and analyzed during the current study are
available from the corresponding author upon reasonable request.

\textbf{Ethics approval and consent to participate}

This study was reviewed and approved by the Changhai Hospital Ethics
Committee. It was conducted in accordance with the Declaration of
Helsinki. Informed consent was obtained from all patients or their legal
proxy before enrollment. All animal experiments were performed according
to the guidelines for the Care and Use of Laboratory Animals (Ministry
of Health, China, 1998).

\textbf{Consent for publication}

Not applicable.

\textbf{Competing interests}

The authors declare that they have no competing interests.

\textbf{Author details}

1.Department of Anesthesiology and Critical Care Medicine, Changhai
Hospital, Naval medical university, Shanghai, China

2.Department of Anesthesiology and Critical Care Medicine, the 980th
Hospital (Bethune International Peace Hospital) of the Joint Logistics
Support Force, Shijiazhuang, China

3. Department of Burn Surgery, Changhai Hospital, Navy Medical
University, Shanghai, China

\textbf{References}

1. Rudd KE, Johnson SC, Agesa KM, Shackelford KA, Tsoi D, Kievlan DR,
Colombara DV, Ikuta KS, Kissoon N, Finfer S et al: Global, regional, and
national sepsis incidence and mortality, 1990-2017: analysis for the
Global Burden of Disease Study. Lancet 2020, 395(10219):200-211.

2. Cecconi M, Evans L, Levy M, Rhodes A: Sepsis and septic shock. Lancet
2018, 392(10141):75-87.

3. Gotts JE, Matthay MA: Sepsis: pathophysiology and clinical
management. BMJ 2016, 353:i1585.

4. Kaukonen KM, Bailey M, Pilcher D, Cooper DJ, Bellomo R: Systemic
inflammatory response syndrome criteria in defining severe sepsis. N
Engl J Med 2015, 372(17):1629-1638.

5. Shime N, Kawasaki T, Saito O, Akamine Y, Toda Y, Takeuchi M, Sugimura
H, Sakurai Y, Iijima M, Ueta I et al: Incidence and risk factors for
mortality in paediatric severe sepsis: results from the national
paediatric intensive care registry in Japan. Intensive Care Med 2012,
38(7):1191-1197.

6. Pepper M, Jenkins MK: Origins of CD4(+) effector and central memory T
cells. Nat Immunol 2011, 12(6):467-471.

7. Nascimento DC, Melo PH, Pineros AR, Ferreira RG, Colon DF, Donate PB,
Castanheira FV, Gozzi A, Czaikoski PG, Niedbala W et al: IL-33
contributes to sepsis-induced long-term immunosuppression by expanding
the regulatory T cell population. Nat Commun 2017, 8:14919.

8. Andrade MMC, Ariga SSK, Barbeiro DF, Barbeiro HV, Pimentel RN,
Petroni RC, Soriano FG: Endotoxin tolerance modulates TREG and TH17
lymphocytes protecting septic mice. Oncotarget 2019, 10(37):3451-3461.

9. Imbratta C, Leblond MM, Bouzourene H, Speiser DE, Velin D, Verdeil G:
Maf deficiency in T cells dysregulates Treg - TH17 balance leading to
spontaneous colitis. Sci Rep 2019, 9(1):6135.

10. Verstappen GM, Corneth OBJ, Bootsma H, Kroese FGM: Th17 cells in
primary Sjogren's syndrome: Pathogenicity and plasticity. J Autoimmun
2018, 87:16-25.

11. Knochelmann HM, Dwyer CJ, Bailey SR, Amaya SM, Elston DM,
Mazza-McCrann JM, Paulos CM: When worlds collide: Th17 and Treg cells in
cancer and autoimmunity. Cell Mol Immunol 2018, 15(5):458-469.

12. Cheng H, Guan X, Chen D, Ma W: The Th17/Treg Cell Balance: A Gut
Microbiota-Modulated Story. Microorganisms 2019, 7(12).

13. Miao M, Hao Z, Guo Y, Zhang X, Zhang S, Luo J, Zhao X, Zhang C, Liu
X, Wu X et al: Short-term and low-dose IL-2 therapy restores the
Th17/Treg balance in the peripheral blood of patients with primary
Sjogren's syndrome. Ann Rheum Dis 2018, 77(12):1838-1840.

14. Qu X, Han J, Zhang Y, Wang Y, Zhou J, Fan H, Yao R: MiR-384
Regulates the Th17/Treg Ratio during Experimental Autoimmune
Encephalomyelitis Pathogenesis. Front Cell Neurosci 2017, 11:88.

