Decreased T-Cell Repertoire Diversity in Sepsis: A Preliminary Study

Article (PDF Available)inCritical care medicine 41(1) · December 2012with43 Reads
DOI: 10.1097/CCM.0b013e3182657948 · Source: PubMed
Abstract
OBJECTIVE:: Septic syndromes are the leading causes of mortality in intensive care units. In patients, the occurrence of sepsis-induced immune suppression is associated with delayed mortality, although the exact role of lymphocyte dysfunctions is not well established. The objective of this study was to investigate T-cell receptor diversity, an important feature of T-cell response, in patients with septic shock. DESIGN:: Preliminary prospective observational study. SETTING:: Adult intensive care units in a university hospital. SUBJECTS:: Patients with septic shock (n = 41) sampled twice after the onset of shock (early after inclusion [day 1] and at the end of the first week [day 7]). MEASUREMENTS AND MAIN RESULTS:: Using a novel molecular biology technique, the combinatorial diversity of human T-cell receptor β-chain (TRB locus) was measured in peripheral blood. Patients with septic shock presented with a marked decreased T-cell receptor diversity after the onset of shock in comparison with normal values. Importantly, in paired samples, a very steep recovery slope of T-cell receptor diversity, never described in other clinical situations, was observed between day 1 and day 7 (p < 0.0001, Wilcoxon's paired test). Decreased T-cell receptor diversity was associated with mortality (log-rank test, p = 0.0058; hazard ratio = 4.48; 95% confidence interval 1.96-53.32), and the development of nosocomial infections (p < 0.05, Mann-Whitney U test). CONCLUSION:: Our results show for the first time that septic patients present with a marked decreased T-cell receptor diversity that returned rapidly toward normal values over time. This opens novel cognitive research perspectives that deserve to be investigated in experimental models of sepsis. After confirmation in larger cohorts of these preliminary results, T-cell receptor diversity measurements may become a crucial tool to monitor immune functions in ICU patients.
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Critical Care Medicine www.ccmjournal.org 1
S
evere sepsis and septic shock are common indications for
admission to ICUs and affect millions of individuals around
the world each year (1). They constitute the leading cause
of morbidity and mortality for critically ill patients worldwide,
although an enormous effort has been expended during the last
20 yrs to find new therapies that decrease the mortality resulting
from this syndrome (1, 2).
It is generally accepted that septic patients develop immune
alterations affecting both innate and adaptive immunity (3). So
far, most work has been devoted to the study of the innate part
of these sepsis-induced immune dysfunctions. Importantly, it has
been shown that the intensity and duration of these alterations
are associated with increased mortality and susceptibility to sec-
ondary/nosocomial infections (3). In contrast, far less work has
been dedicated to the study of lymphocyte alterations, although
a link between a reduced delayed-type hypersensitivity response
(predominantly mediated by T-cells and a hallmark of immune
suppression) in ICU patients and increased risk of nosocomial in-
fections and death has been described >30 yrs ago (4).
In this study, we investigated a novel aspect of sepsis-induced
lymphocyte alterations. Using a novel molecular biology tech-
nique covering the whole T-cell receptor (TCR) repertoire (TRB
Objective: Septic syndromes are the leading causes of mortality in
intensive care units. In patients, the occurrence of sepsis-induced
immune suppression is associated with delayed mortality, although
the exact role of lymphocyte dysfunctions is not well established. The
objective of this study was to investigate T-cell receptor diversity, an
important feature of T-cell response, in patients with septic shock.
Design: Preliminary prospective observational study.
Setting: Adult intensive care units in a university hospital.
Subjects: Patients with septic shock (n = 41) sampled twice after the
onset of shock (early after inclusion [day 1] and at the end of the first
week [day 7]).
Measurements and Main Results: Using a novel molecular biology
technique, the combinatorial diversity of human T-cell receptor β-
chain (TRB locus) was measured in peripheral blood. Patients with
septic shock presented with a marked decreased T-cell receptor di-
versity after the onset of shock in comparison with normal values.
Importantly, in paired samples, a very steep recovery slope of T-cell
receptor diversity, never described in other clinical situations, was
observed between day 1 and day 7 (p < 0.0001, Wilcoxon’s paired
test). Decreased T-cell receptor diversity was associated with mortal-
ity (log-rank test, p = 0.0058; hazard ratio = 4.48; 95% confidence
interval 1.96–53.32), and the development of nosocomial infections
(p < 0.05, Mann-Whitney U test).
Conclusion: Our results show for the first time that septic patients pres-
ent with a marked decreased T-cell receptor diversity that returned rap-
idly toward normal values over time. This opens novel cognitive research
perspectives that deserve to be investigated in experimental models of
sepsis. After confirmation in larger cohorts of these preliminary results, T-
cell receptor diversity measurements may become a crucial tool to moni-
tor immune functions in ICU patients. (Crit Care Med 2013; 41:0–0)
Key Words: divpenia; lymphocyte; septic shock; T-cell receptor
diversity
Decreased T-Cell Repertoire Diversity in Sepsis:
A Preliminary Study
Fabienne Venet, PharmD, PhD
1,2
; Orchidée Filipe-Santos, PhD
3
; Alain Lepape, MD
2,4
Christophe Malcus, PharmD, PhD
1
; Françoise Poitevin-Later, PharmD
1
; Audrey Grives, MSc
3
;
Nadia Plantier, MSc
3
; Nicolas Pasqual, PhD
3
; Guillaume Monneret, PharmD, PhD
1,2
1
Cellular Immunology Laboratory, Hôpital E Herriot, Lyon, France.
2
Hospices Civils de Lyon, Université Claude Bernard Lyon I, Lyon, France.
3
ImmunID Technologies, CEA/iRTSV, Grenoble, France.
