Paediatrica Indonesiana

p-ISSN 0030-9311; e-ISSN 2338-476X; Vol.57, No.3(2017). p. 138-43; doi: http://dx.doi.org/10.14238/pi57.3.2017.138-43

Original Article

CD4+ T-cell, CD8+ T-cell, CD4+/CD8+ ratio,

and apoptosis as a response to induction phase
chemotherapy in pediatric acute lymphoblastic
leukemia
May Fanny Tanzilia1, Andi Cahyadi2, Yetti Hernaningsih3, Endang Retnowati3,
I Dewa Gede Ugrasena2

Abstract correlations between apoptosis and CD4+ (P=0.002; rs=-0.584),

Background Acute lymphoblastic leukemia (ALL) is a neoplastic and CD8+ (rs=-0.556; P=0.004), before chemotherapy. In
disease resulting from somatic mutation in the lymphoid progenitor addition, CD4+-delta and apoptosis-delta also had a significant
cells, often occuring in children aged 2-5 years, predominantly positive correlation (rs=0.478; P=0.016). However, no correlation
in males. Results from the induction phase of chemtherapy are was found between the CD4+/CD8+ ratio and apoptosis, before
used to measure success, but the failure remission rate is still or after chemotherapy.
high. Increased apoptosis of cancer cells, as induced by CD4+ Conclusion There are significantly lower mean CD4+, CD8+,
and CD8+ T-cells, is an indicator of prognosis and response to and CD4+/CD8+ ratio after chemotherapy than before. Also,
chemotherapy. there are significant correlations between CD4+-delta and
Objective To assess for correlations between CD4+, CD8+, or apoptosis-delta, as well as between apoptosis and CD4+, CD8+,
CD4+/CD8+ ratio to the chemotherapy induction phase response and CD4+/CD8+ ratio, before chemotherapy. The CD4+, CD8+,
(i.e., apoptosis) in pediatric ALL patients. and CD4+/CD8+ ratio can be used to predict apoptosis before
Methods This observational analytical cohort study was done chemotherapy. In addition, CD4+-delta can be used to predict
in 25 pediatric ALL patients. Whole blood (3 mL) with EDTA apoptosis-delta as a response to induction phase chemotherapy in
anticoagulant were used to measure absolute counts of CD4+, pediatric ALL. [Paediatr Indones. 2017;57:138-43 doi: http://
CD8+, and CD4+/CD8+ ratio. Peripheral blood mononuclear cells dx.doi.org/10.14238/pi57.3.2017.138-43 ].
(PBMC) were examined for apoptosis. The principle of CD4+,
CD8+ examination was bond between antigens on the surface of Keywords: ALL; CD4+; CD8+; apoptosis
the leukocyte in the blood with fluorochrome labeled antibodies
in the reagents, while the principle of apoptosis examination
was FITC Annexin V will bonds with phosphatidylserine that
moves out of the cell when the cell undergoes apoptosis, then
intercalation with propidium iodide (PI). All examination were
detected by flow cytometry BD FACSCalibur.
From the Clinical Pathology Specialization Program1, Department of Child
Results Subjects were 25 newly-diagnosed, pediatric ALL patients
Health2, and Department of Clinical Pathology3, Airlangga University
(64% males and 36% females). Most subjects were 3 years of age Medical School/Dr. Soetomo Hospital, Surabaya, East Java, Indonesia.
(20%). Numbers of CD4+ and CD8+ cells, as well as CD4+/
CD8+ ratio were significantly decreased after chemotherapy. Reprint requests to: May Fanny Tanzilia, Sukolilo Dian Regency, Makmur
However, apoptosis was not significantly different before and 3/8, Surabaya 60111, East Java, Indonesia. Email: dr.mayfannytanzilia@
after chemotherapy (P=0.689), There were significant negative gmail.com.

