Introduction
Occupational, environmental, accidental, or therapeutic
exposure to ionizing radiation have major impacts on human health and may result in defects in the hematopoietic
and immune systems.[] Lymphocytes
have the highest cell turnover in mammalians, which
makes them extremely radiosensitive.[,] Although the chromosome-aberration assay remains the "gold
standard" for early-response accident biodosimetry
and dose assessment, the lymphocytes are accepted as
the most efficient practical laboratory tool for estimating
exposed dose in population monitoring and they
enable prompt commencement of medical intervention
in case of nuclear accidents.[,]
TBI causes significant immunosuppression, but different
lymphocyte subsets have various radiosensitivities;
B cells are the most sensitive ones and Natural killer
cells are the most resistant ones. T helper/effector cells
(Th) are responsible for the regulation of immune system
and are more radiosensitive than T cytotoxic/suppressor
cells (CTL), which directly destroy target cells.
[] T regulatory (Treg) cells are crucial for self-tolerance
and are the potent suppressors of antitumor immunity.
[7,8] They are found to be resistant to IR compared with
Th and CTL cells in both in vivo and in-vitro studies,
but there is limited data on this.[,-] Developments
in flow cytometry provide us with a detailed analysis
of lymphocyte subsets using monoclonal antibodies
(MAbs) specific to lymphocyte differentiation antigens.
[] In addition, recent developments and rising questions
in the context of combining immunotherapy with
RT compelled us to revisit the concept of radiation-induced
quantitative and functional changes in lymphocyte
subsets by flow cytometry using animal models with
the aim of transitioning the findings to clinical studies.
Methods
Twenty-three Swiss albino rats of both sexes weighing
200?250 g at 4?8 weeks of age were evaluated. Animals
were housed in a dedicated animal room maintained at
22°C±2°C and a relative humidity of 50%±20%. Animals
remained on 12:12-h light and dark cycles with
free access to food and water.
Experimental Design
Immediately prior to irradiation, 1 cc. heparinized
blood sample was drawn via cardiac puncture under
superficial anesthesia. Next, flow cytometric analysis
was performed within two hours by the following
fluorescent labeled MAbs designed specifically for rats:
CD3-FITC (Fluorescein isothiocyanate), CD4-PE (phycoerythrin),
CD8-A PE, CD25 IL-2, Mouse Anti-Rat
CD3-IF4, and Mouse Anti-Rat CD25-OX39 at the immunology
laboratory. First, antibodies were added and
after waiting for 15 min under room temperature and
darkness, erythrocytes were lysed using FACS Lysing
solution (Beckton and Dickinson, San Jose, CA), cen-trifuged, and washed with Phosphate Buffer Solution.
After washing, flow cytometric analysis was performed
using FACSCAN flow cytometry system (Beckton and
Dickinson, San Jose, CA) and read on simulset software
program. Using CD3-FTIC, lymphocytes were
gated by isotope match negative control antibodies and
were differentiated into FITC and PE channels. Analysis
was performed by assaying 15,000 spheres per tube.
Next, the rats were exposed to TBI under ketamine
anesthesia (75 mg/kg-IM) at a single fraction of 5 Gy.
Irradiation (IR) was performed using Co60 ?-rays and
administered to a midline tissue. Following this, the IR
rats were taken back to their home cages. The peripheral
blood samples were redrawn at time points of 1
day (acute period) and 7 and 14 days (latent period)
post-TBI and flow cytometric analyses were performed
again. In addition, complete blood counts with differentials
were performed in pediatric hematology laboratory
using a clinical hematology analyzer.
CD3+CD19- represent T lymphocytes, CD3+CD4+
represent T helper/inducer cells (Th), CD3+CD8+
represent T cytotoxic/suppressor cell (CTL), and
CD4+CD25+represent Treg cells.[]
The ratio of lymphocyte subsets are calculated by
dividing the total T lymphocyte count by absolute
lymphocyte subset numbers. The lymphocyte subset
depletion and recovery kinetics by time were analyzed.
None of the animals were euthanized.
Statistical Analysis
Statistical analysis was performed using SPSS 21.0 software
(SPSS Inc., Chicago, IL). Continuous variables
were expressed as mean±standard deviation and categorical
variables were expressed as n (%). Comparisons
were done using Repeated Measures ANOVA or Fischer"s
exact test where appropriate. A p value of <0.05 was
considered statistically significant.