4
Hospices Civils de Lyon, Intensive Care Units, Lyon-Sud University Hospital,
Pierre-Bénite, Grenoble, France.
5
ImmunID Technologies, CEA/iRTSV, Grenoble, France.
Supported, in part, by funds from the Hospices Civils de Lyon, by DHOS-
Inserm “Recherche Clinique Translationnelle 2009” (to Dr. Monneret) and by
the French Ministry of Health (PHRC 2008, to Dr. Monneret and Dr. Lepape).
These funding sources had no role in the study design; in the collection, analy-
sis, and interpretation of data; in the writing of the report; and in the decision
to submit the paper for publication.
This work was performed at Hospices Civils de Lyon, Cellular Immunology
Laboratory, Hôpital E. Herriot, Lyon, France.
The authors have not disclosed any potential conflicts of interest.
For information regarding this article, E-mail: guillaume.monneret@chu-lyon.fr
Critical Care Medicine
0090-3493
10.1097/CCM.0b013e3182657948
00
00
2012
Copyright © 2013 by the Society of Critical Care Medicine and Lippincott
Williams & Wilkins
DOI:10.1097/CCM.0b013e3182657948
Saranya devi
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Venet et al
2 www.ccmjournal.org January 2013 • Volume 41 • Number 1
locus), we studied in patients with septic shock the combinatorial
diversity of the TCR β-chain directly at the genomic level (5–7).
Considering the importance of having a large and diverse TCR
repertoire for the quality of the immune response to infection, we
reasoned that if TCR diversity was decreased after septic shock,
this might participate in increased mortality and susceptibility to
secondary nosocomial infections in patients.
PATIENTS AND METHODS
Patients
The study group consisted of 41 consecutive patients with septic
shock, according to the diagnostic criteria of the American Col-
lege of Chest Physician/Society of Critical Care Medicine (8). The
exclusion criteria were patients <18 yrs of age and subjects with
aplasia or immunosuppressive disease (e.g., human immunodefi-
ciency virus). Septic shock was defined by an identifiable site of in-
fection, persisting hypotension despite fluid resuscitation requir-
ing vasopressor therapy, and evidence of a systemic inflammatory
response manifested by at least
two of the following criteria: 1)
temperature >38°C or <36°C;
2) heart rate >90 beats/min; 3)
respiratory rate >20 breaths/min;
and 4) white blood cell count
>12,000/mm
3
or <4000/mm
3
.
Severity was assessed by the Sim-
plified Acute Physiologic Score
II calculated at inclusion in the
protocol (9). The development of
organ dysfunction was assessed
by the Sequential Organ Failure
Assessment score (SOFA; range,
0–24), measured after 24 hrs of
ICU stay (10). Mortality was de-
fined as death occurring within
28 days after the onset of shock.
The onset of septic shock was
defined as the beginning of va-
sopressor therapy. None of these
patients was infected by superan-
tigen-producing bacteria. Sec-
ondary ICU-acquired infections
were defined according to Euro-
pean definitions of the European
Centre for Disease Prevention
and Control (11).
Biological analyses were per-
formed on residual blood after
completing routine follow-up
performed in the ICU. EDTA-an-
ticoagulated blood was collected
from patients at two time points:
days 1–3 (D1) and days 5–8 (D7)
after diagnosis of septic shock. Be-
cause the laboratory did not oper-
ate on Saturdays and Sundays, patients were included on weekdays
during working hours. In addition to patients who died or left the
ICU before the second sampling time, this accounted for the major-
ity of missing samples.
This work belongs to a global study on ICU-induced immune
dysfunctions. It has been approved by our Institutional Review
Board for ethics (Comité de Protection des Personnes) and reg-
istered at French Ministry of Research and Enseignement (#DC-
2008–509). It is also recorded at the Commission Nationale de
l’Informatique et des Libertés.
Normal values for TCR β-chain combinatorial diversity were
defined based on a cohort of 27 healthy volunteers extracted from
a larger study (SuSa Study, AFSSAPS clinical trial number 2010-
A00428-31, ImmunID; age range, 39–70 yrs; 11 females and 16
males). Normal median TCR diversity was 68% (95% confidence
interval [CI] 65–69). For the purpose of this preliminary study,
we defined 56% (mean − 2 SD) as the lower limit of normal TCR
diversity (Fig. 1A).
Figure 1. Reduced T-cell receptor (TCR) diversity in patients with septic shock. A, T-cell receptor β-chain diversity
(TCR diversity) was measured by molecular biology in 41 patients with septic shock between day 1 and day 3 (D1,
n = 38) and day 5 and day 8 (D7, n = 26) after the onset of shock (gray boxes) and in 27 healthy volunteers (HV,
open box). Results are presented as box plots and individual values for the percentage of TCR diversity.
Dotted line represents inferior limit of normal TCR diversity value (56%) defined based on values measured in HV.
B, Representative examples of TCR diversity measurements are shown for one septic patient at D1 and D7 and one
HV. x-axis represents 23 T-cell receptor β V (TRBV) genes (Vβ); y-axis represents 12 T-cell receptor β J (TRBJ)
genes (Jβ); and z-axis represents the relative intensity of each Vβ–Jβ combinatorial TCR chain amplified. C, TCR
diversity was monitored in 23 septic patients with paired samples at D1 and D7. Individual values and medians are
shown. D, The number of circulating T-cells (10
3
cells/µL) in 23 septic patients with paired samples at D1 and D7
are shown. Individual values and medians are presented. Nonparametric Wilcoxon’s paired test was used to compare
overtime evolution of TCR diversity and number of T-cells in patients.