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 May F. Tanzilia et al: Response to induction phase chemotherapy in acute lymphoblastic leukemia

A
cute lymphoblastic leukemia (ALL) is a who had never received chemotherapy, and planned
neoplastic disease resulting from somatic to undergo induction phase chemotherapy regularly
mutation in the lymphoid progenitor for 6 weeks. The exclusion criteria were ALL patients
cells in the bone marrow, often occurring with a history of HIV/AIDS or had infections as
in children aged 2-5 years, and predominantly in indicated by fever and other signs. Healthy patients
males. Diagnosis of ALL is based on finding ≥ control were obtained from healthy volunteers aged 1
25% lymphoblasts of blood smear or bone marrow month to 16 years who had investigated no history of
aspiration (BMA) evaluation. Outcome criteria was certain diseases and seem good condition during the
made as follows, < 5% lymphoblasts was belonging to examination was done. This study was approved by
remission, and > 5% was belonging to non remission, the Ethics Committee of our institution and written
including partial remission.1-5 informed consent was obtained from all patients’
Failure of apoptosis and the immune response parents.
causes uncontrolled growth of cancer cells. The Clinical characteristics including age, sex,
progression of ALL has been correlated with hemoglobin (Hb) level, platelet count, and leukocyte
quantitative and qualitative (function) host immune count were obtained at diagnosis (before chemotherapy)
responses. Cluster differentiation 8+ (CD8+) T-cell and after induction phase chemotherapy. Whole blood
are an effector cell resulting an apoptosis tumor (3 mL) with EDTA anticoagulant were used to measure
cells by cytotoxic or cytolytic effects. Anti-tumor absolute counts of CD4+, CD8+, and CD4+/CD8+
mechanisms of CD4+ T-cells are not fully understood. ratio. Reagents used for CD4+, CD8+ examination
The CD4+ T-cells have no cytotoxic or cytolytic was BD Multitest CD3FITC/CD8PE/CD45PerCP/
effects. Many cytokines produced by CD4+ T-cells are CD4APC. The principle of CD4+, CD8+examination
needed for the development of CD8+ effector cells. was to detect bonds between antigens on the surface of
Tumor necrosis factor (TNF) and interferon-gamma the leukocyte present in the blood with fluorochrome
(IFN-gamma) secreted by activated CD4+ T cells labeled antibodies present in the reagents. Peripheral
will induce expression of major histocompatibility blood mononuclear cells (PBMC) were examined
complex I (MHC I) and induce CD8 + T-cell for apoptosis. The FITC Annexin V and propidium
activity to lyse tumor cells.6-10 The induction phase iodide (PI) were used for apoptosis examination. The
of chemotherapy is usually used as a measure of principle of apoptosis examination was FITC Annexin
successful chemotherapy, but remission failure rates V will bonds with phosphatidylserine that moves
are still high.10 Increased apoptosis of cancer cells out of the cell when the cell undergoes apoptosis,
induced by CD4+ or CD8+ T-cells can be used as then intercalation with propidium iodide (PI). The
one indicator of prognosis and response to induction FITC Annexin V can identify early apoptosis, late
phase chemotherapy in pediatric ALL patients.9 The apoptosis and necrosis based on nucleus changes.
aim of this study was to assess for correlations between Propidium Iodide (PI) was used to distinguish late
apoptosis and CD4+, CD8+, and CD4+/CD8+ ratio, apoptosis and necrosis with early apoptosis. All
before and after the induction phase of chemotherapy examination were detected by flow cytometry BD
in pediatric ALL patients. FACSCalibur performed before and after induction
phase chemotherapy, at the Department of Clinical
Pathology, Airlangga University Medical School/
Methods Dr. Soetomo Hospital, Surabaya.
Comparison of Hb levels before and after
This observational, analytical, cohort study included induction phase chemotherapy was evaluated by
25 new cases of pediatric ALL patients who were paired T-test, while leukocyte counts, platelet counts,
diagnosed based on bone marrow aspiration at the CD4+, CD8+ cells, CD4+/CD8+ ratio, and apoptosis
Department of Pediatrics, Airlangga University before and after induction phase chemotherapy were
Medical School/Dr. Soetomo Hospital, Surabaya, from evaluated by Wilcoxon Signed Rank test. Correlations
July to December 2016. The inclusion criteria were of absolute count of CD4+, CD8+ cells, CD4+/CD8+
new cases of pediatric ALL aged 1 month to 16 years ratio with apoptosis were evaluated by Spearman’s

