Differentiation of Cytotoxic CD8⁺ T Cell Subsets Under Tumor Progression: Can CD69 Be a New Therapeutic Target?

ABSTRACT

Tumor‐specific CD8 ⁺ T cells play a pivotal role in anti‐tumor immunity. Here, we review the heterogeneity of CD8 ⁺ T cell subsets during tumor progression. While both acute and chronic viral infections induce distinct CD8 ⁺ T cell responses, chronic responses are also observed during tumor development. Chronic immune responses have traditionally been considered to represent a dysfunctional state of CD8 ⁺ T cells, whereas the identification of TCF1 ⁺ stem‐like CD8 ⁺ T cells has highlighted their importance in anti‐tumor immunity. During tumor progression, TCF1 ⁺ stem‐like CD8 ⁺ T cells differentiate into cytotoxic Tim‐3⁺ terminally differentiated CD8 ⁺ T cells through mechanisms that remain largely unknown. We recently identified CD69 as an important regulator of chronic CD8 ⁺ T cell responses and showed that blocking CD69 function, either through the administration of anti‐CD69 antibody (Ab) or genetic knockout, enhanced the generation of cytotoxic Tim‐3⁺ terminally differentiated CD8 ⁺ T cells in both tumor‐draining lymph nodes (TDLNs) and the tumor microenvironment (TME), thereby enhancing the anti‐tumor immune response. These findings suggest that CD69 is an attractive therapeutic target that controls the chronic anti‐tumor CD8 ⁺ T cell response.

This review summarizes the current understanding of cytotoxic CD8⁺ T cell subset differentiation under tumor progression and highlights the crucial role of chronic CD8⁺ T cell responses in anti‐tumor immunity. CD69 is an important regulator of chronic CD8⁺ T cell response that contributes to the induction of the Tox gene via calcineurin/NFAT signaling.

Abbreviations

Ab

antibody

CTLA‐4

cytotoxic T‐lymphocyte‐associated protein 4

DCs

dendritic cells

HNSCC

head and neck squamous cell carcinoma

ICB

immune checkpoint blockade

IFN

interferon

LCMV

lymphocytic choriomeningitis virus

MHC

major histocompatibility complex

Myl

myosin light chain

NK

natural killer

PD‐1

programmed cell death 1

PD‐L1

programmed cell death‐ligand 1

S1P₁

sphingosine‐1‐phosphate receptor 1

TCF1

T cell factor 1

TCR

T cell receptor

TDLNs

tumor‐draining lymph nodes

Tim‐3

T cell immunoglobulin and mucin domain‐containing 3

TME

tumor microenvironment

TOX

thymocyte selection‐associated high mobility group box

Treg

regulatory T cells

T_RM

resident memory CD8⁺ T cells

1. Introduction

Cancer immunotherapy, which utilizes the immune system to eliminate cancer cells, has emerged as a promising treatment strategy. Tumor antigens can elicit robust immune responses and contribute to tumor eradication [1]. While various immune cell types are involved in anti‐tumor immune responses, tumor‐specific cytotoxic CD8 ⁺ T cells, which recognize tumor antigens loaded on major histocompatibility complex (MHC) class I molecules, are particularly crucial targets for immunotherapeutic approaches. Indeed, the efficacy of most successful cancer immunotherapies depends on CD8 ⁺ T cell effector functions [2].

In this review, we discuss recent advances in our understanding of tumor‐specific CD8 ⁺ T cell subset differentiation during tumor progression, including their biological functions and therapeutic potential. We especially focus on CD69, a novel regulator of chronic CD8 ⁺ T cell responses, and its promise as a therapeutic target in cancer treatment.

2. CD69 Expression in the Tumor Microenvironment and Tumor‐Draining Lymph Nodes

CD69 was first reported in the late 1980s as a membrane protein whose expression is induced upon activation of T cells, B cells, and natural killer (NK) cells [3, 4, 5, 6]. Since then, CD69 has been recognized as an early activation marker of leucocytes, including lymphocytes, granulocytes, macrophages, and dendritic cells (DCs) [7, 8]. While CD69 is almost undetectable at steady state on most leucocytes, it is constitutively expressed in tissue‐resident memory T cells [9]. Furthermore, CD69 expression is highly detectable in the tissues under inflammation [9], suggesting that CD69 functions in certain immune responses.