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Laboratory Investigation
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Human ImmunTraCkeRβ Test/Combinatorial Diversity of
TCR β-Chain
Human T-lymphocyte repertoire diversity was measured us-
ing Human ImmunTraCkeRβ test (ImmunID Technologies,
Grenoble, France), a novel molecular biology technique on ge-
nomic DNA. Genomic DNA was extracted using standard tech-
niques from total peripheral mononuclear blood cells of pa-
tients and control subjects after informed consent. Multi-N-plex
polymerase chain reaction (PCR) was performed using an up
stream primer specific of all functional members of a given T-cell
receptor β V (TRBV) family and a downstream primer specific of
a given T-cell receptor β J (TRBJ) segment. This assay allows for
the simultaneous detection of several V–J rearrangements in the
same reaction. Using this technique, it is possible to detect 276 dif-
ferent TRBV–TRBJ rearrangements covering 100% of the possible
combinatorial rearrangements. All V–J1, J2, J3, J4, and Jn prod-
ucts were separated as a function of their size with a maximum
amplicon size of ~5 kb. PCR signals were detected and analyzed
using the Constel’ID software developed by ImmunID Technolo-
gies. PCR products were generated using iProof GC rich Master
Mix (Bio-Rad, Hercules, CA). The cycling conditions were as fol-
lows: Touchdown PCR cycle protocol was performed beginning
with 98°C for 3 mins, 98°C for 20 secs, 72°C for 20 secs, and 72°C
for 3 mins 30 secs; the annealing temperature was reduced by 1°
for every cycle until it reached 68°C, which was then repeated 23
times. Finally, one cycle of 10 mins at 72°C was performed. To
perform semiquantitative analysis, PCR reactions were stopped
at the exponential step of the PCR. To normalize DNA quantity
in each reaction, the actin gene was amplified in the same PCR
run. DNA isolated from kidney and/or human embryonic kidney
cell lines, in which no rearrangement takes place, was used as a
negative PCR control. PCR products were separated on a 0.8%
agarose gel, directly stained with SYBR Green I, and quantified us-
ing a charge-coupled device camera equipped with BIO-1D (Vil-
bert Lourmart, France). Constel’ID software was used for further
analytical studies including the generation of three-dimensional
repertoire illustration. Results are expressed as percentages of de-
tected rearrangements among the total 276 possible combinato-
rial rearrangements. A decreased percentage (i.e., a decreased TCR
diversity) defines a state of divpenia.
Flow Cytometry
The following lymphocyte subsets were analyzed: total T lympho-
cytes (CD45
+
CD3
+
), CD4
+
T lymphocytes (CD45
+
CD4
+
CD3
+
),
CD8
+
T lymphocytes (CD45
+
CD8
+
CD3
+
), total B-cells
(CD45
+
CD19
+
), natural killer cells (CD45
+
CD3
CD56
+
), and reg-
ulatory T-cells (CD4
+
CD25
high
CD127
low
), as previously described
(12). Human leukocyte antigen-DR (HLA-DR) expression was
monitored on circulating monocytes (mHLA-DR), as previously
described (13). Results are expressed as number of cells per micro-
liter of blood for lymphocyte subsets, as percentages of regulatory
T-cells among total CD4
+
lymphocyte population, and as percent-
ages of positive cells among total monocyte subset for mHLA-DR
measurements. Age-matched reference values were provided by
the routine flow cytometry laboratory of our institution.
Statistics
Results are presented as box plots with individual values. Lower
limit for the reference values is shown as a dot line. Compari-
sons between groups were made using the nonparametric Mann-
Whitney U test (survivors vs. nonsurvivors). The nonparametric
Wilcoxons paired test was used to assess variations between time
points in septic patients. Spearmans correlation test was used to
assess correlations between TCR diversity values and clinical and
biological variables. Fisher’s exact test was used to compare pro-
portions. A receiver operating characteristic curve was performed
to determine the cutoff values for TCR diversity and number of
T lymphocytes with regard to prediction of mortality. The best
cutoff value was selected based on likelihood ratio and Youdens
index. Using these thresholds, Kaplan-Meier survival curves
were obtained, and differences in survival between groups were
TABLE 1. Demographic, Clinical, and
Immunological Data for Patients With
Septic Shock
Clinical Data
Age at admission (yrs)
a
71 [57–77]
Male gender 29 (71)
Simplified Acute Physiologic Score II at
diagnosis of shock
a
57 [44–67]
Sequential Organ Failure Assessment
score
a
9.5 [8–12]
Main diagnosis category
Medical 17 (41)
Surgery 24 (58)
Comorbidities
0 21 (51)
1
20 (49)
Infection
Bacilli Gram-negative 14 (34)
Cocci Gram-positive 10 (24)
Fungi 3 (7)
Others 10 (24)
Type of infection
Community-acquired 22 (54)
Hospital-acquired 15 (37)
ICU-acquired 4 (10)
Site of infection
Pulmonary 12 (29)
Abdominal 13 (32)
Other 16 (39)
(
Continued
)
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Venet et al
4 www.ccmjournal.org January 2013 • Volume 41 • Number 1
evaluated using log-rank test. Univariate analysis through the Cox
model was used for the estimation of the hazard ratio and 95%
CI. Statistical analyses were performed using Prism software (ver-
sion 4.03; GraphPad Software, La Jolla, CA). A p value < 0.05 was
considered statistically significant.
RESULTS
Cohort Description
Forty-one patients with septic shock were included. Patients were
sampled twice after the onset of shock: first between day 1 and day
3 (D1, n = 38 samples) and then between day 5 and day 8 (D7, n =
26 samples). Twenty-three patients were sampled at the two time
points.
Clinical and immunological data are presented in Table 1.
Twenty-eight-day mortality was 39% (n = 16 nonsurvivors), and
four patients developed a nosocomial infection (10%).