Results
White blood cell and lymphocytes counts in the peripheral
blood at baseline and at 1, 7, and 14 days after
TBI are presented in Table 1 and Figure 1.
Table 1: Mean absolute leucocyte and lymphocyte levels at baseline and 1, 7, and 14 days after Total-Body-Irradiation (TBI)
Fig 1: Mean absolute leucocyte and lymphocyte levels at
baseline and 1, 7, and 14 days after Total-Body-
Irradiation (TBI).
At 1, 7, and 14 days after TBI, a statistically significant
decrease was noted in all T lymphocyte subsets
compared with the baseline value both in terms of absolute
number and ratio (Table 2-3 and Fig. 2). The decrease
in Th subset was more pronounced compared
with that in CTL.
Table 2: Mean lymphocyte subset ratios at baseline and 1, 7, and 14 days after Total-Body-Irradiation (TBI) according to
flow cytometric analysis
Table 3: Mean absolute lymphocyte subset levels/?l blood at baseline and 1, 7, and 14 days after Total-Body-Irradiation
(TBI) according to flow cytometric analysis
Fig 2: Mean lymphocyte and subset ratios at baseline
and 1, 7, and 14 days after Total-Body-Irradiation
(TBI) according to flow cytometric analysis.
Baseline CD4+/ CD8+ ratio was 0.85. This ratio significantly
decreased to 0.29 of the baseline value one day after TBI, then steadily increased in subsequent
measurements and reached a near normal value (Table
4 and Fig. 3).
Table 4: The changes in CD4+/ CD8+ ratio at baseline and 1, 7, 14 days after Total-Body-Irradiation (TBI) according to flow
cytometric analysis
Fig 3: CD4+/CD8+ ratio at baseline and 1, 7, 14 days
after Total-Body-Irradiation (TBI) according to
flow cytometric analysis.
Treg lymphocyte level markedly declined to 6.5%
of baseline value one day after TBI, and then steadily
increased during follow-up. By the end of 14 days, it
reached half of its baseline value (Table 5 and Fig. 4).
Table 5: Mean absolute number of Treg cells/?l blood at baseline and 1, 7, 14 days after Total-Body-Irradiation (TBI) according
to flow cytometric analysis
Fig 4: Mean absolute number of Treg cells/?l blood at
baseline and 1, 7, 14 days after Total-Body-Irradiation
(TBI) according to flow cytometric analysis.
Discussion
Our study showed that a statistically significant decrease
in absolute leucocyte and lymphocyte subset
number levels in peripheral blood was observed as early
as one day after exposure to 5 Gy TBI; lymphocyte
subsets have different radiosensitivities and monitoring
T lymphocyte subset depletion and recovery kinetics
as a biodosimetry has potential utility for predicting
the dose, timing, and outcome of radiation exposure.
Also, we have found that Treg cell recovery was faster
than that of Th and CTL. This finding may contribute
to Treg-augmented immunosuppression in addition to
direct immunosuppressive effects of ionizing radiation.
The mean Lethal Dose (LD50) is defined as a dose
that results in 50% mortality in a population within
30?60 days after TBI.[] LD50 is accepted at 7.6-7.8
Gy in rats.[] Because lymphocytes are extremely sensitive
to IR, doses close to LD50 may cause significant
lymphocyte depletion (<200cells/mm); as a result their
utility in assessing the exposed dose may be challenging.[] Therefore, TBI dose of 5 Gy was preferred in
this study since it is a sublethal dose.
