Immunological effect of Zeusion X
J. Akagi (✉)
Tamana Regional Health Medical Center, 2172 Tamana, Kumamoto, Japan

Corresponding author: J. Akagi
Telephone: +81-968-72-5111
Fax: +81-968-73-4919
E-mail: jnjakagi@gmail.com

key word; Zeusion X、PD-1、CD8+ T cell differentiation、cancer patients、prognosis

1

Abstract
It is very important to understand CD8+ T-cell dynamics to determine the immune
status of cancer patients: CD8+ T-cell populations, i.e., early-differentiated CD8+ T
cells (CD27+CD8+CD57− T cells, hereafter referred to as early-CD8+ T cells),
intermediate-differentiated CD8+ T cells (CD27+CD8+CD57+ T cells, hereafterreferred to as intermediate-CD8+ T cells), and terminally differentiated CD8+ T cells(CD27−CD8+CD57+ T cells, , hereafter referred to as terminal-CD8+ T cells). We
conducted univariate analysis of CD8+ T-cell populations related to CD8+ T-celldifferentiation in a culture of activated self lymphocyte cells and peripheral blood of
100 stage IV cancer patients and reported that early-CD8+ T cells are involved in a poor
prognosis and terminal-CD8+ T cells are involved in a favorable prognosis. In addition,
we reported that there is an inverse correlation between early-CD8+ T cells andterminal-CD8+ T cells (Spearman correlation coefficient, 0.919; p < 0.0001). The CD57
ratio (terminal-CD8+ T cells/early-CD8+ T cells) was significantly more correlated withprognosis than early-CD8+ T cells and terminal-CD8+ T cells alone in multivariate
analysis and was a good, independent prognostic factor. The CD57 ratio is a good
indicator of the dynamic state of CD8+ T-cell differentiation, and we believe that it is2highly likely to be the best indicator of CD8+ T-cell differentiation. In the present study,
we examined the immunological effect of Zeusion X, consisting of plant lactic acidbacteria, organic mineral fulvic acid, and golden oyster mushroom (β-1,3 D-glucan).
Zeusion X was administered to 15 patients with progressive stage IV cancer for 3
months. Peripheral blood was collected before and 3 months after administration.
Early-CD8+ T cells, intermediate-CD8+ T cells, terminal-CD8+ T cells, PD-1+
terminal-CD8+ T cells, and the CD57 ratio were measured by flow cytometry andsubjected to analysis. In 8 of 15 patients (53.3%), the CD57 ratio increased and PD-1+
terminal-CD8+ T cells decreased. However, in all 4 patients with a CD57 ratio of >4.0,
the CD57 ratio decreased and PD-1+ terminal-CD8+ T cells increased, but all of these
had a good prognosis. Conversely, 8 of 11 patients (72.7%) with a CD57 ratio of <4.0
showed an increase in the CD57 ratio, and 7 patients had a good prognosis. Five of the 6patients in whom the CD57 ratio increased and PD-1+ terminal-CD8+ T cells decreased
had a good prognosis. Conversely, all 3 patients in whom the CD57 ratio decreased andPD-1+ terminal-CD8+T cells increased had a poor prognosis. These findings suggestthat Zeusion X regulates CD8+ T-cell immunity in both positive and negative directions
by controlling the CD57 ratio and PD-1+ terminal-CD8+ T cells.
3Introduction
We have long focused on CD57+ T cells, particularly CD8+CD57+ T cells, as
prognostic factors for cancer patients. CD57+ T cells are independent poor prognosis
factors in advanced stomach cancer (HR = 3.136, 95% CI 1.142–8.16, p = 0.027), andthe CD57+ T-cell elevation group (>18%) has a significantly poorer prognosis than the
low value group (Kaplan–Meyer log rank p = 0.011) [1]. In melanoma patients, thegroup with low levels of CD8+CD57+ T cells in peripheral blood (<23%) before IFN-α
administration has been found to have a better prognosis than the high-level group [2],but in advanced renal cancer patients, the outcome has been the opposite. That is, theprognosis in the group of patients with peripheral blood CD8+CD57+ T-cell elevation(>30%) before IFN-α administration has been found to be good [3]. IFN-α has the effectof increasing CD8+CD57+ T cells when they are low in numbers before administration.
Conversely, IFN-α decreases CD8+CD57+ T cells when they are high in numbers
before administration. In melanoma patients, the CD8+CD57+ T-cell level, which is low
before administration, increases by IFN-α administration and the survival time isprolonged. Conversely, in advanced renal cancer patients, the CD8+CD57+ T-cell level,
which is high before administration, is decreased by IFN-α administration and the4

