Journal of Cancer Therapy, 2013, 4, 1382-1390
http://dx.doi.org/10.4236/jct.2013.48164 Published Online October 2013 (http://www.scirp.org/journal/jct)
Is the CD4/CD8 Ratio an Effective Indicator for Clinical
Estimation of Adoptive Immunotherapy for
Cancer Treatment?
Goki Shindo1, Takayoshi Endo1, Masamitsu Onda1, Shigenori Goto2, Yoju Miyamoto3, Toru Kaneko3
1Cardiothoracic Department, Meditopia Numazu Clinic, Numazu, Japan; 2Department of Immunotherapy, Seta Clinic Tokyo, Tokyo,
Japan; 3Department of Immunotherapy, Seta Clinic Shin-Yokohama, Yokohama, Japan.
Received September 12th, 2013; revised October 10th, 2013; accepted October 18th, 2013
Copyright © 2013 Goki Shindo et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Background: The importance of immunotherapy in cancer treatment has been increased owing to its non-toxicity and
application to personalized medicine. However, precise estimation indices of immunotherapy have yet to be established.
To determine effective evaluation indices of immunotherapy for cancer treatment, we analyzed the CD4/CD8 ratio un-
der various conditions in clinical patients with advanced cancer. Patients and Methods: Thirty-four patients who un-
derwent one course of adoptive activated immunotherapy with or without additional conventional chemotherapy were
enrolled. Before and after one course of immunotherapy, changes in the CD4/CD8 ratio were estimated by flow cyto-
metry. Results: All patients showed a tendency toward a decrease in the CD4/CD8 ratio during a 3-month period after
one course of adoptive activated T lymphocyte immunotherapy. Patients who had undergone prior surgery showed a
remarkable increase in CD8 T cell number. Thus, adoptive activated T lymphocyte immunotherapy improves immu-
nological ability against cancer invasion. The Eastern Cooperative Oncology Group’s performance status during one
course of immunotherapy was significantly improved in the antecedent surgery group, with no evidence of improved PS
in the non-antecedent surgery group. Patients with an increased CD4/CD8 ratio (n = 6) may have a worse outcome dur-
ing adoptive activated T lymphocyte immunotherapy even with an additional course of immunotherapy. Improved ac-
tuarial survival rate of patients in the antecedent surgery group showed significant long-term benefit compared to those
in the non-antecedent surgery group (p = 0.0298), as previously reported. Conclusion: The CD4/CD8 ratio is a signifi-
cant indicator of outcome of adoptive activated T lymphocyte immunotherapy.
Keywords: Immunotherapy; Adoptive Activated T Lymphocyte therapy; Flow Cytometry; Performance Status;
Advanced Cancer
1. Introduction
Immunotherapy plays an important role in cancer treat-
ment because of its non-toxic therapeutic effect and be-
cause it can be highly personalized by using autologous
activated lymphocytes obtained from patients. Recent
technological progress in flow cytometry using laser
beams directed onto a hydrodynamically focused stream
of liquid has made it possible to count suspended cells
and molecular particles on the cell surface at a rate of
thousands of particles per second.
Since Rosenberg et al. first applied lymphokine-acti-
vated killer (LAK) cell immunotherapy with direct injec-
tion of interleukin 2 (IL-2) to patients for clinical use in
1986 [1], despite subsequently observed adverse effects
of the original procedure, immunotherapy has been de-
veloped steadily over the past 20 years as a personalized
non-toxic therapy for cancer patients.
Egawa established modified adoptive immunotherapy
for clinical use in 1999 without direct injection of IL-2
into the human body [2]. Since then, supportive evidence
of the clinical use of immunotherapy for various types of
cancers has been reported [3-6]. However, the effective
response rate according to the complete/partial response
outlined in the RECIST criteria was determined to be low,
adverse reactions were rarely observed, and patients
generally showed a good Eastern Cooperative Oncology
Group (ECOG) performance status (PS) during the
course of immunotherapy. A recent phase 3 trial using
Egawa’s modified adoptive immunotherapy in combina-

Is the CD4/CD8 Ratio an Effective Indicator for Clinical Estimation of Adoptive Immunotherapy for Cancer Treatment? 1383
tion with chemotherapy also showed important signifi-
cant additive effects of immunotherapy compared to che-
motherapy alone in lung cancer patients at all clinical
stages, as well as a positive effect on quality of life (QOL)
until near the time of death in advanced lung cancer pa-
tients [7]. To date there have been few reports on the
efficacy of immunotherapy using accurate estimation
indices [8,9], especially for modified adoptive immuno-
therapy. On the basis of recent advances in flow cytome-
try, we aimed to identify effective estimation indices for
evaluating the efficacy of immunotherapy, either alone or
combined with chemotherapy, in the course of cancer
treatment. We chose the CD4/CD8 ratio as an indicator
of immunological response before and after one course
of adoptive immunotherapy treatment in patients with
advanced clinical stages of disease.