As shown in Table 1, patients presented with usual immune
alterations as described in the course of septic shock: a major lym-
phopenia associated with an increased percentage of circulating
regulatory T-cells as well as a decreased percentage of monocytes
expressing HLA-DR (12–14).
TCR Diversity Is Decreased in Septic Patients
TCR β-chain combinatorial diversity (TCR diversity) was mea-
sured on peripheral blood mononuclear cells in patients using
a novel molecular biology technique. According to this indica-
tor, the patients can or cannot be qualified as divpenic (low TCR
diversity).
As shown in Figure 1A, TCR diversity was markedly decreased
in most septic patients (95% values of individual TCR diversity
below control values) at D1 and remained low at D7 in the major-
ity of the cohort (77% below control values). Indeed, in patients,
median TCR diversity was 15% at D1 (95% CI, 12–25) and 43% at
D7 (95% CI, 36–49). Therefore, most septic patients in this cohort
were divpenic. Representative examples of TCR diversity data for
one septic patient at D1 and D7 and for one healthy volunteer are
given in Figure 1B.
Interestingly, in the subgroup of 23 patients who were sampled
twice, there was a significant increase in TCR diversity over time
(p < 0.0001, Wilcoxons paired test) (Fig. 1C). In fact, TCR diver-
sity decreased in only one patient between D1 and D7 and the
patient died 9 days after the onset of shock (of note, the number of
circulating T-cells increased in this patient between D1 and D7).
Meanwhile, in these patients, the number of circulating T-cells
increased as well between D1 and D7 (Fig. 1D; p = 0.0026; Wil-
coxons paired test). However, in contrast to TCR diversity, this
increase was not systematic in every septic patient.
Correlations Between TCR Diversity and Clinical and
Immunological Parameters
No significant correlations were found between TCR diversity and
SOFA score or Simplified Acute Physiologic Score II either at D1
or at D7 (Spearmans correlation test). No significant differences
were observed between patients with or without comorbidities
(the Mann-Whitney U test, data not shown).
TABLE 1. Demographic, Clinical, and
Immunological Data for Patients With
Septic Shock (Continued)
Clinical Data
Mortality
Nonsurvivors 16 (39)
Nosocomial infections
Occurrence 4 (10)
Total lymphocytes
a
D1 0.6 × 10
3
[0.2–0.9]
D7 0.8 × 10
3
[0.6–1.3]
B lymphocytes
a
D1 80 [40–200]
D7 110 [70–260]
Natural killer cells
a
D1 60 [30–100]
D7 80 [50–130]
CD3
+
T lymphocytes
a
D1 400 [100–500]
D7 500 [300–900]
CD3
+
CD4
+
T lymphocytes
a
D1 200 [100–400]
D7 400 [200–600]
CD3
+
CD8
+
T lymphocytes
a
D1 70 [40–200]
D7 160 [70–260]
Regulatory T-cells (%)
a
D1 8.5 [6–12.5]
D7 11 [8–13]
mHLA-DR (%)
a
D1 31 [18–53]
D7 42 [26–60]
D = day.
a
Continuous data as well as biological parameters are presented as
medians and interquartile ranges [Q1–Q3]. Simplified Acute Physiologic
Score II calculated at inclusion in the protocol; Sequential Organ Failure
Assessment score measured after 24 hrs of intensive care unit stay.
Blood samples were obtained from 41 patients with septic shock at two
time points after the onset of shock. Patients were sampled within the first 3
days (D1) and between day 5 and day 8 (D7) after the onset of shock.
For clinical parameters, categorical data are presented as number
of cases and percentages respective to the total patient’s population,
within brackets.
The following leukocyte subsets were analyzed: total lymphocyte
number, CD3
+
T lymphocytes, CD3
+
CD4
+
T lymphocytes, CD3
+
CD8
+
T
lymphocytes, B lymphocytes, and natural killer cells, CD4
+
CD25
high
CD127
low
regulatory T-cells (Treg). Results are expressed as number of cells/μL and as
percentages of Treg among total CD4
+
lymphocyte population.
Expression of human leukocyte antigen-DR (HLA-DR) was monitored on
circulating monocytes (mHLA-DR). Results are presented as percentages of
monocytes expressing HLA-DR. Normal values from our laboratory for age-
matched individuals are 1.3 × 10
3
–3.2 × 10
3
total lymphocytes/µL, 150–400
B-cells/µL, 50–600 natural killer cells/µL, 900–1900 T-cells/µL, 500–1250
CD4+ T-cells/µL, 350–900 CD8+ T-cells/µL, 5%–7% Treg, and >90%
mHLA-DR.
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Interestingly, a significant correlation was observed between
TCR diversity and percentage of HLA-DR expressing monocytes
when measured at D1 (r = 0.3388, p = 0.0066, Spearmans cor-
relation test). However, no significant correlations were found be-
tween TCR diversity and percentage of regulatory T-cells either at
D1 or at D7.
A correlation was found between TCR diversity and the num-
ber of circulating T-cells at D1 (r = 0.5095, p = 0.0011, Spearmans
correlation test), but this correlation was lost at D7 showing that
TCR diversity evolves independently of T-cell count after septic
shock. No correlation was observed in healthy volunteers.
Low TCR Diversity May Possess a Predictive Value on
Mortality.Sixteen patients died within 28 days after the onset of
shock in this cohort. Five patients died before D8 and 11 patients
died after D10. Mean time of death was 13 days postshock.
No significant differences were observed in TCR diversity be-
tween survivors and nonsurvivors either at D1 or at D7. The per-
centage of monocytes expressing HLA-DR was significantly lower
in nonsurvivors than in survivors at D7 but not at D1 (p = 0.0109
and 0.2442, respectively, Mann-Whitney U test).