In anti‐tumor immunity, CD69 has been reported to be highly expressed on CD8⁺ T cells not only in the TME of murine tumor model [10], but also in human cancers, such as lung cancer, head and neck squamous cell carcinoma (HNSCC), ovarian cancer, and melanoma [11, 12, 13, 14, 15]. Studies using tumor‐specific or tumor‐unrelated MHC tetramers indicate that CD69 is expressed in both tumor‐specific and bystander CD8⁺ T cells [11]. This result suggests that while CD69 is mainly induced by T cell receptor (TCR) signaling, signals other than TCR stimulation may also induce CD69 expression in CD8⁺ T cells in the TME. Indeed, some cytokines, such as Type I interferon (IFN) and Tumor necrosis factor, can induce CD69 expression on activated T cells [16, 17, 18]. CD69 is also expressed on T cells in TDLNs in oral squamous cell carcinoma patients, although tumor‐specific and bystander T cells could not be distinguished in this study [19]. Thus, CD69 is expressed on T cells in both the TME and TDLNs, suggesting that CD69 might play a role in anti‐tumor immunity.

3. Blockade of CD69 Function Enhances Anti‐Tumor Immunity

Several groups, including ours, have reported that both CD69 deficiency and the administration of anti‐CD69 Ab reduced tumor growth by enhancing anti‐tumor immune responses (Table 1) [20, 21, 22, 23, 24]. We have demonstrated that the growth of murine tumors such as 4 T1 (breast carcinoma), A20 (B cell lymphoma), and CT26 (colon carcinoma) was significantly reduced in Cd69 ⁻/⁻ mice [23, 24]. CD69 deficiency also reduced spontaneous lung metastasis following inoculation of 4 T1 cells into the mammary fat pad [23]. Notably, this retardation of tumor growth was associated with enhanced anti‐tumor immune responses, as the number of intratumoral T cells was significantly increased in Cd69 ⁻/⁻ mice compared with Cd69 ⁻/⁻ mice, and IFN‐γ production and Granzyme B expression by intratumoral CD8⁺ T cells were increased in Cd69 ⁻/⁻ mice [23, 24].

TABLE 1.

Studies based on CD69‐deficient mice and anti‐CD69 Ab treatment.

Cancer cells	Cancer types	CD69‐deficient mice	Anti‐CD69 Ab treatment	Anti‐CD69 Ab clones	Target	Combination	Reference
RMA‐S	T cell lymphoma	Enhanced anti‐tumor immunity	Enhanced anti‐tumor immunity	CD69.2.2	NK cells		[20, 21]
RM‐I	Prostatic carcinoma	Enhanced anti‐tumor immunity	Enhanced anti‐tumor immunity	CD69.2.2	NK cells		[20, 21]
Renca	Renal cell carcinoma	Not tested	Enhanced anti‐tumor immunity	no description	T cells	Dendritic cell‐based vaccine	[22]
4 T‐1 (expressing luc2)	Breast carcinoma	Enhanced anti‐tumor immunity	Enhanced anti‐tumor immunity	H1.2F3	T cells		[23]
A20	B cell lymphoma	Enhanced anti‐tumor immunity	Not tested	—	T cells		[23]
CT26	Colorectal carcinoma	Enhanced anti‐tumor immunity	Enhanced anti‐tumor immunity	H1.2F3	T cells; CD8+ T cells		[23, 24]
B16 (expressing OVA)	Melanoma	Not tested	Enhanced anti‐tumor immunity	H1.2F3	CD8+ T cells	Anti‐PD‐1 Ab	[24]

CD69 has a large extracellular region that can be targeted with Abs. Indeed, we showed that in vivo treatment with anti‐CD69 Ab resulted in a significant reduction of tumor growth and metastasis of 4 T1, with a significant increase in the number of tumor‐infiltrating T cells [23]. Notably, the combined use of anti‐CD69 and anti‐PD‐1 Abs resulted in a significant reduction of immune‐refractory tumors such as B16 (melanoma) [24]. Additionally, the combination of a DC‐based vaccine and anti‐CD69 Ab significantly reduced tumor volume in mice with Renca (renal cell carcinoma) [22]. This phenomenon may be attributed to the upregulation of CD69 expression in T cells after DC‐based vaccination. These data strongly suggest that CD69 can be a therapeutic target for cancer treatment, specifically targeting T cells. We have summarized studies using anti‐CD69 Ab that support the therapeutic value of targeting CD69 (Table 1) [20, 22, 23, 24].