2. Patients and Methods
2.1. Patient Characteristics
Thirty-four patients with advanced stage III or IV cancer
who underwent one course of adoptive activated immu-
notherapy with measurement of their CD4/CD8 ratio
before and after immunotherapy at Meditopia Numazu
Clinic, Seta Clinic Tokyo, and Seta Clinic Shin-Yoko-
hama between December 2008 and June 2012 were en-
rolled. Written informed consent was obtained from all
patients before starting immunotherapy.
The primary sites of disease in the 34 patients are
shown in Table 1(A). Fourteen (41.2%) patients had can-
cer of the lung and 9 (26.5%) had cancer of the ovary;
cancers in the remaining patients included colorectal and
stomach. Patient characteristics are summarized in Table
1(B). To clarify the effect of antecedent surgery which
was reported previously to be one of the most important
benefits for patients with advanced cancer [10], patients
were divided into two groups—antecedent surgery and
non-antecedent surgery groups—and the CD4/CD8 ratio
was analyzed separately. All patients in the antecedent
surgery group underwent curative surgery in accordance
with the Japanese guidelines for surgical procedures for
cancer in the organ affected and were found to have Un-
Table 1. Primary cancer sites and patient characteristics.
(A) Primary cancer sites
Cancer site Total (%) Antecedent surgery group Non-antecedent surgery group
lung 14(41.2) 3 11
ovary 9(26.5) 9 0
uterus 2(5.9) 1 1
colorectal 2(5.9) 2 0
melanoma 2(5.9) 2 0
stomach 1(3.0) 0 1
pancreas 1(3.0) 0 1
kidney 1(3.0) 1 0
throat 1(3.0) 1 0
Multiple myeloma 1(3.0) 0 1
total 34(100) 19 15
(B) Patient characteristics in each group
Total (n = 34) Antecedent surgery group
(n = 19)
Non-antecedent surgery
group (n = 15)
P value (surg. vs
non-surg.)
age(y/o) 58.6(28 - 80) 60’ (41.2%) 58.6(28 - 78) 58.5(30 - 80) N. S.
gender M(%)/F(%) 12(35.3)/22(64.7) 2(10.5)/17(89.5) 10(66.7)/5(33.3) 0.0025
stage III (%)/IV(%) 8(22.5)/26(76.5) 5(26.3)/14(73.7) 4(26.7)/11(73.3) N. S
PS 0(%)/1(%) 24(70.6)/10(29.4) 14(73.7)/5(26.3) 10(66.7)/5(33.3) N. S.
time interval (mos)
OP(Rec)-IT 28.16 2.7 0.0002
combination IT + CT 25/34(73.5%) 12/19(63.2) 13/15(86.7) N. S.
I
T: immunothrapy, CT: chemotherapy, OP-IT: surgery to IT, Rec-IT: recurrence of cancer to IT.

Is the CD4/CD8 Ratio an Effective Indicator for Clinical Estimation of Adoptive Immunotherapy for Cancer Treatment? 1384
ion for International Cancer Control TNM stage 3 or 4
disease.
The ECOG PS at the first, fourth, and sixth admini-
stration of αβ Τ cell infusion was estimated. When pa-
tients were stable and tolerated one course of immuno-
therapy, immunotherapy was continued periodically with
a 2- to 4-week interval for as long as possible, according
to patients’ request and their condition.
There were no significant differences in age, ECOG
PS before starting immunotherapy, or proportion of pa-
tients undergoing combination therapy involving che-
motherapy between the two groups. By contrast, there
were significant differences in gender (more female pa-
tients in the antecedent surgery group, especially in the
case of ovarian cancer) and the time interval before ini-
tiation of one course of immunotherapy (16.0 vs. 2.7
months).