survival time is prolonged. This indicates that the characteristics of peripheral blood
CD8+CD57+ T cells of melanoma patients and peripheral blood CD8+CD57+ T cells ofadvanced renal cancer patients are different. Cytotoxic CD8+CD57+ T cells are
dominant in melanoma patients, and immunosuppressive CD8+CD57+ T cells are
dominant in advanced renal cancer patients. We recently reported that CD8+CD57+ T
cells (equivalent to terminally differentiated CD8+ T cells, hereafter referred to as
terminal-CD8+ T cells) are involved in a good prognosis in activated autologous
lymphocyte therapy [4]. Thus, both a cytotoxic population and an immunosuppressive
population exist in CD8+CD57+ T cells in peripheral blood, and which population
proliferates dominantly seems to depend on the cancer type.

5CD8+CD57+ T cells appear in the pathway of differentiation of CD8+ T cells (Fig. 1)

At least the following 2 types of CD8+CD57+ T cells are present in the CD8+ T-celldifferentiation pathway: intermediate-differentiated CD8+ T cells (CD27+CD8+CD57+
T cells, hereafter referred to as intermediate-CD8+ T cells) and terminal-CD8+ T cells(CD27−CD8+CD57+ T cells). The properties of these 2 CD8+CD57+ T cells are quite
different. Intermediate-CD8+ T cells are CD27 positive, are GB+Perlo, and have low
cytotoxic activity. Terminal CD8+ T cells are CD 27 negative, are GBhighPerhigh, andhave high cytotoxic activity. CD57+ T cells (CD8+CD57+ T cells), which increased inperipheral blood of advanced stomach cancer and advanced renal cancer patients and6were involved in a poor prognosis, are thought to be intermediate-CD8+ T cells.
Moreover, terminal-CD8+ T cells are thought to increase in melanoma patients andactivate autologous lymphocyte cells. It has been reported that differentiation fromearly-differentiated CD8+ T cells (hereafter referred to as early-CD8+ T
cells)/intermediate-CD8+ T cells to terminal-CD8+ T cells and cytotoxic activity are
inhibited by TGF-β [5], IL-10, and PD-1 [6] in cancer patients. Because activatedautologous lymphocyte therapy causes lymphocyte proliferation using CD3 antibody
and IL-2 in vitro, this suppression of differentiation is lost and terminal-CD8+ T cellswith high cytotoxic activity are increased. This is believed to contribute to a good
prognosis [4]. Thus, it is considered that the prognosis of cancer patients is defined bywhich population increases during the differentiation of CD8+ T cells. Therefore, it isimportant in cancer treatment to always consider the stage of cytotoxic T lymphocyte(CTL) differentiation of CD8+ T cells. The CD57 ratio is a very important index
because it accurately expresses the process of its differentiation.

We conducted univariate analysis of CD8+ T-cell populations related to CD8+ T-celldifferentiation in a culture of activated self lymphocyte cells [4] and peripheral blood of7

100 stage IV cancer patients [7,8] and reported that early-CD8+ T cells are involved in apoor prognosis and terminal-CD8+ T cells are involved in a favorable prognosis [7,8].
Furthermore, the CD57 ratio (terminal-CD8+ T cells/early-CD8+ T cells) calculatedusing early-CD8+ T cells and terminal-CD8+ T cells, which show an inverse correlation,
was significantly more correlated with prognosis than early-CD8+ T cells andterminal-CD8+ T cells alone and was a good, independent prognostic factor [7,8]. The
CD57 ratio is considered to be an important indicator of the dynamic state ofdifferentiation of CD8+ T cells. When the CD57 ratio is high, the differentiation of
CD8+ T cells is smooth and cytotoxic terminal-CD8+ T cells are sufficiently induced.
However, when the CD57 ratio is low, there is some obstacle to the differentiation of
CD8+ T cells, which is considered to be a state in which immunosuppressive early-CD8+ T cells are induced. In advanced cancer patients, this CD57 ratio declines in many
patients, and we think that raising the CD57 ratio, i.e., activating the immunity of CD8+
T cells, is an important cornerstone of cancer treatment. When the CD57 ratio is low,i.e., when immunity is not sufficiently induced, treatment is not always effective. We
were looking for a therapeutic agent capable of increasing the CD57 ratio and activating
CD8+ T-cell immunity. We found Zeusion X in such a situation.
8