Peripheral blood samples were collected one day
after TBI because after 24-48 hours of IR exposure,
a predictable decrease in absolute lymphocyte counts
occurs that leads to the use of lymphocyte depletion
kinetics as a part of biodosimetric model in case of radiation
accident and this effectively helps in the management
of mass casualty incidents.[,] Also, early
and rapid hematologic changes caused by over exposure
to radiation, for example, neutrophil and lymphocyte
depletion and a decrease in CD4+/CD8+ ratio may
also help in discriminating exposed and non-exposed
individuals and estimate the dose exposed.[]
Samples after 7 and 14 days were collected for predicting
the amount of exposure in the latency period in
case of a mass casualty incident wherein victims may
not have immediate access to medical care for various
reasons until days or even weeks after exposure. Decreased
T lymphocyte subset levels (especially CTL) in
latency period may predict the severity of exposure.[]
We observed that leucocyte and lymphocyte counts
reached nadir level at one day after TBI similar to other studies [,]. In concordance with the findings by
Ossetrova et al., the lymphocyte numbers decreased to
15% of pre-TBI levels one day after TBI.[] This rapid
depletion may be the result of extreme radiosensitivity
of mature lymphocytes.[]
One day after TBI, the decrease in Th cells was more
pronounced than in CTL cells and this resulted in a significant
decrease in the CD4+/CD8+ ratio. This finding
is also in concordance with the literature and may be
used to predict the severity of immunosuppression that
may develop in exposed individuals.[]
After 7 and 14 days, although some amount of recovery
was noted in all T lymphocyte subsets, leucopenia
and lymphopenia persisted in all subsets and this
findingshows similarity with findings in the literature.
[] In a study by Hu, after 3.3 Gy, 60 days were required
for neutrophils to return to 95% of their normal
values and for lymphocytes to return to 55% of their
normal levels.[] Also, Inoue et al. showed radioresistant
stem cell subfraction nine days after exposure to
4?6 Gy in mice.[] Similarly, we found that 7 and 14
days after 5 Gy TBI, almost 20% of leucocytes and lymphocytes
were radioresistant, possibly by means of radioresistant
stem cells. This finding may be used as the
rationale to opt for cytokine therapy after exposure to
IR and the explanation of hematological recovery seen
in these individuals. Conversely, this result may inform
us about the need for the prolonged use of hematopoietic
growth factors, blood product supplies, and antibiotherapy
in case of nuclear accidents.[]
In the latency period, recovery in CTL was slower;
7 days after TBI, Th levels increased by 2.7 times, but
CTL levels increased by only 1.007 times. This result reflected
an increase in CD4+/CD8+ ratio, almost reaching
the baseline level. Thus, CD4+/CD8+ ratio in the latency
period may be insignificant in estimating the severity of
exposure, and persistently low CTL level, a finding similar
to that in our study, may be more predictive.[]
The regenerative capacity of Treg cells in the latency
period was higher than in all other subsets; at the end
of the study, Treg levels almost reached half of the pre-
TBI levels. This rapid recovery of Treg cells may have
further suppressed Th and CTL recovery.
Data from in-vitro and in-vivo studies showed that
Treg cells were radioresistant, although the underlying
mechanism and dependency on dose and fractionation
are not fully understood.[,-] Intrinsic radioresistance,
radiation-induced activation of TGF-Β promoting
Treg cells or the increased output of Treg cells from
thymus and spleen, or the increased functional activation
of Treg cells in response to local IR or TBI are possible mechanisms.[,] In this study, we showed
that Treg cells were radiosensitive but their replicative
capacity was high and 5 Gy TBI was not enough to suppress
Treg cells. Our finding about the radioresistance
of Treg cells has two opposing effects. First it makes
host favorable for tolerance after TBI to avoid rejection
which is desirable. On the other hand it has a negative
impact on tumor immunity in terms of tumor control
which is an undesirable outcome.[,]
Radiotherapy (RT) has the reputation of being immunosuppressive.[,] But it has recently been established
that RT has a dual effect on tumor immunity
in the tumor microenvironment; while increasing immunostimulation
by the infiltration of CTL and natural
killer (NK) cells which inhibit tumor growth on one
hand, on the other hand it causes immunosuppression
by Treg, tumor-associated macrophages, and myeloidderived
suppressor cell infiltration that may result in
tumor growth.[,-] Related to this subject, we
have found that immunosuppressive Treg cell number
significantly decreased one day after TBI, but their selfrenewal
was faster than that of Th cells and CTL. This effect
may result in additional suppressive effect on antitumor
immunity and generalized immunosuppression in
case of TBI. Also, single fraction 5 Gy high IR dose may
have caused an increase in circulating Treg cells, which
is also depicted in literature.[] These results may warn
us in clinical studies about the use, dose, fractionation,
technique, timing, and sequencing of RT adjunct to anti-
CTLA-4 and anti-PD-L1 immunotherapy.[,-] In
the future, the immunosuppressive effect of Treg cells on
tumor immunity may be overcome by using monoclonal
antibodies targeting CD25 as a part of multimodality
therapy concurrent with RT and tumor vaccines; in an
animal model, Treg cell depletion reportedly resulted in
tumor rejection and longer tumor immunity in numerous
tumor-inovulated mice.[,,]
Limitations of the Study
A major limitation of this study is the unavailability
of multiple blood samples in shorter intervals, the use
range of doses, and different dose rates. The absence of
sham-irradiated group and the repeating of experiment
to show the reproducibility are the other limitations of
our study. Also, caution is warranted in the interpretation
of these results because the lysis method used
to remove red blood cells prior to immunophenotypic
analysis of white blood cells may have had an influence
on the results.[]
The relatively higher number of animals evaluated
in our experiment is the strength of our study. In ad dition, we preferred blood samples from rats instead
of patients treated with TBI to exclude the effects of
cancer and previous chemotherapy on the immune
system.