2.2. Adoptive Activated T Lymphocyte
Immunotherapy
Adoptive activated T lymphocyte immunotherapy is a
non-specific therapy without sensitization by cancer-
specific peptides. During the culture period there is pro-
moted killer activities and a decrease in the number of
regulatory T lymphocytes which suppress tumor immu-
nity. Peripheral blood lymphocytes and mononuclear
cells are harvested by centrifugation and >1 × 106 har-
vested cells are cultured with an immobilized anti-CD3
antibody and IL-2 for 14 days, obtaining >5 × 109 lym-
phocytes on average. This method of therapy is termed
αβ T immunotherapy. The cultured lymphocytes consist
of 61% ± 15% CD8 T cells and 30% ± 15% CD4 T cells,
an average CD4/CD8 ratio of 0.8 (0.66 - 2.9), and a small
percentage of natural killer (NK) cells and NK T cells,
indicating that CD8 T lymphocytes proliferate more at a
greater rate than CD4 T lymphocytes during the 2-week
culture period [11,12]. The precise processing of these
cells is described elsewhere [10-12]. Expanded CD8-rich
T lymphocytes were infused intravenously once every 2
weeks up to 6 times for 3 months during one course of
immunotherapy.
2.3. Flow Cytometry
Heparinized peripheral blood samples were collected for
each patient before and after one course of immunother-
apy and β, CD4+ and CD8+ T cell counts, and the CD4/
CD8 ratio were measured by flow cytometry [13,14].
Prior to this study, we determined the influence of stor-
age temperature and time between peripheral blood col-
lection and assay initiation on the results of flow cy-
tometry, and we established that consistent results were
obtained when peripheral blood was stored at 18˚C -
22˚C for 1 day before phenotypic analysis. Phenotypic
analysis of peripheral blood mononuclear cells was car-
ried out by whole-blood staining with Optlyse C lysis
solution. The absolute numbers of αβ, CD4 and CD8 T
cells were determined using Flow-Count™ fluorosphere
internal standard beads. These reagents and monoclonal
antibodies against CD3, CD4, CD8 and TCR pan αβT
cells were purchased from Beckman Coulter (Brea, CA).
Cytomics FC 500 and/or Gallios flow cytometer (Beck-
man Coulter) was used for data acquisition and the data
were analyzed with CXP and/or Kaluza software (Beck-
man Coulter).
2.4. Statistical Analysis
The chi-squared test was used to compare patient demo-
graphic, clinical, and laboratory variables, with the Mann-
Whitney U test used for qualitative variables and the
Mantel-Haenszel procedure used for analyzing multiple 2
× 2 contingency tables. Fisher’s Student t test was used
for quantitative variables in the case of equal variance of
the two comparable populations, and Welch’s t test was
used when the assumption of equality of the variances
was dropped. Patient survival was analyzed using the
Kaplan-Meier method. Differences in survival were de-
termined using the log-rank test for univariate analysis.
Statcel Ver. 2 for Windows Excel software was used for
analysis.
3. Results
During one course of immunotherapy, six intravenous
infusions of αβ T cells (3 - 10 × 109 cells) were infused at
intervals of approximately 2 weeks. Overall, 73% of all
patients received additional conventional therapy when
indicated, with an appropriate interval between immuno-
therapy and chemotherapy to avoid the adverse cytocidal
or cytostatic effects of immunotherapy. There were no
significant differences in the proportion of patients with
combination therapy involving chemotherapy between
the antecedent and non-antecedent surgery groups (Table
1B).
Among a group of Japanese healthy adults (n = 32)
serving as control subjects, the absolute number (mean ±
SD) of CD3+αβTCR+, CD4+, and CD8+ T lymphocytes
measured by cytometry was 1247 ± 320，806 ± 199, and
508 ± 181 cells/μL, respectively, and the CD4/CD8 ratio
was 0.8 (0.66 - 2.90). The proportions of CD4+ and
CD8+ T cells were 64.6% and 40.7%, respectively. The
phenotypic proportions of CD4+ and CD8+ T cells in the
total cohort of the 34 patients before the start of adoptive
immunotherapy were similar to (66.2%) and lower than
(28.6%) the values of the healthy controls, respectively
(Table 2(A)).