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 May F. Tanzilia et al: Response to induction phase chemotherapy in acute lymphoblastic leukemia

correlation test. CD4+ -delta was defined as difference (P=0.014) but platelet count was signigficantly higher
between number of CD4+ before and after induction (P=0.000), between before and after induction phase
phase chemotherapy, as well as CD8+-delta, CD4+/ of chemotherapy. Mean numbers of CD4+ and CD8+
CD8+-delta, and apoptosis delta. Results with P cells, as well as CD4+/CD8+ ratio were significantly
values ≤ 0.05 were considered to be statistically decreased after induction phase chemotherapy.
significant. Mean numbers of CD4+ before induction phase
chemotherapy was 3,060.24 (SD 4660.03), after
induction phase chemotherapy was 887.64 (1531.33).
Results Mean numbers of CD8+ before induction phase
chemotherapy was 3,084.76 (SD 4535.51), after
During the study period, 41 new cases of induction phase chemotherapy was 1,647.28 (SD
pediatric ALL patients fulfilled the inclusion criteria, 3644.99). Mean numbers of CD4 +/CD8 + ratio
but 16 patients dropped out, of whom 11 patients before induction phase chemotherapy was 1.12 (SD
died and 5 patients did not complete the induction 0.67), after induction phase chemotherapy was 0.65
phase of chemotherapy. Hence, our cohort study of (SD 0.61). However, apoptosis did not significantly
25 new cases of pediatric ALL patients consisted of decrease after chemotherapy (P=0.689), before
16 males (64%) and 9 females (36%), with a 1.78:1 induction phase chemotherapy was 18.19 (SD 19.82)
ratio (Table 1). Many of our subjects were 3 years of and after induction phase chemotherapy was 14.09
age (20%). Leucocyte count was significantly lower (SD 10.85) (Table 2).

Table 1. Clinical characteristics of study subjects before and after

induction phase chemotherapy
Characteristics N=25 P value
Gender, n
Male 16
Female 9
Age, n
1-5 years 13
6-10 years 7
≥ 10 years 5
Leucocyte count, x103 μL 0.014*
Before
Mean (SD) 52.68 (120.45)
Median (range) 8.81 (1.20 – 585.00)
After
Mean (SD) 7.58 (10.78)
Median (range) 5.12 (0.79 – 56.00)
Hb concentration, g/dL 0.165
Before
Mean (SD) 10.30 (3.13)
Median (range) 9.48 (5.00 – 16.00)
After
Mean (SD) 11.35 (2.02)
Median (range) 11.30 (7.60 – 15.40)
Thrombocyte count, x103 μL 0.000*
Before
Mean (SD) 62.26 (54.17)
Median (range) 42.6 (5.00 – 204.00)
After
Mean (SD) 190.33 (119.07)
Median (range) 177.00 (18.80 – 447.00)
*significant if P ≤ 0.05, Before=before induction phase chemotherapy, After=after induction
phase chemotherapy

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 May F. Tanzilia et al: Response to induction phase chemotherapy in acute lymphoblastic leukemia

Table 2. Comparison between CD4+, CD8+ cells, CD4+/CD8+ ratio, and apoptosis, before and after
induction phase chemotherapy in pediatric ALL patients
Variables Time Mean (SD) Median (range) P value
CD4+, cells Before 3,060.24 (4660.03) 1751.00 (210.0 – 23,016.0 0.000*
After 887.64 (1531.33) 199.00 (4.0 – 5,966.0)
CD8+, cells Before 3,084.76 (4535.51) 1820.00 (341.0 – 18,541.0) 0.004*
After 1,647.28 (3644.99) 423.00 (52.0 – 17,859.0)
CD4+/CD8+ ratio Before 1.12 (0.67) 1.03 (0.33 – 2.91) 0.004*
After 0.65 (0.61) 0.44 (0.06 – 2.31)
Apoptosis Before 18.19 (19.82) 10.83 (0.26 – 80.58) 0.689
After 14.09 (10.85) 10.34 (2.86 – 49.17)
*significant if P ≤ 0.05, Before=before induction phase chemotherapy, After=after induction phase chemotherapy