CD69 has also been reported to regulate anti‐tumor immunity in a T cell‐independent manner. Natural killer (NK) cells are cytotoxic lymphocytes crucial for prompt anti‐tumor immune responses, preferentially targeting MHC‐I‐deficient cancer cells [25]. Studies suggest that CD69 deficiency causes the augmentation of NK cell‐mediated responses in anti‐tumor immunity [20, 21]. Research has shown that CD69 deficiency suppressed the growth of MHC‐I‐deficient tumors, such as RMA‐S (T cell lymphoma) and RM‐1 (prostatic carcinoma), and improved the survival of tumor‐bearing mice, with an increase in NK and T cell numbers and a decrease in transforming growth factor‐β production by NK and T cells. Furthermore, the application of anti‐CD69 Ab can activate resting NK cells against RMA‐S and lung metastases of RM‐1 by increasing cytolytic activity and IFN‐γ production [20, 21]. Thus, CD69 functional blockade results in tumor retardation with enhanced anti‐tumor immunity, establishing CD69 as a novel therapeutic target for cancer immunotherapy.

4. Two Lineages in CD8⁺ T Cell Differentiation

CD8⁺ T cells mediate two types of immune responses: acute response and chronic response, the latter including anti‐tumor immune responses (Figure 1). These immune responses have been well characterized using lymphocytic choriomeningitis virus (LCMV) strains—the Armstrong strain for acute response and the Clone 13 strain for chronic response [26]. In the acute response using LCMV‐Armstrong, naïve virus‐specific CD8⁺ T cells undergo strong proliferation and clonal expansion to differentiate into cytotoxic effector CD8⁺ T cells that directly kill infected cells within 10 days [26]. After virus clearance, only a small subset differentiates into long‐lived memory CD8⁺ T cells. In contrast, in the chronic response using LCMV‐Clone 13, virus‐specific CD8⁺ T cells are exposed to persistent antigen stimulation and are unable to efficiently acquire a memory state. This phenomenon was generally referred to as “T cell exhaustion,” commonly used to describe a state of dysfunction that arises during chronic viral infections and cancer development [27]. “Exhausted” CD8⁺ T cells generated under chronic response are functionally distinct from effector and memory T cells generated under acute response, with features such as reduced effector function, high and persistent expression of inhibitory receptors such as programmed cell death protein 1 (PD‐1), and altered epigenetic and transcriptional profiles [27].

FIGURE 1.

Differentiation of CD8⁺ T cell subsets. Upon activation by DCs, naïve CD8⁺ T cells differentiate into two main lineages. The acute immune response is medicated by effector CD8⁺ T cells, whereas the chronic immune response is mediated by TOX⁺ stem‐like and terminally differentiated CD8⁺ T cells.

Although “exhaustion” has been considered a dysfunctional state of CD8⁺ T cells, serial studies using single‐cell transcriptome analysis and flow cytometry have revealed the heterogeneity of exhausted CD8⁺ T cells [28, 29, 30, 31, 32]. Particularly significant is the discovery of the TCF1⁺ CD8⁺ T cell subset that contributes to sustaining a virus‐specific T cell population in chronic infection [28, 29, 30, 31]. TCF1⁺ CD8⁺ T cells maintain the expression of the inhibitory receptor PD‐1 and lack many common effector functions but have the ability to persist long‐term through self‐renewal. Importantly, TCF1⁺ CD8⁺ T cells can serve as a source of the other subpopulation, PD‐1⁺ Tim‐3⁺ TCF1⁻ CD8⁺ T cells, which possess cytotoxic function, express cytotoxic molecules such as Granzyme B and perforin, and have the ability to eliminate cancer cells [33, 34] (Figure 1). Therefore, the coordination between TCF1⁺ CD8⁺ T cells and Tim‐3⁺ CD8⁺ T cells is crucial for optimal anti‐tumor immune responses.