In both groups and in patients who were sampled twice, TCR
diversity increased significantly between the two time points
(p = 0.0002 in survivors, p = 0.0195 in nonsurvivors, Wilcoxons
paired test). Importantly, although most patients in the survi-
vors group (91%) and every patient in the nonsurvivors’ group
(100%) had a decreased TCR diversity at D1, only 67% of sur-
vivors remained with a decreased TCR diversity, whereas most
of the nonsurvivors still presented with a decreased value at D7
(91%; Table 2).
Subsequently, the best thresholds for prediction of mortality
were calculated for TCR diversity at D1, then at D7 using receiv-
er operating characteristic curve analyses. At D1, the area under
the curve was close to 0.5, illustrating the absence of predictive
value for this parameter at this time point. However, at D7, the
area under the curve was 0.7 (95% CI 0.5–0.9). The best cutoff
value based on Youdens index and likelihood ratio was found to
be 33% (Youdens index = 0.32; likelihood ratio = 3.4). Using this
value, the Kaplan-Meier survival curves were obtained. Impor-
tantly, based on this threshold, a significantly different survival
was observed between patients with or without low TCR diversity
(Fig. 2A; log-rank test, p = 0.0058; hazard ratio = 4.48; 95% CI
1.96–53.32).
Of note, similar analyses were performed to determine the
prognostic value for the number of circulating T-cells. The
best cutoff value at D7 was found to be 730 cells/µL. How-
ever, based on this threshold, no significantly different sur-
vival was observed between the groups (data not shown).
This illustrates that divpenia by itself possesses predictive
value on mortality independently of the number of circulat-
ing lymphocytes.
TCR Diversity May Present With Predictive Value on Noso-
comial Infections. Only four patients developed a nosocomial
episode in our cohort. Every nosocomial infection was diagnosed
later than 14 days after the onset of shock. Average diagnosis time
was 16 days after shock. Interestingly, and despite the less number
of patients in our cohort, patients who will develop a nosocomial
episode present with significantly reduced TCR diversity at D1 in
comparison with the patients who remain free of secondary infec-
tion (Fig. 2B). Furthermore, at D7, every patient who developed
a nosocomial episode remained with a reduced diversity, whereas
only 73% of the patients free of any nosocomial infection present
with a low TCR diversity (Table 2).
Of note, neither at D1 nor at D7 did we observe any difference
regarding the number of circulating T-cells between patients who
will or will not develop a nosocomial episode (data not shown).
Predictive Value of Numeration Diversity
Lymphocyte Scoring
Finally, to test whether the combination of TCR diversity and
number of circulating T-cells was more informative than each
parameter considered alone, patients were stratified based on
these parameters at D1 and at D7 using thresholds previously
calculated through receiver operating characteristic curve analy-
ses (TCR diversity < 33%, number of circulating T-cells < 730
cells/µL). Based on these two parameters, patients sampled at D1
were classified into four groups (Fig. 3A; n = 38). The numera-
tion diversity lymphocyte (NDL)1 group (presenting with low
TCR diversity and low number of T-cells, n = 26) was compared
TABLE 2. T-Cell Receptor Diversity in Patients With Septic Shock
Survivors Nonsurvivors Noninfected Infected
D1 D7 D1 D7 D1 D7 D1 D7
Number of samples 23 15 15 11 34 22 4 4
Median 13 49 18 40 18 41 1 49
Quartile 1 2 37 2 20 2 29 1 31
Quartile 3 29 64 34 52 32 58 12 51
Divpenia (proportion <56%
a
)
91 67 100 91 94 73 100 100
D = day.
a
Lower limit of normal T-cell receptor diversity based on the evaluation of 27 healthy volunteers. T-cell receptor diversity (%) was evaluated on peripheral blood
mononuclear cells extracted from 41 patients with septic shock at day (D) 1 (between D1 and D3 after the onset of shock, n = 38 samples) and D7 (between
D5 and D8 after the onset of shock, n = 26 samples).
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Venet et al
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with NDL2 (higher TCR diversity and low number of T-cells,
n = 5), NDL3 (low TCR diversity and higher number of T-cells, n =
4), and NDL4 (higher TCR diversity and higher number of T-cells,
n = 3). At this time point, the majority of patients belonged to group
NDL1 (68% patients), whereas group NDL2 represented 13%,
group NDL3 11%, and group NDL4 8% of the total population.
The NDL1 group showed a significantly lower percentage of
mHLA-DR expressing monocytes than the other three groups
(median, 26%; 95% CI, 21–39 for group NDL1; median, 55%;
95% CI 35–67 for the other groups; p < 0.05, Mann-Whitney U
test). However, no differences between NDL1 and the other groups
were observed for Simplified Acute Physiologic Score II and SOFA
scores. Interestingly, every nosocomial infection occurred in the
NDL1 group, and mortality was 38% vs. 25% in the other pooled
groups.
At D7 (n = 26; Fig. 3B), patients’ distribution according to
these NDL groups was NDL1 (n = 6), NDL2 (n = 11), NDL3
(n = 1), and NDL4 (n = 8). Therefore, the majority of patients
now belonged to group NDL2 (42% patients), whereas group
NDL1 represented 23%, group NDL3––4%, and group NDL4–
–31% of the total population. Interestingly, at this time point,
initial SOFA score was significantly higher in group NDL1 as
compared with the other three groups (median, 13; 95% CI 11–15
in NDL1; median, 9; 95% CI 9–11 in the other groups; p < 0.01,
Mann-Whitney U test), whereas the percentage of mHLA-DR
Figure 2. Reduced T-cell receptor (TCR) diversity in patients with septic
shock is associated with increased risk of death and nosocomial infection.