Zeusion X consists of plant lactic acid bacteria, organic mineral fulvic acid, andgolden oyster mushroom (β-1,3 D-glucan). It has been reported that lactic acid bacteria
have a tumor-suppressive effect in colon cancer patients [9] and breast cancer patients[10]. In our study, Zeusion X showed a tumoricidal effect equivalent to that of cisplatin
at a concentration of 12.5 mg/ml in a colon-26 cultured cell line. Because Zeusion X
contains plant lactic acid bacteria and golden oyster mushroom (β-1,3 D glucan), in
addition to the direct tumor-killing effect, an immune-mediated mechanism is assumed
to contribute to this tumor-suppressing effect.
In the present study, we administered Zeusion X to 15 stage IV progressive cancer
patients for 3 months. Peripheral blood was collected before administration and 3
months after administration, and each cell fraction of the CD8+ T-cell differentiation
process (early-CD8+ T cells, intermediate-CD8+ T cells, terminal-CD8+ T cells, and theCD57 ratio) was measured and analyzed.

Patients and Methods
We evaluated 100 patients (45 males, 55 females) diagnosed with stage IV cancer at
the Tamana Regional Health and Medical Center from July 2013 to March 20169

(stomach cancer, 9; colorectal cancer, 23; pancreatic cancer, 10; liver cancer, 6; lungcancer, 14; breast cancer, 9; others, 29). In total, 10 ml of peripheral blood of thesepatients was collected, and FACScan was performed. The antibodies used wereanti-CD3 FITC (HIT3a), anti-CD57 FITC (NK-1), anti-CD 27 PE (M-T 271), anti-Fox
P3 PE (259 D/C7), anti-CD 57 PE (NK-1), and anti-CD8 PE-Cy7 (RPA-T8) (BD
Pharmingen, San Diego, CA, USA). Lymphocytes were analyzed with BD
FACSCalibur, and BD CellQuest software was used for data analysis. All these tests
were conducted at SRL Inc. (Tokyo, Japan).

Fifteen patients took 8 ml of Zeusion X 4 times a day (morning, noon, night, andbefore bed) for 3 months. Peripheral blood was collected twice: before administration
and after 3 months of administration. CD57+ T cells, CD8+CD57+ T cells, early-CD8+
T cells, intermediate-CD8+ T cells, and terminal-CD8+ T cells in peripheral blood were
measured by FACScan. The cases were as follows: stomach cancer, 1; colon cancer, 5;breast cancer, 2; pancreatic cancer, 1; liver cancer, 1; lung cancer, 1; and other cancers,
4.

10Statistical analysis was performed using SPSS version 13.0 for Windows (SPSS Inc.,
Chicago, IL, USA). Mann–Whitney U test was used to compare the numerical values ofthe 2 groups, which were not normally distributed, and the χ2 test was used forcomparison of categories between the 2 groups. The Kaplan–Meier method was used
for analysis of the survival rate, and the log-rank test was used to test for significant
difference in the survival rate. The Cox regression model was used for multivariate
analysis.

Results

Univariate and multivariate analyses
Univariate and multivariate analyses were conducted to determine the association
between progression-free survival (PFS) and early-CD8+ T cells, intermediate-CD8+ T
cells, and terminal-CD8+ T cells, which are the groups of cells involved in the
differentiation of CD8+ T cells. We found that CD57+ T cells, CD8+CD57+ T cells, andterminal-CD8+ T cells were associated with a favorable prognosis (Table 1).