Conclusion
Radiation-induced immunodeficiency may not be explained
only by the decrease in lymphocyte cell number
but also by the relative increase in Treg cell number
that may present an additional role.
In spite of the underpowered nature of our study,
our findings might represent an empirical approach by
using lymphocyte subset counts as a biodosimetry to
predict the severity of the exposed radiation dose.
The next step for us may be the use of other established
serum protein combinations in addition to
lymphocyte counts, such as acute-phase C-reactive
protein, serum amylase activity, Flt3L, p53, p21, IL-6
to create a multiparametric radiation model for biodosimetry.
Also, attempts to find out whether there is any
functional change in Treg cells induced by IR should
be evaluated.
Peer-review: Externally peer-reviewed.
Conflict of Interest: The authors declare that there is no
conflict of interest.
Financial Support: None.
Acknowledgments: We thank Ege University Immunology
Laboratory and Laboratory Animals Research and Zafer
Karaguler for their valuable contributions to our study.
Authorship contributions: Everyone who is listed as an
author in this article has made a substantial, direct, intellectual
contribution to the work and takes public responsibility
for it.
References
Ossetrova NI, Condliffe DP, Ney PH, Krasnopolsky
K, Hieber KP, Rahman A, et al. Early-response
biomarkers for assessment of radiation exposure in
a mouse total-body irradiation model. Health Phys
2014;106(6):772-86.
Hall EJ. Acute effects of total-body irradiation. In: Hall
EJ, editor. Radiobiology for the Radiologist. Philadelphia,
PA: Lippincott Williams & Wilkins; 2000:124-35.
Goans RE, Holloway EC, Berger ME, Ricks RC. Early
dose assessment in criticality accidents. Health Phys
2001;81(4):446-9.
IAEA. Biological Dosimetry. In: Cytogenetic Analysis
For Radiation Dose Assessment: A Manual. Vienna:
International Atomic Energy Agency; 2001.
Hu S, Smirnova OA, Cucinotta FA. A biomathematical
model of lymphopoiesis following severe radiation
accidents-potential use for dose assessment. Health
Phys 2012;102(4):425-36.
Pecaut MJ, Nelson GA, Gridley DS. Dose and dose rate
effects of whole-body gamma-irradiation: I. Lymphocytes
and lymphoid organs. In Vivo 2001;15(3):195-208.
Qu Y, Zhang B, Liu S, Zhang A, Wu T, Zhao Y. 2-Gy
whole-body irradiation significantly alters the balance
of CD4+ CD25- T effector cells and CD4+ CD25+
Foxp3+ T regulatory cells in mice. Cell Mol Immunol
2010;7(6):419-27.
Nishikawa H, Sakaguchi S. Regulatory T cells in cancer
immunotherapy. Curr Opin Immunol 2014;27:1-7.
Qu Y, Jin S, Zhang A, Zhang B, Shi X, Wang J, et al. Gamma-
ray resistance of regulatory CD4+CD25+Foxp3+
T cells in mice. Radiat Res 2010;173(2):148-57.
Schaue D, Ratikan JA, Iwamoto KS, McBride WH.
Maximizing tumor immunity with fractionated radiation.
Int J Radiat Oncol Biol Phys 2012;83(4):1306-10.