A, T-cell receptor β-chain diversity (TCR diversity) was measured by molecular
biology in 41 patients with septic shock between day 1 and day 3 (D1,
n = 38) and day 5 and day 8 (D7, n = 26) after the onset of shock. The
Kaplan-Meier survival curves were obtained after patients with septic shock
were stratified based on TCR diversity value at 33% at D7 determined by
receiver operating characteristic curve analysis. Log-rank test was used to
analyze the difference in 28-day survival between patient groups. B, TCR
diversity was monitored in 41 septic patients sampled at D1 and/or D7.
Results are presented as box plots and individual values for patients who
remained free of any nosocomial episode (open boxes, n = 34 at D1, n = 22
at D7) and patients who developed a nosocomial infection (gray boxes,
n = 4 at D1 and at D7). The nonparametric Mann-Whitney U test was used to
compare TCR diversity between groups of patients.
Figure 3. Numeration diversity scoring at day (D1) and day 7 (D7). T-cell
receptor (TCR) diversity and number of circulating T-cells (10
3
/µL) were
monitored in (A) 38 septic patients at D1 and (B) 26 septic patients at
D7. Based on thresholds previously determined (33% TCR diversity, 730
T-cells/µL), four groups were created: numeration diversity lymphocyte
(NDL)1 score corresponding to lymphodivpenic patients (low TCR diversity,
low number of T-cells, n = 26), NDL2 score corresponding to lymphopenic
patients (higher TCR diversity, low T-cells, n = 5), NDL3 score corresponding
to divpenic patients (low TCR diversity, higher T-cells, n = 4), and NDL4
score corresponding to normality (higher TCR diversity, higher T-cells, n = 3).
Results are presented as individual values.
Copyright (c) Society of Critical Care Medicine and Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited
Laboratory Investigation
Critical Care Medicine www.ccmjournal.org 7
expressing monocyte was lower (median, 25%; 95% CI 12–
41 in NDL1; median, 51; 95% CI 39–58 in the other groups;
p < 0.05, Mann-Whitney U test). Furthermore, mortality in the
NDL1 group was 67% (four nonsurvivors among six patients),
whereas only seven patients among 20 died in the other groups
(mortality = 35%).
DISCUSSION
As a result of their ability to interact with the cells of the innate
immune system and with the other cells of the adaptive immune
system, lymphocytes play a central role in the anti-infectious
immune response not only as effectors, but also as regulators of
this response. This has been illustrated by the observation of a dys-
regulated, proinflammatory immune response after polymicrobial
septic challenge in mice lacking T-cells and B-cells (15, 16). With-
out showing any clear association with the increased risk of death
or nosocomial infections in patients, some alterations of the
lymphocytic response have been previously reported after septic
shock. They comprise the following: 1) phenotypic alterations in-
cluding an increase in inhibitory coreceptor expression associated
with a decreased expression of activating coreceptors and CD3;
2) functional alterations mainly characterized by a decreased ex
vivo lymphocyte proliferation associated with decreased Th1 and
increased Th2 immune responses; 3) induction of regulatory
T-cell populations; and 4) increased apoptosis participating in
the initial lymphopenia (1, 3). Such lymphocyte dysfunctions
were recently observed in patients’ lymphoid organs in a post-
mortem study (17). The authors observed that patients who died
of sepsis present with features consistent with the development
of immunosuppression after sepsis (reduced cytokine produc-
tion after TCR stimulation of splenocytes, increased expression of
inhibitory receptors and ligands as well as expansion of suppres-
sor cell populations in both lung and spleen cells). These results
illustrate that sepsis-induced immune suppression is a profound
mechanism that can be observed not only in circulating blood
cells, but also in lymphoid organs.
In the current study, we reinforce and expand these results
by investigating at the molecular level a novel aspect of sepsis-
induced lymphocyte alterations: the diversity of the TCR β-chain
repertoire. To the best of our knowledge, we observe for the first
time that septic patients present with a dramatic decrease of
peripheral T-cell repertoire. This was associated with other usual
immune dysfunctions described in such cohorts of patients (lym-
phopenia, decreased percentage of HLA-DR expressing mono-
cytes) and most importantly with increased risk of death and
nosocomial infections. The diversity of T-cells plays a central role
in the host’s ability to mount protective anti-infectious immune
responses (18, 19) and an optimal level of TCR diversity allows for
efficient protection against pathogens (19). Indeed, because the
organism cannot predict the precise pathogen-derived antigens
that will be encountered, the immune system relies on the genera-
tion and maintenance of a diverse TCR repertoire (19). It follows
that the size and diversity of the available T-cell repertoire are cru-
cial in shaping the immune response to a given antigen.
One physiological (aging) and several pathological/therapeutic
(HIV infection/highly active antiretroviral therapy, T-cell leuke-
mia, and bone marrow transplantation) conditions have yielded
reduced TCR diversity in humans (19, 20). Importantly, decreased
TCR repertoire diversity has been associated with viral escape in
chronic viral infections such as HCV (21), and changes in the TCR
repertoire have been correlated with outcomes of renal transplan-
tation (22, 23). In ICU patients, such reduced TCR repertoire, as
observed in our study, might participate in increased susceptibil-
ity to nosocomial infections in the context of use of invasive de-
vices (intubation, urinary catheter, central venous catheter, etc.).
Furthermore, this might play a role in the reactivation of viral
infections (cytomegalovirus, Epstein–Barr virus), normally solely
occurring in immunocompromised hosts and also observed in
ICU patients (24, 25).