Peripheral blood samples were obtained before the
first, fourth, and sixth infusions of αβ Τ cells. The num-

Is the CD4/CD8 Ratio an Effective Indicator for Clinical Estimation of Adoptive Immunotherapy for Cancer Treatment? 1385
ber of patients with αβ Τ cell numbers <0.3 × 107 in pe-
ripheral blood at the beginning of each infusion was
compared between the two groups, and a statistically
significant difference was observed at the first and sixth
infusion time using Fisher’s exact probability test and the
Mantel-Haenszel procedure (p = 0.0363) (Table 2(B)).
This finding suggests that patients’ immunological con-
dition was superior in the antecedent surgery group fol-
lowing one course of immunotherapy, even though the
same amount of augmented αβ Τ cells ((3 - 10) × 109)
was infused each time in both groups.
The change in the CD4/CD8 ratio before and after one
course of immunotherapy is shown in Tabl e 3. Among
the total cohort of 34 patients there was no significant
difference in the CD4/CD8 ratio (p = 0.0766). However,
the antecedent surgery group showed a significant de-
crease in the ratio (p = 0.0177), although the non-antece-
dent surgery group showed no significant change (p =
0.3050) despite a tendency for decrease after one course
of immunotherapy (Table 3(A), Figure 1). Further analy-
sis of the number of patients with a decrease or increase
in CD4 and CD8 T cell numbers after one course of im-
munotherapy revealed that the antecedent surgery group
had a significant increase in CD8 T cell number (p =
0.0002) but no change in CD4 T cell number compared
with the non-antecedent surgery group (p = N. S.),
whereas the non-antecedent surgery group showed a sig-
nificant increase in CD8 T cells number (p = 0.0134)
(Table 3(B)). These results (Table 3) reveal that the de-
creasing tendency of the CD4/CD8 ratio in the antece-
dent surgery group depended mostly on an increase in
CD8 T cells and not on CD4 T cell number. As CD8 T
cells are cytotoxic and kill cancer cells, a significant in-
crease in the number of CD8 T lymphocytes may act to
improve the anticancer suppressive activity of the pa-
tient’s immune system during immunotherapy.
Many physicians including Egawa, the founder of
Japanese clinical immunotherapy who conducted the first
clinical investigation of immunotherapy for cancer on
Figure 1. Change in the CD4/CD8 ratio before and after one
course of immunotherapy.
Table 2. Phenotypes of healthy control and advanced cancer patients.
(A) Comparison of peripheral blood immunocyte phenotypes between health control and advanced cancer patients.
phenotypes healthy control (N = 32) patients before IT (N = 34) (% of control)patients after one course of IT (N = 34) (% of control)
CD3+ T 1247 ± 320 cells/μL 927.5 ± 387 cells/μL (74.4%) 1032 ± 356 cells/μL (82.8%)
CD4+ T 806 ± 199 614.5 ± 252(76.2%) 611.5 ± 248(75.9%)
CD8+ T 508 ± 181 266 ± 158(52.3%) 358.9 ± 204.7(70.6%)
CD4/CD8 0.6(0.66 ~ 2.9） 2.43 ± 1.7(300%) 1.80 ± 1.1(225%)
(B) Number of patients with αβ Τ cells number less than 0.3 × 107 at the beginning of each infusion time.
IT infusion Antecedent surgery group
(n = 19) Non-antecedent surgery group (n = 15) P value
1st 4(21.1%) 7(46.7%) 0.0068
4th 3(15.8%) 1(6.7%) 0.5014
6th 2(10.5%) 7(46.7%) 0.00005
Mantel-Haenszel procedure among 3 levels 0.0363
IT, immunotherapy.

Is the CD4/CD8 Ratio an Effective Indicator for Clinical Estimation of Adoptive Immunotherapy for Cancer Treatment? 1386
Table 3. Change in CD4/CD8 ratio.
(A) Change in the CD4/CD8 ratio before and after immunotherapy
Total (n = 34) Antecedent surgery group (n = 19)Non-antecedent surgery group (n = 15)
before after before after before after
mean 2.4293 1.8029 2.1078 1.56 2.8364 2.091
SD 1.7293 1.1112 0.8099 0.5605 2.4259 1.5183
P value 0.0766 (N. S.) 0.0177 0.3050 (N. S.)