11

Table 1 Univariate and multivariate analyses(Univariate analysis)

HR

HR95%CI

P-value

CD57+ T cells

0.362

0.186~0.704

0.003

CD8+CD57+T cells

0.424

0.220~0.820

0.011

Early-CD8+ T cells

5.937

2.589~13.6<0.0001

2.325

1.216~4.447

0.011

0.154

0.072~0.329<0.0001(CD27+CD8+CD57− T cells)Intermidiate-CD8+ T cells(CD27+CD8+CD57+ T cells)
Terminal-CD8+ T cells(CD27−CD8+CD57+ T cells)
0.337

End-CD8+ T cells(CD27−CD8+CD57+ T cells)
CD57− ratio

0.143

0.065~0.314<0.0001(multivariate analysis)

HR

HR95%CI

P-valueCD57− ratio

0.143

0.065~0.314<0.0001

Early-CD8+ T cells and intermediate-CD8+ T cells were associated with a poor
prognosis (Table 1). As reported previously [7, 8], early-CD8+ T cells andterminal-CD8+ T cells have a strong inverse correlation, and each has both poor andgood prognostic factors. Therefore, in order to determine additional potent prognostic
immunological parameters, the CD57 ratio (=terminal-CD8+ T cells/early-CD8+ T
cells) was calculated. This is an immunological indicator that well reflects thedifferentiation status of CD8+ T cells. If it is high, CD8+ T-cell differentiation is smooth12and terminal-CD8+ T cells, showing cytotoxic activity, are sufficiently induced.
Conversely, if it is low, there is some obstacle to CD8+ T-cell differentiation, andterminal-CD8+ T cells are not sufficiently induced. In this state, early-CD8+ T cells are
involved in immunosuppression [7, 8]. Results of multivariate analysis indicated that
the CD57 ratio is an independent favorable prognostic factor that correlates moresignificantly with PFS than early-CD8+ T cells or terminal-CD8+ T cells alone. The
CD57 ratio is considered to be an important parameter in ascertaining theimmunological status of cancer patients.

Effect of Zeusion X on CD57 ratio
Early-CD8+ T cells decreased in 8 of 15 patients (53.3%), and terminal-CD8+ T cells
increased in 8 of 15 patients (53.3%) after the administration of Zeusion X (Fig. 2).

13

The

CD57

ratio

increased

in

8

of

14

15

patients(53.3%)(Fig.

3)

In approximately half of the patients, the administration of Zeusion X led to an increasein the CD57 ratio, i.e., a decrease in early-CD8+ T cells and an increase interminal-CD8+ T cells. This suggests that Zeusion X can activate CD8+ T-celldifferentiation and induce the production of terminal-CD8+ T cells, which are effector
cells. Further detailed examination revealed that of patients with a CD57 ratio of >4.0,
compared with before administration, all 4 patients (100%) showed a decrease in the
CD57 ratio after the administration of Zeusion X. Conversely, of patients with a CD57
ratio of <4.0, compared with before administration, 8 of 11 patients (72.7%) showed anincrease in the CD57 ratio after the administration of Zeusion X (Fig. 4).

15

Effect of Zeusion X on PD-1+ terminal-CD8+ T cells
The proportion of PD-1+ terminal-CD8+ T cells was decreased in 8 of 15 patients by
the administration of Zeusion X (Table 2).
Table 2 The change in PD-1+ terminal-CD8+ T cells before and after the administration of Zeusion X using the CD57 ratio
CD57 ratio
PD-1+terminal-CD8+ T cells

Increase

Decrease

Increase2

5

Decrease

62

Pearson χ２test p=0.030

Further, we examined the change in PD-1+ terminal-CD8+ T cells before and after theadministration of Zeusion X using the CD57 ratio. We found that PD-1+ terminal-CD8+
T cells were decreased in 6 of 8 patients (75%) in whom the CD57 ratio was increasedby the administration of Zeusion X (Table 2). In 5 of patients in whom the CD57 ratio
was not increased by the administration of Zeusion X, PD-1+ terminal-CD8 + T cells
increased. Although the CD57 ratio decreased after the administration of Zeusion X andPD-1+ terminal-CD8+ T cells increased in the 4 patients with a CD57 ratio of >4.0, theprognosis was good in all the patients. Of the 11 patients with a CD57 ratio of <4.0, theprognosis was good in 5 of 6 patients in whom the CD57 ratio increased and PD-1

16

decreased after the administration of Zeusion X. However, all 3 patients in whom theCD57 ratio decreased and PD-1 increased showed a poor prognosis (Table 3).
Table 3 The relationship between the change in CD57 ratio and PD-1+ terminal-CD8+ T cells after the administration of Zeusion
and the prognosis
No