Table 3. Comparison of apoptosis between pediatric ALL patients and healthy children before
and after induction phase chemotherapy
Apoptosis
Variables P value
Mean (SD) Median (range)
ALL patients (before chemotherapy) 18.19 (19.82) 10.83 (0.26-80.58) 0.683
Healthy control patients 16.21 (3.52) 17.73 (12.19-18.71)
ALL patients (after chemotherapy) 14.09 (10.85) 10.34 (2.86-49.17) 0.316
Healthy control patients 16.21 (3.52) 17.73 (12.19-18.71)

Table 4. Correlation between apoptosis and CD4+, CD8+ cells, and CD4+/CD8+ ratio before
and after induction phase chemotherapy
Correlation with apoptosis before Correlation with apoptosis after
Variables chemotherapy chemotherapy
rs P value rs P value
CD4+ cells - 0.584 0.002 0.081 0.701
CD8+ cells - 0.556 0.004 - 0.105 0.619
CD4+/CD8+ ratio - 0.117 0.579 0.040 0.849

A comparison of apoptosis was done in ALL

patients and healthy control subjects. We found no Discussion
significant differences in apoptosis between the two
groups either before or after chemotherapy (Table Acute lymphoblastic leukemia is a hematological
3). malignancy that often occurs in children, and
We also assessed for correlations between comprises 25–30% of all pediatric malignancies.
apoptosis, before and after chemotherapy, and The highest incidence is in 2–5-year-olds, and
CD4+, CD8+ absolute counts, and CD4+/CD8+ predominantly in boys. Our cohort study of 25 new
ratio. There were significant, negative correlations cases of pediatric ALL patients consisted of 16 (64%)
between apoptosis and CD4+, as well as apoptosis and males and 9 (36%) females, with 1.78 : 1 ratio. Many
CD8+, before chemotherapy. However, there was no subjects were 3 years of age (20%) (Table 1).
significant correlation between CD4+/CD8+ ratio and There were significant differences in leukocyte
apoptosis, before or after chemotherapy (Table 4). (P=0.014) and thrombocyte (P=0.000) counts, before
There was a significant correlation between and after induction phase chemotherapy. Uncontrolled
+
CD4 -delta and apoptosis-delta after induction phase lymphocyte proliferation and defective apoptosis
chemotherapy. However, we observed no significant in ALL patients caused leukocytosis dominated by
correlation between apoptosis-delta and CD8+-delta lymphoblasts. Infiltration of hematopoetic cells by
or CD4+/CD8+-delta (Table 5). leukemic cells accumulated in the bone marrow

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 May F. Tanzilia et al: Response to induction phase chemotherapy in acute lymphoblastic leukemia