(B) Comparison between patients who decreased or increased their CD4/CD8 cell counts or
the CD4/CD8 ratio after one course of immunotherapy
Antecedent surgery group Non-antecedent surgery group
CD4
decrease 10 7
increase 9 8
P value N. S. N.S.
CD8
decrease 3 4
increase 16 11
P value 0.0002 0.0134
CD4/CD8
decrease 14 13
increase 5 2
P value 0.0042 0.00007
his brother, have suggested that patients can maintain
ordinary daily life activities during immunotherapy until
close to the end of their life. To clarify this postulation,
in the present study we analyzed PS during one course of
adoptive activated immunotherapy [7,11,12]. Table 4(A)
shows analysis of the number of patients in whom PS
improved after one course of immunotherapy (Figure 2).
Among all patients there was a significant improvement
in PS (p = 0.0077). However, in separate analyses, the
non-antecedent surgery group did not show any signifi-
cant improvement, whereas PS improved in the antece-
dent surgery group; there was a significant difference
between the two groups (p = 0.0179).
Next, the changing pattern of the CD4/CD8 ratio was
analyzed by subdividing all patients into two groups: the
“downward” group in which the CD4/CD8 ratio de-
creased while CD4 T cells were almost unchanged and
CD8 T cells increased after immunotherapy and the
“upward” group in which there was an increased CD4/
CD8 ratio. PS in the upward group showed a signifi-
cantly worse tendency (p = 0.025), whereas it was not sig-
nificantly changed in the downward group (p = 0.2393)
during immunotherapy (Table 4(B)). Therefore, the in-
crease in the CD4/CD8 ratio after immunotherapy may
imply worse outcome during immunotherapy.
Median survival time in the antecedent and non-ante-
cedent surgery groups were 33.2 months and 8.9 months,
respectively (Figure 3). Analysis of the actuarial survival
rate of the two groups after one course of immunotherapy
with subsequent immunotherapy alone or in combination
with chemotherapy revealed that the antecedent surgery
group had a significantly superior response, as deter-
mined by the log-rank test (p = 0.0298), even though
there was a discrepancy in gender distribution between
the two groups. This finding confirmed the result of a
previous study showing that surgery before immuno-
therapy confers an important benefit even for patients
with advanced cancer [10].
4. Discussion
Several indices have been used to evaluate the effective-
ness of immunotherapy for cancer treatment. Most of
these, however, estimate the suppression of cancer ef-
fects by tumor markers, reflect the toxicity of interferon-γ

Is the CD4/CD8 Ratio an Effective Indicator for Clinical Estimation of Adoptive Immunotherapy for Cancer Treatment? 1387
Table 4. Performance status and changing pattern of the CD4/CD8 ratio.
(A) Increase in performance status after one course of immunotherapy
Total (n = 34) Antecedent surgery group (n = 19)Non-antecedent surgery group (n = 15)
increase decrease increase decrease increase decrease
30 4 18 1 12 3
P value 0.0077 0.0005 0.0613
P = 0.01794 (Surg. vs non-surg.)
(B) Changing pattern of the CD4/CD8 ratio vs increase in ECOG performance status after one course of immunotherapy
ECOG PS
CD4/CD8 Pattern
increase decrease
P value
Downward group (n = 28) 26 2 0.2393 (N. S.)
Upward group (n = 6) 4 2 0.0253
P=0.0005 P=0.0613
P=0.0179
SURG. GROUPNON-SURG. GROUP
Before After Before After
PS 0
PS 1
PS 2
Figure 2. Change of ECOG performance status (P) before
and after one course of immunotherapy.