Only two studies previously investigated the TCR repertoire
in sepsis by using flow cytometry: one in patients and one in the
murine model of cecal ligation and puncture. The first study in-
cluded nine patients with Staphylococcus aureus septic shock (26),
and no specific T-cell repertoire modifications were observed. In
the second study, Unsinger et al (27) showed that after cecal liga-
tion and puncture, mice presented with lymphopenia but that the
TCR repertoire measured 21 days after cecal ligation and puncture
was not skewed toward any Vβ type but resembled the repertoire
found in normal mice. The authors concluded that, after sepsis,
T-cells were not primed to any antigen resulting from the infec-
tion. Importantly, in both studies, TCR repertoire was measured
by flow cytometry. Using this technique, a maximum of 25 differ-
ent Vβ clones could be detected (28). In contrast, 276 TCR Vβ–Jβ
rearrangements could be identified by the present molecular biol-
ogy technique. Furthermore, in both previously published studies,
TCR repertoire was evaluated at late time points after the onset
of shock (after 21 days in mice and within 10 days in patients).
Considering the fast recovery slope observed in our cohort, this
could explain the discrepancy with our results. However, a study
comparing these two techniques of TCR repertoire measurement
should be conducted. To our knowledge, such study has never
been done yet.
This fast increase in TCR repertoire observed in patients be-
tween D1 and D7 is of the utmost importance in this study. In-
deed, to our knowledge, such a rapid recovery of TCR diversity has
not been described previously in any other clinical context. This
argues against a novel thymus-based lymphocyte generation but
rather suggests a systemic liberation of lymphocytes trapped in
peripheral compartments. This assumption is in agreement with
the reduced percentage of CD45RA
+
(naive) but not of CD45RO
+
(memory) T-cells previously described in septic patients (14, 29,
30). This is further confirmed by our observation that lymphocyte
reconstitution after septic shock occurs mainly through a switch
from NDL1 to NDL2 group (i.e., through increased diversity) and
not through a switch from NDL1 to NDL3 group (i.e., through an
increase in cell number). This suggests a uniform reconstitution
of TCR diversity through rapid mobilization from lymphocyte
peripheral stores rather than immune reaction-based active oli-
goclonal expansion. These results open novel cognitive research
perspectives that deserve to be further investigated in experimen-
tal models of sepsis. Similarly, the evaluation of TCR diversity of
memory vs. naive T-cells and its overtime evolution in patients
Copyright (c) Society of Critical Care Medicine and Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited
Venet et al
8 www.ccmjournal.org January 2013 • Volume 41 • Number 1
with septic shock should be evaluated in a specifically designed
study. Indeed, the dynamic measurement of TRB diversity and
the composition of T-cell subpopulations (i.e., naive/memory/
regulatory) over time in peripheral tissues as well as in spleen
and thymus would help in understanding the mechanisms of the
rapid recovery of TCR diversity in patients with septic shock and
of sepsis-induced immune suppression. More generally, this fast
increase in TCR diversity after the onset of shock suggests that
this temporal immune suppression belongs to the homeostatic re-
sponse that takes place after septic shock. It is the lack of (or slow)
recovery of immune functions that is associated with unfavorable
outcome (as observed with decreased mHLA-DR). This is in favor
of the initiation of immunotherapy in sepsis to facilitate this res-
toration in selected patients with persistent immune alterations.
Our study has some limitations. First, this preliminary study
included a small cohort of patients. Therefore, no multivariate
analysis could be performed to test the independent predictive
value of TCR diversity on mortality vs. clinical variables (Simpli-
fied Acute Physiologic Score II and SOFA scores) or other im-
mune parameters (mHLA-DR or number of circulating T-cells).
Similarly, the low number of nosocomial infections clearly weak-
ens the statistical analysis of our data because it is quite difficult to
obtain significant results with such unbalanced groups of patients.
This may be explained by the rather high mortality rate (i.e., 39%)
in our cohort knowing that, by definition, patients who died can-
not develop nosocomial infections. Second, the impact of thera-
peutic interventions on TCR diversity was not evaluated. Finally,
the absence of an exact age-matched control group prevents us
to definitively conclude on the sepsis-induced decrease in TCR
diversity. With that said, such a low percentage of TCR variability
observed at D1 in septic patients could not solely be explained by
their increasing age, considering results for TCR repertoire diver-
sity in elderly published in the literature (18) as well as the fact
that no correlation with age was measured at D7 in patients or
within the cohort of healthy volunteers (maximum age = 70 yrs,
data not shown).
In total, in a cohort of patients with well-documented immune
dysfunctions (lymphopenia, decreased HLA-DR expression on
monocytes), we describe for the first time an additional aspect of
sepsis-induced immune suppression. Our results add to the cur-
rent knowledge of lymphocyte dysfunctions in sepsis and of their
association with increased mortality and risk of nosocomial in-
fections (3, 31, 32). Furthermore, our results show that the mea-
surement of TCR repertoire diversity, as a novel immune response
indicator, may provide predictive information regarding risk of
death and nosocomial infection independently of the number of
circulating lymphocytes. As a consequence, immunostimulating
therapies are now proposed in the treatment of sepsis. One poten-
tial candidate could be recombinant human IL-7 because, in addi-
tion to its capacity to increase circulating lymphocyte number and
function (33), its in vivo injection in healthy volunteers was able to
increase TCR diversity without inducing any major side effect (34).
Furthermore, in mice, initial studies showed that recombinant hu-
man IL-7 was able to reduce mortality after cecal litigation and
puncture (35, 36).
CONCLUSION
In this study, we describe for the first time at the molecular level
a novel aspect of sepsis-induced lymphocyte dysfunctions. We
observed that patients with septic shock present with a severely
decreased TCR β-chain diversity after the onset of shock. Impor-
tantly, independent of the number of circulating T-cells, patients
who did not restore rapidly their diversity presented with both
increased risk of death and of nosocomial infections. On valida-
tion in a large multicentric clinical study, divpenia assessment
may become an interesting tool for immunomonitoring. Further-
more, on a more general basis, the observation of a rapid recovery
of TCR diversity in peripheral lymphocytes, never described in
other clinical situations, opens a novel and interesting cognitive
research avenue to understand the underlying mechanisms of im-
mune homeostasis after septic shock.