Since several groups independently identified these CD8⁺ T cell subsets, PD‐1⁺TCF1⁺ CD8⁺ T cells are referred to as “stem‐like,” “memory‐like,” “precursor exhausted,” “progenitor exhausted,” “progenitor‐like,” or “stem cell‐like progenitor” CD8⁺ T cells [35, 36], whereas PD‐1⁺Tim‐3⁺TCF1⁻ CD8⁺ T cells are referred to as “terminally differentiated” or “terminally exhausted” CD8⁺ T cells. In this review, we use the term “stem‐like” CD8⁺ T cells for TCF1⁺ CD8⁺ T cells and “terminally differentiated” CD8⁺ T cells for Tim‐3⁺ CD8⁺ T cells, because TCF1⁺ CD8⁺ T cells have the ability to self‐renew and produce Tim‐3⁺ CD8⁺ T cells that have cytotoxic function, with features distinct from effector CD8⁺ T cells generally generated in acute response.

5. The Transcription Factor TOX Is Crucial for Chronic Immune Responses Including Anti‐Tumor Responses

Several groups have demonstrated that the transcription factor thymocyte selection‐associated high mobility group box (TOX) is required for CD8 ⁺ T cells to acquire an exhausted phenotype by inducing the expression of inhibitory receptors such as PD‐1 and Tim‐3 in response to chronic antigen stimulation in tumor‐bearing or chronic virus‐infected mice [37, 38, 39, 40, 41]. In contrast, effector CD8 ⁺ T cells generated under acute infection did not express TOX, indicating that TOX expression is induced specifically under chronic conditions and might be associated with the “dysfunction” of CD8 ⁺ T cells. However, interestingly, TOX‐deficient tumor‐specific CD8 ⁺ T cells with reduced expression of inhibitory receptors did not recover their cytotoxic function. Instead, TOX‐deficient tumor‐specific CD8 ⁺ T cells became overstimulated in the TME and could not persist due to activation‐induced cell death [37]. Furthermore, TOX was found to be critical for the differentiation and persistence of TCF1 ⁺ stem‐like CD8 ⁺ T cells [39, 40, 41].

What is the relationship between TOX expression levels and anti‐tumor immunity? Although complete deletion of the Tox gene leads to a defect in the persistence of tumor‐specific CD8 ⁺ T cells, tumor‐specific CD8 ⁺ T cells with a heterozygous deletion in the Tox gene show enhanced anti‐tumor effects compared with wild‐type CD8 ⁺ T cells that recognize the same antigen [41]. This result indicates that partial downregulation of TOX has a beneficial effect on anti‐tumor immunity [37, 38, 39, 40, 41]. Since TOX is known to be critical for the maintenance of TCF1 ⁺ stem‐like CD8 ⁺ T cells, reduced expression of TOX may render stem‐like CD8 ⁺ T cells more susceptible to differentiation into terminally differentiated CD8 ⁺ T cells.

Thus, TOX ⁺ exhausted CD8 ⁺ T cells play a pivotal role in anti‐tumor immune responses, and we suggest that “CD8 ⁺ T cell exhaustion” is no longer appropriate terminology to describe these functional cells. We therefore propose using the term “chronic CD8 ⁺ T cell response” to describe immune responses mediated by TOX ⁺ stem‐like and terminally differentiated CD8 ⁺ T cells in cancer and chronic infection. Likewise, “acute CD8 ⁺ T cell response” should be used to describe immune responses mediated by TOX ⁻ effector CD8 ⁺ T cells in acute infection (Figure 1).

6. CD69 Regulates TOX Expression in Tumor‐Specific CD8 ⁺ T Cells

We recently conducted a study focusing on the function of CD69 in the regulation of tumor‐specific CD8 ⁺ T cells [24]. CD69 was found to be expressed in both stem‐like and terminally differentiated CD8 ⁺ T cells in the TME of tumor‐bearing mice. Our single‐cell transcriptomic analysis defined CD69 as a critical regulator for chronic CD8 ⁺ T cell responses in anti‐tumor immunity, as Cd69 ^−/− tumor‐specific CD8 ⁺ T cells showed reduced levels of TOX. We demonstrated that the generation of Tim‐3⁺ terminally differentiated CD8 ⁺ T cells was increased in Cd69 ^−/− mice, most likely due to lower levels of TOX compared to Cd69 ^+/+ mice, leading to the enhancement of anti‐tumor immunity in the TME. This increased generation of Tim‐3⁺ terminally differentiated CD8 ⁺ T cells was also observed following the administration of anti‐CD69 Ab [24]. These findings suggest that CD69 functional blockade may promote differentiation of stem‐like CD8 ⁺ T cells into terminally differentiated CD8 ⁺ T cells.