0.2
030
1
0.8
0.6
5101520
0.4
A
log-rank p=0.02984
B
25 35
ACTUARIAL SURVIVAL RATE
MONTHS
A: n=19
B: n=15
Figure 3. Actuarial survival curve of the antecedent surgical
group (A) and the antecedent non-surgical group (B).
or tumor necrosis factor-α Τ [15], or have an indirect
influence on cancer invasiveness (e.g., the number of
white blood cells, lymphocytes, or monocytes) [16,17]
rather than provide a direct anticancer immunological
index. Recent technological progress in flow cytometry
has opened a new field of precise counting of immu-
nological cells and revealed their detailed character in
individual patients. Immunological cells in the blood,
such as lymphocytes and monocytes, with fluorochrome-
labeled antibodies that bind specifically to lymphocyte
surface antigens travel past a detector with an analogue-
to-digital conversion system under laser light. These
scattered cells and fluorescence signals from cellular par-
ticles detected by the flow cytometer provide information
about cell size, internal complexity, and relative fluores-
cence intensity. Lymphocyte subset percentages and ab-
solute counts can be calculated using flow data from the
beads and the sample. Flow cytometry can provide pre-
cise data on several types of immunological variables in-
cluding CD3, CD4, CD8, and TCR pan αβ Τ cells and
the CD4/CD8 ratio.
In a previous study [12] involving 678 patients treated
with autologous activated lymphocyte immunotherapy at
our institute, we cultured their cells ex vivo under stimu-
lation with immobilized anti-CD3 antibody and IL-2 and
analyzed them by flow cytometry using monoclonal an-
tibodies against CD3, CD4, CD8, CD56, and TCRγδ [12].
The percentage (means ± SD) of CD3+CD4+ helper T
cells and CD3+CD8 cytotoxic T cells in cultured cells
were 31% ± 15% and 61% ± 15%, respectively. Thus, αβ
T lymphocytes in these cultured lymphocytes contained
more CD8+ lymphocytes compared with the total cohort
of 34 patients in the present study. CD8+ T cells have a
cytotoxic function in cancer cells and a suppressive abil-
ity against regulatory T lymphocytes [8,11,17]. Expand-
ed CD8-rich T lymphocytes were infused intravenously
once every 2 weeks up to 6 times during one course of
adoptive immunotherapy. As a result, the CD4+ T cell
numbers were maintained at almost the same level com-
pared with levels before immunotherapy, whereas the
CD8 T cell number increased significantly (P = 0.05).
Takayama et al. [4] presented a clinical report of the

Is the CD4/CD8 Ratio an Effective Indicator for Clinical Estimation of Adoptive Immunotherapy for Cancer Treatment? 1388
feasibility and efficacy of adoptive immunotherapy to
prevent recurrence of hepatocellular carcinoma in pa-
tients who underwent hepatectomy. From peripheral
blood mononuclear cells cultured for 2 weeks with ex
vivo IL-2 and immobilized anti-CD3 antibody, they ob-
tained uncontaminated and viable activated T lympho-
cytes. The phenotype analysis of their activated T lym-
phocytes showed 87% CD3, 25% CD8, and 72% CD8 T
cells. Autologous activated lymphocytes were infused
five times during the first 6 months. Compared with no
adjuvant treatment, adoptive immunotherapy significant-
ly lowered the risk of recurrence after a median follow-
up of 4.4 years.
There are several different kinds of immunotherapy
using different phenotypic proportions of CD4 and CD8
T cells and differing CD4/CD8 ratios. For instance,
Recchia et al. [18] presented the findings of maintenance
immunotherapy in 100 patients with metastatic breast
cancer who had received direct subcutaneous injection of
low-dose IL-2 and administration of oral retinoic acid to
prevent tumoricidal activity. Patients were followed for 3
years, and lymphocytes, NK cell numbers, and the CD4/
CD8 ratio were found to increase significantly (p = 0.05)
each year compared with baseline. However, since there
was an interval of 1 year between each measurement
point, it is difficult to analyze the factors that may have
contributed to increasing the CD4/CD8 ratio.
The results of the analysis of the CD4/CD8 ratio in the
present study clearly show that within 3 months from the
start of one course of adoptive activated T lymphocyte
immunotherapy, it was possible to estimate the effec-
tiveness of immunotherapy even for patients with ad-
vanced cancers treated with not only immunotherapy
alone but also combination therapy involving other con-
ventional therapies such as chemotherapy or radiotherapy.
Overall, 73.5% of our patients received combination
therapy with chemotherapy.
In this study, a downward tendency of the CD4/CD8
ratio during one course of immunotherapy suggested an
active immunological response against cancer invasion
while an upward tendency suggested a worse immu-
nological response during immunotherapy, indicating the
possible timing of the next therapy for further improve-
ment of the patient’s immunological ability.