CD57

PD-1

prognosis

ratio
CD57ratio

betterpoorer

4

↓

↑

4

0

2

↑

↑

2

0

6

↑

↓

5

1

3

↓

↑

0

3

0

↓

↓

0

0

>4.0

CD57ratio<4.0

Discussion
As reported to date, the results of univariate analysis confirmed thatintermediate-CD8+ T cells in peripheral blood of cancer patients are involved in a poor
prognosis. In addition, we found that early-CD8+ T cells (CD27+CD8+CD57− T cells)
are involved in a poor prognosis (Table 1). Although it has been reported thatintermediate-CD8+ T cells are involved in a poor prognosis, the fact that early-CD8+ T
cells are involved in a poor prognosis is a new finding that has never been reported todate. Terminal-CD8+ T cells are involved in a good prognosis (Table 1). In addition,17

because we showed a strong inverse correlation between early-CD8+ T cells andterminal-CD8+ T cells (Spearman correlation coefficient, 0.919; p < 0.0001) [7, 8], theCD57 ratio (=terminal-CD8+ T cells/early-CD8+ T cells) was calculated. This isthought to reflect the differentiation state of CD8+ T cells from early-CD8+ T cells toterminal-CD8+ T cells. When the CD57 ratio is low (early-CD8+ T cells are high andterminal-CD8+ T cells are low), it is suggested that CD8+ T-cell differentiation issuppressed. Conversely, when the CD57 ratio is high (early-CD8 + T cells are low andterminal-CD8+ T cells are high), it is suggested that the differentiation of CD8+ T cells
is smooth and the production of effector terminal-CD8+ T cells is smooth. Therefore,
this CD57 ratio is considered to be an indicator directly reflecting the dynamic state ofthe differentiation of CD8+ T cells, which is directly linked to the prognosis of cancer
patients. As a result of multivariate analysis, we found that the CD57 ratio most strongly
reflects a patient’s prognosis among any cell groups in the CD8+ T-cell differentiation
process (HR = 0.143, 95% CI 0.065–0.314, p < 0.0001) (Table 1). From the ROC curve,
the cutoff value of the CD57 ratio was 1.45 (AUC 0.810, sensitivity 85.5%, specificity
78.9%, p < 0.0001) [7, 8]. The high value group in terms of the CD57 ratio (>1.45) had

18

a significantly better prognosis than the low value group (<1.45) (Kaplan–Meyer log
rank p < 0.0001) [7, 8].

We investigated the immunological effect of Zeusion X using early-CD8+ T cells,
terminal-CD8+ T cells, PD-1+ terminal-CD8+ T cells, and the CD57 ratio. The results
showed that Zeusion X increased the CD57 ratio in 8 of 15 patients (53.3%). In otherwords, it is thought that Zeusion X activated cytotoxic terminal-CD8+ T cells by
activating the differentiation of CD8+ T cells in approximately half of the patients.
Further, as shown in Fig. 3, the CD57 ratio decreased after the administration of
Zeusion X in all 4 patients with a CD57 ratio of 4.0 or more. Conversely, the CD57 ratio
increased in 8 of 11 patients (72.7%) with a CD57 ratio of <4.0 (Table 3). The prognosis
was good in all 4 patients in whom the CD57 ratio was high (>4.0) before theadministration of Zeusion X. Of 11 patients with a low CD57 ratio (<4.0) before theadministration of Zeusion X, the CD57 ratio increased in 8 after administration, andtheir prognoses were good, except in 1 patient. However, 3 of 4 patients in whom theCD57 ratio decreased after administration had a poor prognosis (Table 3).

19We also measured the expression of PD-1 in terminal-CD8+ T cells in the present
study. The expression of PD-1 in CD8+ T cells is most common in early-CD8+ T cells
and decreases as CD8+ T-cell differentiate into intermediate-CD8+ T cells and further
into terminal-CD8+ T cells [11]. Normally, there is little expression of PD-1 interminal-CD8+ T cells. However, it has been reported that in cancer patients, theexpression of PD-1 in CD8+ T cells persists without decreasing, despite the progress ofdifferentiation of CD8+ T cells [12,13]. In case of cancer patients with a high CD57
ratio (CD8+ T-cell differentiation is normal), the expression of PD-1 in CD8+ T cells
decreased with differentiation and PD-1 in terminal-CD8+ T cells was almost
undetectable(Fig.5).