Furthermore, we found that CD69 contributes to signaling through the TCR/NFAT pathway, which is required for Tox gene transcription, as nuclear NFAT2 was found to be decreased in CD8 ⁺ T cells from TDLNs of Cd69 ^−/− mice [24]. CD69 has a large extracellular domain with a C‐type lectin‐like domain [42] and four ligand candidates have been identified that interact with CD69: Myl9/12, Galectin‐1, S100A8/9, and oxLDL [ 9, 12, 43, 44, 45, 46, 47, 48, 49]. The association between CD69 and these ligands may play a role in CD69 function (Figure 2). Therefore, we propose a model wherein CD69 may cooperate with certain CD69 ligands to control TCR/NFAT signaling in tumor‐specific CD8 ⁺ T cells, thereby regulating TOX induction and chronic immune responses, including anti‐tumor immunity (Figure 2). However, further studies are required to elucidate the detailed mechanisms.

FIGURE 2.

CD69 structure and function. CD69 is a type‐2 transmembrane protein expressed as a disulfide‐linked homodimer. CD69 is an activation marker of leukocytes that is rapidly i nduced induced after stimulation through the TCR. CD69 contributes to induction of the Tox gene via calcineurin/NFAT signaling. The extracellular domain of CD69 interacts with ligand proteins, such as Myl9/12, Galectin‐1, S100A8/9, and oxLDL. These interactions are thought to regulate the functional roles of CD69.

7. Where Does CD69 Function in the Differentiation of Tumor‐Specific CD8 ⁺ T Cells?

During tumor progression, tumor antigens are presented by DCs in TDLNs, initiating anti‐tumor T cell responses. Therefore, the TDLNs are potential targets for successful immunotherapy [50]. Indeed, two independent reports suggest that TCF1 ⁺ stem‐like CD8 ⁺ T cells are generated in TDLNs, migrate into the TME, and then give rise to cytotoxic Tim‐3⁺ terminally differentiated CD8 ⁺ T cells [51, 52]. Our current study showed that CD69 was expressed in both stem‐like and terminally differentiated CD8 ⁺ T cells in TDLNs [ 24]. Moreover, CD69 deficiency led to decreased expression of TOX in tumor‐specific CD8 ⁺ T cells not only in the TME but also in TDLNs [ 24]. Furthermore, Cd69⁻/⁻ mice showed higher numbers of cytotoxic Tim‐3⁺ terminally differentiated CD8 ⁺ T cells in TDLNs (Figure 3), suggesting that CD69‐mediated TOX regulation occurs at least in the TDLNs and thereby controls tumor‐specific CD8 ⁺ T cell differentiation within TDLNs [ 24].

FIGURE 3.

CD69 action on tumor‐specific CD8⁺ T cells. Tumor antigens are captured by DCs in the TME. DCs migrate into TDLNs and prime naïve tumor‐specific CD8⁺ T cells to differentiate into stem‐like CD8⁺ T cells in TDLNs. In wild‐type mice, stem‐like CD8⁺ T cells are recruited to the TME and give rise to cytotoxic terminally differentiated CD8⁺ T cells (upper). In CD69‐defecient mice, cytotoxic terminally differentiated CD8⁺ T cells are generated in TDLNs and both stem‐like and terminally differentiated CD8⁺ T cells are recruited to the TME, thereby enhancing anti‐tumor immunity (lower).

The importance of TDLNs is highlighted by recent studies showing that TDLNs, rather than the TME, play a crucial role in the efficacy of immune checkpoint blockade (ICB) [53, 54]. CTLA‐4 blockade is involved in T cell priming in TDLNs [ 55, 56, 57], and TDLNs are also essential for PD‐1/PD‐L1 blockade efficacy [53, 54]. These data clearly demonstrate that TDLNs serve as a critical site for CD8 ⁺ T cell differentiation and, therefore, are a crucial determinant of immunotherapy outcomes. CD69 is one of the key molecules that target CD8 ⁺ T cell differentiation in TDLNs.