Kachikwu EL, Iwamoto KS, Liao YP, DeMarco JJ, Agazaryan
N, Economou JS, et al. Radiation enhances regulatory
T cell representation. Int J Radiat Oncol Biol
Phys 2011;81(4):1128-35.
Kusunoki Y, Kyoizumi S, Hirai Y, Suzuki T, Nakashima
E, Kodama K, et al. Flow cytometry measurements
of subsets of T, B and NK cells in peripheral blood
lymphocytes of atomic bomb survivors. Radiat Res
1998;150(2):227-36.
Frey O, Bräuer R. Regulatory T cells: magic bullets for
immunotherapy? Arch Immunol Ther Exp (Warsz)
2006;54(1):33-43.
Zeman E. The Biological Bases of Radiation Oncology.
In: Gunderson L, editor. Clinical Radiation Oncology.
Elsevier: Phliedelphia; 2016. p. 30.
Vodicka I, Hanus J, Hradil J. Use of the adaptive classifier
for determination of LD50 in the acute radiation
disease. Radiobiol Radiother (Berl) 1989;30(2):143-9.
Ossetrova NI, Sandgren DJ, Blakely WF. Protein biomarkers
for enhancement of radiation dose and injury
assessment in nonhuman primate total-body irradiation
model. Radiat Prot Dosimetry 2014;159(1-4):61-76.
Goans RE, Holloway EC, Berger ME, Ricks RC. Early
dose assessment following severe radiation accidents.
Health Phys 1997;72(4):513-8.
Blakely WF, Salter CA, Prasanna PG. Early-response
biological dosimetry-recommended countermeasure
enhancements for mass-casualty radiological incidents
and terrorism. Health Phys 2005;89(5):494-504.
Ossetrova NI, Sandgren DJ, Gallego S, Blakely WF. Combined approach of hematological biomarkers and
plasma protein SAA for improvement of radiation
dose assessment triage in biodosimetry applications.
Health Phys 2010;98(2):204-8.
Dainiak N, Waselenko JK, Armitage JO, MacVittie
TJ, Farese AM. The hematologist and radiation casualties.
Hematology Am Soc Hematol Educ Program
2003:473-96.
Hu S, Cucinotta FA. Characterization of the radiation-
damaged precursor cells in bone marrow based
on modeling of the peripheral blood granulocytes response.
Health Phys 2011;101(1):67-78.
Inoue T, Hirabayashi Y, Mitsui H, Sasaki H, Cronkite
EP, Bullis JE Jr, et al. Survival of spleen colony-forming
units (CFU-S) of irradiated bone marrow cells in mice:
evidence for the existence of a radioresistant subfraction.
Exp Hematol 1995;23(12):1296-300.
Derer A, Frey B, Fietkau R, Gaipl US. Immune-modulating
properties of ionizing radiation: rationale for
the treatment of cancer by combination radiotherapy
and immune checkpoint inhibitors. Cancer Immunol
Immunother 2016;65(7):779-86.
Anacak Y, Kamer S, Sert F. Combining radiotherapy
with immunotherapy. Marmara Med J 2015;28(Special
Issue 1):40-4.
Walle T, Martinez Monge R, Cerwenka A, Ajona D,
Melero I, Lecanda F. Radiation effects on antitumor
immune responses: current perspectives and challenges.
Ther Adv Med Oncol 2018;10:1758834017742575.
Twyman-Saint Victor C, Rech AJ, Maity A, Rengan
R, Pauken KE, Stelekati E, et al. Radiation and dual
checkpoint blockade activate non-redundant immune
mechanisms in cancer. Nature 2015;520(7547):373-7.
Barker CA, Postow MA. Combinations of radiation
therapy and immunotherapy for melanoma: a review
of clinical outcomes. Int J Radiat Oncol Biol Phys
2014;88(5):986-97.
Kudo-Saito C, Schlom J, Camphausen K, Coleman
CN, Hodge JW. The requirement of multimodal therapy
(vaccine, local tumor radiation, and reduction of
suppressor cells) to eliminate established tumors. Clin
Cancer Res 2005;11(12):4533-44.
Einwallner E, Subasic A, Strasser A, Augustin D, Thalhammer
R, Steiner I, et al. Lysis matters: red cell lysis
with FACS Lyse affects the flow cytometric enumeration
of circulating leukemic blasts. J Immunol Methods
2013;390(1-2):127-32.