15. Ratajczak P, Janin A, Peffault de Latour R, Leboeuf C, Desveaux A,
Keyvanfar K, Robin M, Clave E, Douay C, Quinquenel A et al: Th17/Treg
ratio in human graft-versus-host disease. Blood 2010, 116(7):1165-1171.

16. Sun L, Fu J, Lin SH, Sun JL, Xia L, Lin CH, Liu L, Zhang C, Yang L,
Xue P et al: Particulate matter of 2.5 mum or less in diameter disturbs
the balance of TH17/regulatory T cells by targeting glutamate
oxaloacetate transaminase 1 and hypoxia-inducible factor 1alpha in an
asthma model. J Allergy Clin Immunol 2020, 145(1):402-414.

17. Esensten JH, Helou YA, Chopra G, Weiss A, Bluestone JA: CD28
Costimulation: From Mechanism to Therapy. Immunity 2016, 44(5):973-988.

18. Mikami N, Kawakami R, Chen KY, Sugimoto A, Ohkura N, Sakaguchi S:
Epigenetic conversion of conventional T cells into regulatory T cells by
CD28 signal deprivation. Proc Natl Acad Sci U S A 2020,
117(22):12258-12268.

19. Wakamatsu E, Omori H, Ohtsuka S, Ogawa S, Green JM, Abe R:
Regulatory T cell subsets are differentially dependent on CD28 for their
proliferation. Mol Immunol 2018, 101:92-101.

20. Singer M, Deutschman CS, Seymour CW, Shankar-Hari M, Annane D, Bauer
M, Bellomo R, Bernard GR, Chiche JD, Coopersmith CM et al: The Third
International Consensus Definitions for Sepsis and Septic Shock
(Sepsis-3). JAMA 2016, 315(8):801-810.

21. Gogishvili T, Langenhorst D, Luhder F, Elias F, Elflein K, Dennehy
KM, Gold R, Hunig T: Rapid regulatory T-cell response prevents cytokine
storm in CD28 superagonist treated mice. PLoS One 2009, 4(2):e4643.

22. Shrum B, Anantha RV, Xu SX, Donnelly M, Haeryfar SM, McCormick JK,
Mele T: A robust scoring system to evaluate sepsis severity in an animal
model. BMC Res Notes 2014, 7:233.

23. Zhang H, Zhai JY, Du HB, Zhang LM, Li LF, Bian AQ, Jiang LN, Zhao
ZG: Mesenteric lymph drainage alleviates hemorrhagic shock-induced
spleen injury and inflammation. Acta Cir Bras 2019, 34(9):e201900903.

24. Fang F, Li Y, Chang L: Mechanism of autophagy regulating
chemoresistance in esophageal cancer cells. Exp Mol Pathol 2020:104564.

25. Hu Q, Ren H, Li G, Wang D, Zhou Q, Wu J, Zheng J, Huang J, Slade DA,
Wu X et al: STING-mediated intestinal barrier dysfunction contributes to
lethal sepsis. EBioMedicine 2019, 41:497-508.

26. Zou Q, Yang M, Yu M, Liu C: Influences of Regulation of miR-126 on
Inflammation,Th17/Treg Subpopulation Differentiation, and Lymphocyte
Apoptosis through Caspase Signaling Pathway in Sepsis. Inflammation
2020, 43(6):2287-2300.

27. Dinesh P, Rasool M: Berberine mitigates IL-21/IL-21R mediated
autophagic influx in fibroblast-like synoviocytes and regulates
Th17/Treg imbalance in rheumatoid arthritis. Apoptosis 2019,
24(7-8):644-661.

28. Volkmann N, Marassi FM, Newmeyer DD, Hanein D: The rheostat in the
membrane: BCL-2 family proteins and apoptosis. Cell Death Differ 2014,
21(2):206-215.

29. Camperio C, Muscolini M, Volpe E, Di Mitri D, Mechelli R, Buscarinu
MC, Ruggieri S, Piccolella E, Salvetti M, Gasperini C et al: CD28
ligation in the absence of TCR stimulation up-regulates IL-17A and
pro-inflammatory cytokines in relapsing-remitting multiple sclerosis T
lymphocytes. Immunol Lett 2014, 158(1-2):134-142.