20However, in patients with a low CD57 ratio (patients in whom CD8+ T-celldifferentiation is compromised), PD-1 expression in CD8+ T cells did not decrease andPD-1 was highly expressed in the cells (Fig. 5). Terminal-CD8+ T cells expressing PD-1
are thought to be CD8+ T cells exhausted by continuous stimulation with cancer
antigens. Exhausted CD8+ T cells have extremely low cytotoxic activity, and it isthought that the prognosis will be poor in cancer patients with increased exhausted
CD8+ T cells. Therefore, by measuring PD-1+ terminal-CD8+ T cells, it is possible topredict the prognosis of cancer patients. Although the CD57 ratio decreased and PD-1+
terminal-CD8+ T cells increased with the administration of Zeusion X in patients with a21high CD57 ratio (>4.0), the prognosis was good in all the patients. This is considered to
be a normal biological reaction when the immune reaction is excessive (CD57 ratio >4.0). In case of a low CD57 ratio (<4.0), there is a good prognosis when the CD57 ratio
increases and PD-1+ terminal-CD8+ T cells decrease after the administration of Zeusion
X. These cases are considered to be cases compatible with Zeusion X. Conversely, evenwhen Zeusion X is administered, the CD57 ratio decreases and PD-1+ terminal-CD8+ T
cells increase in some cases. The prognosis of those cases is obviously poor. Therefore,
these cases are considered to be incompatible with Zeusion X.

These results suggest that Zeusion X reduces the CD57 ratio and increases PD-1 interminal-CD8+ T cells to suppress immunity in patients in whom immunity induction of
CD8+ T cells is excessive, such as those with a CD57 ratio of > 4.0. On the other hand,in patients in whom CD8+ T-cell immunity induction is insufficient, such as those witha CD57 ratio of <4.0, Zeusion X is suggested to increase the CD57 ratio and decreasePD-1 in terminal-CD8+ T cells to activate immunity. We speculate that patients with a
low CD57 ratio, which are often cancer patients, the administration of Zeusion X
increases the CD57 ratio, decreases PD-1+ terminal-CD8+ T cells, activates CD8+ T-cell22differentiation, and induces cytotoxic terminal-CD8+ T cells. However, in cancer
patients with a high CD57 ratio, we speculate that Zeusion X decreases the CD57 ratio
and increases the PD-1+ terminal-CD8+ T cells to adjust the immune system against
acting excessively. However, in cancer patients in whom immunity cannot be controlled
successfully, i.e., cancer patients who do not show any improvement with Zeusion X
administration despite having a low CD57 ratio, cytotoxic terminal-CD8+ T cells may
be insufficiently induced and PD-1+ terminal-CD8+ T cells that suppress cytotoxic
activity may increase. Therefore, the prognosis is considered to be poor. Conversely, inpatients in whom the CD57 ratio does not decrease and PD-1+ terminal-CD8+ T cells do
not increase despite a high CD57 ratio, autoimmune disease may occur. Thus, Zeusion
X suppresses the immunity induction of CD8+ T cells when it is excessive and activatesthe immunity induction of CD8+ T cells when it is deficient, suggesting the possibilityof regulating CD8+ T-cell immunity. These findings suggest that Zeusion X could be
effective not only in cancer patients but also in patients with autoimmune diseases.
However, because the presence of cases that are incompatible with Zeusion X was alsopresumed in this study, we need to consider countermeasures against these cases in the
future.
23

Conclusion
Our results suggest that Zeusion X increases the CD57 ratio by inducing
terminal-CD8+ T cells and reducing PD-1+ terminal-CD8+ T cells so that overall
immunity is activated in patients with a low CD57 ratio. On the other hand, when the
CD57 ratio is high, it is suggested that Zeusion X decreases the CD57 ratio bydecreasing the induction of terminal-CD8+ T cells and increasing PD-1+ terminal-CD8+
T cells so that overall immunity is suppressed. These results demonstrate that Zeusion X
could improve various diseases by normalizing the body in a properly maintained
immune state.

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