As previously reported [10], prior surgery improved
the outcome of immunotherapy. Actuarial survival curves
in the antecedent and non-antecedent surgery groups
showed an additional long-term supportive effect of
adoptive activated T lymphocyte immunotherapy in the
former group (p = 0.0298). Patients then have the chance
to choose more effective therapy 3 months after the first
immunotherapy, and there are several options of immu-
notherapy treatment for patients proceeding to the next
step. Options include γδ Τ cell therapy [19], NK cell the-
rapy [20], and dendritic cell vaccine therapy [21,22] with
additional selection tests such as HLA typing. Immuno-
histochemical staining using pathological tissue speci-
mens obtained in the operating theater in cases of ante-
cedent surgery indicates NK cell therapy for MHC1-
negative patients. NK cell therapy is also provided for
patients with a peripheral blood NK cell number that is
diminished or patients who use antibody-dependent cel-
lular cytotoxicity-containing drugs such as cetuximab or
trastuzumab as combination immunotherapy [23,24]. NK
cell therapy is also effective for patients who have not
shown an effective response to αβ Τ, γδ Τ, cytotoxic T
lymphocytes, and dendritic cell vaccine immunotherapy.
There is some evidence of an association between im-
munological ability and PS [7]. The present study show-
ed a statistically significant association between change
in the CD4/CD8 ratio before and after one course of
adoptive immunotherapy and PS among different groups;
the antecedent surgery group showed an important bene-
fit of immunotherapy compared with the non-antecedent
surgery group (p = 0.0179). Moreover, downward or
upward change in the pattern of the CD4/CD8 ratio be-
fore and after one course of adoptive activated T lym-
phocyte immunotherapy revealed a long-term trend in PS.
Indeed patients with good PS under immunotherapy
maintained good QOL nearly to the end of their life.
Change in the pattern of the CD4/CD8 ratio suggests
reasonable correlation with these phenomena based upon
patient QOL.
Regulatory T (Treg) lymphocyte number is not gener-
ally measured as a commercially available index but has
been experimentally studied in the laboratory and in sev-
eral clinical trials to date [25-28]. Treg cells represent 5%
- 10% of the peripheral CD4 T lymphocytes and are im-
portant supportive regulators of immune tolerance. In
anticancer immunity, Treg cells may disturb beneficial
tumor-specific responses. When Treg cell numbers in-
crease to some extent, immune suppression appears to be
inactivated [25]. Shirabe et al. [26] reported an important
subpopulation balance between CD8+ T cells and Treg
cells in autologous tumor-infiltrating T lymphocyte im-
munotherapy for hepatocellular carcinoma using immu-
nohistochemical analysis of paraffin-embedded tumor
tissue.
Although the mechanisms involved in immune sup-
pression are not fully understood in this condition, the
decrease in the CD4/CD8 ratio clearly appears before the
increase in Treg cell count presenting with an active in-
crease in CD8 T cells while CD4 T cells showed consis-
tently stable cell numbers. An unchanged and stable CD4
T cell count may imply that the increase in Treg cells ap-
pears at a later stage of immunological response com-
pared with the decrease in the CD4/CD8 ratio during the
initial 3 months of adoptive immunotherapy. The time

Is the CD4/CD8 Ratio an Effective Indicator for Clinical Estimation of Adoptive Immunotherapy for Cancer Treatment? 1389
delay between the CD4/CD8 ratio decrease and Treg cell
increase is beneficial for clinical use of the CD4/CD8
ratio as an index of immunological response, although
the immunosuppressive action itself is explained more
precisely by the Treg cell number.
5. Conclusion
In conclusion, in this preliminary clinical study, we have
confirmed that the CD4/CD8 ratio provides a valuable
decision-making index for adoptive immunotherapy with
or without other conventional therapies such as chemo-
therapy and/or radiotherapy at any stage of cancer in-
cluding advanced disease, by measurement within 3
months of one course of immunotherapy. Patients may
then proceed with more precise immunotherapy or other
treatment according to the changing pattern of the CD4/
CD8 ratio. Further studies with a focus on different kinds
of immunological response of cancer in different organs
under several different kinds of immunotherapy are nec-
essary to understand the underlying mechanisms and to
develop new therapeutic strategies.
6. Acknowledgements
We thank Dr Atsumi Noguchi for providing technical
assistance on flow cytometry.
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