ACKNOWLEDGMENTS
We thank Anne Portier and Caroline Guignant, PharmD, PhD,
from the Immunology Laboratory of Hopital E. Herriot, Lyon,
France, for their help in performing preanalytical handling of
samples; Hélène Thizy and Marion Provent (Clinical Research
Center, Lyon-sud) for their work on patients’ inclusion and clini-
cal data acquisition; and Gilles Parmentier, PhD, Tyoka Rabéony et
Anais Couturier, for assistance in statistical analyses.
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    • "Low doses of IL-7 have been shown to preferentially activate effector T cells (Teff) as compared to Tregs in peripheral blood from septic patients [111]. Sepsis patients exhibit an extensive loss of T cell receptor (TCR) diversity, which is linked with an increased incidence of nosocomial infection and associated mortality [112]. IL-7 treatment has the potential to enhance TCR diversity and thereby help to mount an effective immune response against a variety of infecting pathogens [113] . "
    [Show abstract] [Hide abstract] ABSTRACT: Sepsis is defined as life-threatening organ dysfunction caused by dysregulated host responses to infection (Third International Consensus definition for Sepsis and septic shock). Despite decades of research, sepsis remains the leading cause of death in intensive care units. More than 40 clinical trials, most of which have targeted the sepsis-associated pro-inflammatory response, have failed. Thus, antibiotics and fluid resuscitation remain the mainstays of supportive care and there is intense need to discover and develop novel, targeted therapies to treat sepsis. Both pre-clinical and clinical studies over the past decade demonstrate unequivocally that sepsis not only causes hyper-inflammation, but also leads to simultaneous adaptive immune system dysfunction and impaired antimicrobial immunity. Evidences for immunosuppression include immune cell depletion (T cells most affected), compromised T cell effector functions, T cell exhaustion, impaired antigen presentation, increased susceptibility to opportunistic nosocomial infections, dysregulated cytokine secretion, and reactivation of latent viruses. Therefore, targeting immunosuppression provides a logical approach to treat protracted sepsis. Numerous pre-clinical studies using immunomodulatory agents such as interleukin-7, anti-programmed cell death 1 antibody (anti-PD-1), anti-programmed cell death 1 ligand antibody (anti-PD-L1), and others have demonstrated reversal of T cell dysfunction and improved survival. Therefore, identifying immunosuppressed patients with the help of specific biomarkers and administering specific immunomodulators holds significant potential for sepsis therapy in the future. This review focusses on T cell dysfunction during sepsis and discusses the potential immunotherapeutic agents to boost T cell function during sepsis and improve host resistance to infection.
    Full-text · Article · Jul 2016
    • "The concept of immune suppression in sepsis is further exemplified in a study involving 41 patients with sepsis [11]. In this study, there was a marked reduction in the T cell receptor β chain diversity that was associated with increased mortality and a higher risk of developing nosocomial infection [11]. It has also been demonstrated that there is decreased ex vivo proliferation of Th1 and an increased Th2 immune response [12,13]. "
    [Show abstract] [Hide abstract] ABSTRACT: Purpose: The aim of this study was to evaluate the efficacy of Mycobacterium w (Mw), an immunomodulator in severe sepsis. Methods: Patients 18 years or older with severe sepsis were randomized within 48 hours of first organ dysfunction to receive either intradermal Mw or saline. The primary end point was 28-day mortality, whereas the secondary end points were ventilator days, intensive care unit (ICU) and hospital length of stay, and delta Sequential Organ Failure Assessment (SOFA) score. Results: Fifty patients with severe sepsis (25 Mw, 25 control) were included in the study. There were 7 and 8 deaths in the Mw and control groups, respectively (P = 0.51). The days on mechanical ventilator were significantly lesser in the Mw group compared with control (median, 6 vs 9; P = 0.025). The median ICU and hospital length of stay was significantly less in the Mw arm (7 vs 12 days [P =0.006] and 10 vs 16 [P = 0.007], respectively). The delta SOFA score was significantly higher in the control arm (P =0.027). There was a higher incidence of secondary bacterial infections in the control group (P = 0.009). Conclusion: The use of Mw in severe sepsis was associated with significant reduction in days on mechanical ventilation, ICU and hospital length of stay, lower incidence of nosocomial infection, and delta SOFA score.
    Article · Aug 2014
    • "Finally, an increased percentage of circulating CD4 + CD25 + regulatory T cells has been repeatedly observed in septic patients [52]. In some studies, a link between the intensity and duration of these alterations and subsequent mortality/nosocomial infections risk was observed [48,50,51]. However, as opposed to mHLA-DR measurement, to date, no consensually defined marker of sepsis-induced lymphocyte alterations has emerged in the literature. "
    [Show abstract] [Hide abstract] ABSTRACT: Preliminary studies suggest that a subgroup of septic patients with severe immune alterations is at high risk of death or nosocomial infection and therefore could benefit from adjunctive immune stimulating therapies. There is thus an urgent need for robust biomarkers usable in routine conditions evaluating rapidly evolving immune status in patients. Although functional testing remains a gold standard, its standardization remains challenging. Therefore, surrogate markers such as monocyte HLA-DR expression, are being developed. Such biomarkers of immune functionality will enable a novel approach in the design of clinical trials evaluating immunostimulating therapies in sepsis at the right time and in the right patient.
    Full-text · Article · May 2013
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