8. Roles of Resident Memory CD8 ⁺ T Cells in Anti‐Tumor Immune Response

Intratumoral CD8⁺ T cells include a subset that resembles tissue‐resident memory T cells (T_RM) [58, 59], and their increased presence correlates with improved patient outcomes [60]. While traditional T_RM are defined as memory T cells that persist after antigen clearance, CD8⁺ T cells with T_RM‐like features exist in a state of chronic antigen stimulation within the TME, distinguishing them from classical T_RM. These T_RM‐like intratumoral CD8⁺ T cells express characteristic residency markers such as CD69, CD103, and CD49a, with their residency program confirmed through parabiosis studies [61]. Several studies have indicated that the abundance of intratumoral CD103⁺CD8⁺ T_RM is a better predictor of patient survival [62, 63, 64]. Following ICB, increased numbers of CD103⁺ CD8⁺ T_RM have been observed in various cancers [65], although the exact mechanism of T_RM expansion after ICB remains unclear. While CD69 is known to be constitutively expressed on T_RM [9], it is also expressed on recently activated T cells, as well as T cells under chronic stimulation driven by tumor antigens within the TME. Thus, further studies are needed to clarify whether CD69 serves not only as a marker of T_RM but also plays a functional role in T_RM‐mediated anti‐tumor immunity.

9. Benefits of Targeting the CD69 Molecule for Future Cancer Therapy

We have demonstrated that administration of anti‐CD69 Ab results in the retardation of tumor growth with enhanced anti‐tumor immunity through promoted generation of Tim‐3⁺ terminally differentiated CD8 ⁺ T cells [24]. Notably, our data using murine models inoculated with CT26 or B16 showed significant enhancement of anti‐tumor immune responses upon combination therapy with anti‐CD69 and anti‐PD‐1 Abs [24], suggesting that the combination therapy appears to be a compatible approach. While anti‐PD‐1 Ab therapy relies on the proliferation of TCF1 ⁺ stem‐like CD8 ⁺ T cells [33, 34], anti‐CD69 therapy appeared to increase the generation of Tim‐3⁺ terminally differentiated CD8 ⁺ T cells through a different mechanism, rather than promoting the proliferation of TCF1 ⁺ stem‐like CD8 T cells.

While CD69 is expressed on almost all leukocytes, suggesting potential side effects from anti‐CD69 Ab administration [45], whole‐body CD69‐deficient mice are healthy and show no serious defects. Therefore, adverse events associated with anti‐CD69 Ab treatment are expected to be minimal, although careful consideration of side effects is still required. Together, we and others have shown that CD69 plays an important role in anti‐tumor immune responses. These studies thus provide the rationale for clinical evaluation of humanized Abs targeting human CD69 as a new and feasible strategy for cancer immunotherapy.

10. Conclusions

In this review, we have summarized the current understanding of cytotoxic CD8 ⁺ T cell subset differentiation under tumor progression and highlighted the crucial role of chronic CD8 ⁺ T cell responses in anti‐tumor immunity. We and others have shown that CD69 deficiency enhances anti‐tumor immune responses, with our studies specifically revealing CD69's role in controlling chronic CD8 ⁺ T cell responses. Mechanistically, CD69 appears to regulate TCR signaling and consequently modulates the expression of the transcription factor TOX in tumor‐specific CD8 ⁺ T cells within the TDLNs. When CD69 function is blocked, tumor‐specific CD8 ⁺ T cells express low levels of TOX, promoting the differentiation of stem‐like CD8 ⁺ T cells into functional terminally differentiated CD8 ⁺ T cells and thereby enhancing anti‐tumor immune responses. Given that TDLNs are major sites for the development of anti‐tumor immunity, therapeutic approaches targeting CD8 ⁺ T cell subsets in TDLNs, such as anti‐CD69 Ab therapy, represent a promising strategy for cancer treatment. Notably, targeting CD69 introduces a novel paradigm in cancer immunotherapy through its unique mechanism of action: controlling chronic CD8 ⁺ T cell differentiation states to enhance anti‐tumor immunity.

Author Contributions

Ryo Koyama‐Nasu: conceptualization, funding acquisition, writing – original draft, writing – review and editing. Yangsong Wang: writing – review and editing. Hinata Miyano: writing – review and editing. Motoko Y. Kimura: conceptualization, funding acquisition, supervision, writing – original draft, writing – review and editing.

Ethics Statement

All research protocols, including animal experiments, were approved by the Chiba University Review Board.

Consent

The authors have nothing to report.

Conflicts of Interest

The authors declare no conflicts of interest.