30. Hotchkiss RS, Opal SM: Activating Immunity to Fight a Foe - A New
Path. N Engl J Med 2020, 382(13):1270-1272.

31. Lee GR: The Balance of Th17 versus Treg Cells in Autoimmunity. Int J
Mol Sci 2018, 19(3).

32. Attias M, Al-Aubodah T, Piccirillo CA: Mechanisms of human FoxP3(+)
Treg cell development and function in health and disease. Clin Exp
Immunol 2019, 197(1):36-51.

33. Mohr A, Atif M, Balderas R, Gorochov G, Miyara M: The role of
FOXP3(+) regulatory T cells in human autoimmune and inflammatory
diseases. Clin Exp Immunol 2019, 197(1):24-35.

34. Dolff S, Witzke O, Wilde B: Th17 cells in renal inflammation and
autoimmunity. Autoimmun Rev 2019, 18(2):129-136.

35. Yasuda K, Takeuchi Y, Hirota K: The pathogenicity of Th17 cells in
autoimmune diseases. Semin Immunopathol 2019, 41(3):283-297.

36. Hu Y, Chen Z, Zeng J, Zheng S, Sun L, Zhu L, Liao W: Th17/Treg
imbalance is associated with reduced indoleamine 2,3 dioxygenase
activity in childhood allergic asthma. Allergy Asthma Clin Immunol 2020,
16:61.

37. Xia H, Wang F, Wang M, Wang J, Sun S, Chen M, Huang S, Chen X, Yao
S: Maresin1 ameliorates acute lung injury induced by sepsis through
regulating Th17/Treg balance. Life Sci 2020, 254:117773.

38. Sun JK, Zhang WH, Chen WX, Wang X, Mu XW: Effects of early enteral
nutrition on Th17/Treg cells and IL-23/IL-17 in septic patients. World J
Gastroenterol 2019, 25(22):2799-2808.

39. Guo J, Tao W, Tang D, Zhang J: Th17/regulatory T cell imbalance in
sepsis patients with multiple organ dysfunction syndrome: attenuated by
high-volume hemofiltration. Int J Artif Organs 2017, 40(11):607-614.

40. Paoli CJ, Reynolds MA, Sinha M, Gitlin M, Crouser E: Epidemiology
and Costs of Sepsis in the United States-An Analysis Based on Timing of
Diagnosis and Severity Level. Crit Care Med 2018, 46(12):1889-1897.

41. Salluh JI, Soares M: ICU severity of illness scores: APACHE, SAPS
and MPM. Curr Opin Crit Care 2014, 20(5):557-565.

42. Machado FR, Cavalcanti AB, Monteiro MB, Sousa JL, Bossa A, Bafi AT,
Dal-Pizzol F, Freitas FGR, Lisboa T, Westphal GA et al: Predictive
Accuracy of the Quick Sepsis-related Organ Failure Assessment Score in
Brazil. A Prospective Multicenter Study. Am J Respir Crit Care Med 2020,
201(7):789-798.

43. Yu ZX, Ji MS, Yan J, Cai Y, Liu J, Yang HF, Li Y, Jin ZC, Zheng JX:
The ratio of Th17/Treg cells as a risk indicator in early acute
respiratory distress syndrome. Crit Care 2015, 19:82.

44. Orabona C, Grohmann U, Belladonna ML, Fallarino F, Vacca C, Bianchi
R, Bozza S, Volpi C, Salomon BL, Fioretti MC et al: CD28 induces
immunostimulatory signals in dendritic cells via CD80 and CD86. Nat
Immunol 2004, 5(11):1134-1142.

45. Jain N, Miu B, Jiang JK, McKinstry KK, Prince A, Swain SL, Greiner
DL, Thomas CJ, Sanderson MJ, Berg LJ et al: CD28 and ITK signals
regulate autoreactive T cell trafficking. Nat Med 2013,
19(12):1632-1637.

46. Haanstra KG, Dijkman K, Bashir N, Bauer J, Mary C, Poirier N, Baker
P, Crossan CL, Scobie L, t Hart BA et al: Selective blockade of
CD28-mediated T cell costimulation protects rhesus monkeys against acute
fatal experimental autoimmune encephalomyelitis. J Immunol 2015,
194(4):1454-1466.

47. Anderton H, Wicks IP, Silke J: Cell death in chronic inflammation:
breaking the cycle to treat rheumatic disease. Nat Rev Rheumatol 2020,
16(9):496-513.

48. Tang D, Kang R, Berghe TV, Vandenabeele P, Kroemer G: The molecular
machinery of regulated cell death. Cell Res 2019, 29(5):347-364.

49. Thommen DS, Schumacher TN: T Cell Dysfunction in Cancer. Cancer Cell
2018, 33(4):547-562.

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