causes anemia and thrombocytopenia. 1,13 After higher than apoptosis in the healthy control group
chemotherapy, leukocyte count decreased, while Hb (Table 3).
level and thrombocyte count increased. Evaluation There was a negative, significant correlation
of bone marrow aspiration after induction phase between apoptosis and CD4+ and CD8+ cells before
chemotherapy showed that all the patients were in chemotherapy (Table 4). This finding indicates that
remission, as determined by leukemic cell clearance greater numbers of CD4+ and CD8+ cells result in
from the bone marrow, mainly in the first 2 weeks after decreased apoptosis. We noted that not only the
induction phase chemotherapy.13 number of CD4+ and CD8+ cells, but their function,
Mean CD4+, CD8+ cells, and CD4+/CD8+ was also important in immune response. The number
ratio were significantly decreased after induction and function of CD4 + and CD8 + cells before
phase chemotherapy (Table 2). Verma et al. reported chemotherapy were correlated with subjects’ response
a significant decrease in B cells, T cells, and NK to chemotherapy and improved survival.10
cells approximately 2 weeks after chemotherapy.14 Apoptosis had no significant correlation
Decreased CD4+/CD8+ ratio after chemotherapy in with CD4 + /CD8 + ratio, either before or after
this study (P=0.004) was due to the larger decrease chemotherapy. Similarly, Dewyer et al. suggested
in CD4+ cells than that in CD8+ cells. Recovery that CD4 + /CD8 + ratio did not correlate with
of CD4+ cells after chemotherapy was slower than tumor response in induction phase chemotherapy.21
recovery of CD8+ cells. Chemotherapy decreases No correlation between CD4+ or CD8+ cells and
the CD4+ and CD8+ cells by increasing regulatory apoptosis after induction phase chemotherapy, may
T cells (T reg), which supress the immune response have been due to decreases of CD4+, CD8+ cells,
by downregulating IL-2, and upregulating IL-10 and and apoptosis after chemotherapy.
TGF-beta. Although IL-10 and TGF-beta are strong, There was a significant correlation between
immunosupressive factors, IL-2 is an important CD4 + - delta (number of CD4 + cells before
immune regulating factor. It is produced by T helper chemotherapy minus the number of CD4+ cells after
cells, which increase T cell proliferation, NK cell chemotherapy) and apoptosis-delta (Table 5). We
activity, and B cell antibody secretion. Past studies found that decreased CD4+ cells lead to decreased
have shown that IL-2 concentration in ALL patients apoptosis. The CD4+ cells had no cytotoxic or cytolytic
is low.15,16 effect on tumor cells, but many cytokines produced by
After chemotherapy, apoptosis was not CD4+ cells are needed for the development of CD8+
significantly decreased (P=0.689). In contrast, into effector cells.6-10
Laane et al. found an increase in apoptosis after The limitations of this study included the lack of
chemotherapy.17 Firstly, our results may have been an extended time to recognize the possibility of future
caused by: 1) sampling time outside the window of relapse or resistance to chemotherapy drugs, apoptosis
maximal effect on apoptosis time (24 - 72 hours). sample preparation was not accompanied by a specific
Liu et al. reported a significant increase in apoptosis marker for lymphocytes, so a series of monocytes may
of lymphoblasts > 24 hours after induction phase have been included, and the sampling time among
chemotherapy in pediatric ALL patients, rather than patients after chemotherapy varied, potentially
in the early hours after chemotherapy.18 Secondly, effecting the decreased apoptosis.
chemotherapy can cause necrosis, so lymphoblasts Comparing the values before and after induction
may not have been detected as apoptotic bodies.19 phase of chemotherapy we conclude that: 1) CD4+
Third, the subjects may have had chemotherapy and CD8+ count cells not significantly higher, 2) there
drug resistance, although 100% of patients entered is no difference in apoptosis, 3) there was a negative
remission,20 and forth, anti-apoptotic proteins levels correlation between apoptosis and CD4+ and CD8+
(e.g., BCL-2, BCL-XL, etc.) in the patients were higher cells before induction phase chemotherapy in pediatric
than pro-apoptotic proteins levels (e.g., BAX, BOK, ALL patients, but no correlation after induction
BCL-Xs, BID, BAD, or Noxa).18 Lymphoblast cells in phase chemotherapy, 4) there is no correlation
ALL patients are more fragile than in healthy children, between CD4+/CD8+ ratio and apoptosis, 5) there
thus, apoptosis in ALL patients was not significantly is a significant correlation between CD4+-delta and

142 • Paediatr Indones, Vol. 57, No. 3, May 2017

 May F. Tanzilia et al: Response to induction phase chemotherapy in acute lymphoblastic leukemia

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ALL patients. significance of immune function parameters (CD4, CD8
Suggestions for future research also include and CD4/CD8 ratio) in Sudanese patients with chronic
determining the best time of sampling after lymphocytic leukemia. Int J Multidisciplinary Curr Res.
chemotherapy (24 hours, 36 hours, 48 hours, or 72 2016;4:650-3.
hours) to obtain results with significantly increased 11. Technical Data Sheet BD Multitest CD3 FITC/ CD8 PE/
apoptosis, compared to what our study showed, and CD45 PerCP/ CD4 APC Reagent, BD Pharmingen 2012.
to assess the profile of the percentage of T reg and 12. Technical Data Sheet FITC Annexin V Apoptosis Detection
expression of cytokines in ALL patients before and Kit I, BD Pharmingen 2008.
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Conflict of Interest in children. PLoS One. 2015;10:1-10.
14. Verma R, Foster RE, Horgan K, Mounsey K, Nixon H,
None declared. Smalle N, et al. Lymphocyte depletion and repopulation after
chemotherapy for primary breast cancer. Breast Cancer Res.
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