Potentiating Immune System by Hyperthermia



Hyperthermia enhances the host immune responses against cancer through several mechanisms; activating immune cells (e.g., natural killer cells, dendritic cells, and cytotoxic T lymphocytes), canceling immune suppression, altering cell-surface molecules on cancer cells, and modifying adhesion molecules on immune cells and endothelial cells. This chapter discusses the positive effects of hyperthermia on the host immune system.


Hyperthermia Fever-range hyperthermia Mild hyperthermia NK cell Dendritic cell Cytotoxic T cell Regulatory T cell 

12.1 Introduction

The immune system distinguishes between self and non-self to identify and remove foreign substances, including cancer cells. Innate immunity, unlike acquired immunity, can immediately respond to foreign substances without prior exposure to antigens. Natural killer (NK) cells are at the front line of innate immune response in attacking cancer cells. Dendritic cells (DCs) capture, process, and display cancer antigens in order to transmit activation signals to the antigen-specific acquired immune response, which cytotoxic T lymphocytes (CTLs) are major players in. Regulatory T cells (Treg) counteract the anti-cancer immune responses described above.

Hyperthermia treatment raises the temperature of target tissues to 42.5 °C or above when destroying cancer cells, and to 38–41 °C (fever-range hyperthermia or mild hyperthermia) when increasing blood flow and activating immune reactions. When a cancer mass is heated over 42.5 °C, both cancer and immune cells in the center part of the lesion are destroyed, whereas the surrounding areas form a thermal gradient to generate a field of 38–41 °C, where immune reactions are activated as fever-range hyperthermia.

The effects of fever-range hyperthermia on immune cells and their networks are discussed in this chapter.

12.2 Enhancing Anti-Cancer Activity of NK Cells Using Hyperthermia

NK cells express many activating and inhibitory receptors, which recognize specific ligands expressed on target cells (Fig. 12.1) [1]. Most activating ligands on target cells are induced by genomic damage from viral infections and malignant transformations. The expression and processing of ligands are enhanced when thermal stress is applied to the cells before they are recognized by NK cell-activating receptors. On the other hand, major histocompatibility complex (MHC) class I antigens, which are expressed constitutively on normal cells, are engaged by NK cell-inhibitory receptors. A balance of these activating and inhibitory receptor signals regulates the effector function of NK cells. Interestingly, the expression of MHC class I antigens frequently becomes reduced or lost when cells become malignant or metastatic (Table 12.1) [1]. This decrease or loss of MHC class I antigens hides malignant cells from CTLs, but exposes them to attacks by NK cells due to the attenuated inhibitory signals [2].
Fig. 12.1

The effector function of NK cells is regulated by a balance of activating and inhibitory signals between NK cells and target cells

Table 12.1

Frequencies of reduced MHC class I expression in surgically removed tumors


Low or negative cases/total cases

Percentage (%)


Melanoma, primary




Melanoma, metastases




Grade 2 astrocytoma




Glioblastoma multiforme




Head & neck cancer




Laryngeal cancer




Breast cancer




Lung cancer




Hepatocellular carcinoma




Pancreatic cancer




Gastric cancer




Colorectal cancer




Renal cell cancer




Bladder cancer




Prostatic cancer




Penile cancer




Cervical cancer




Ovary cancer




Bone and soft tissue sarcoma




Osteosarcoma, primary




Osteosarcoma, metastases




Activated NK cells directly attack cancer cells using cytotoxic molecules (e.g., perforin and granzyme), death receptors (e.g., FasL, TRAIL, TNF-α), and antibody-dependent cell-mediated cytotoxicity (ADCC) [1, 2]. They also secrete cytokines such as IFN-γ, TNF-α, GM-CSF and IL-2 to stimulate other immune cells (i.e., DC, T and B lymphocytes), which reinforce acquired immunity by enhancing antigen-specific CTL induction and immunoglobulin production [1, 2].

Hyperthermia increases the distinct clustering of NK cell-activating receptors such as NKG2D on the surface of NK cells [3]. Hyperthermia also increases the expression of NK cell-activating ligands such as the major histocompatibility complex class I-related chain A (MICA but not MHC class I [3]. We also reported previously that hyperthermia can enhance surface expressions of MICA but not of MHC class I in several cancer cell lines (Fig. 12.2a and b) [4]. Furthermore, the increased expression of MICA/B in heated cancer cells correlates with the increased NK cell cytotoxicity against them (Fig. 12.2c and d) [4].
Fig. 12.2

Frequencies of cells expressing MICA/B (a) and MHC class I (b). Human cancer cell lines were kept at 37 °C (gray column) or exposed to 42 °C (black column) for 16 h. SKBR3 were derived from breast carcinoma. Daudi and Raji were derived from Burkitt lymphoma. C1AK and UB2MT were established by us from carcinomas of colon and uterine body, respectively. Both the cytotoxicity of NK cells to UB2MT (c) and the frequency of MICA/B-positive UB2MT cells (d) were increased by thermal treatment. The number and cytotoxicity of NK cells were increased by 14-day cultivation using BINKIT (Biotherapy Institute of Japan, Tokyo, Japan) [22]. The cytotoxicity of the expanded NK cells was measured against UB2MT cells at an effector-to-target ratio of 6:1 using a calcein-AM release assay measured by TERASCAN VP (Minerva Tech., Tokyo, Japan) as previously described [22]. Representative results are shown; similar results were obtained in three independent experiments. Based on [4]

Hyperthermia also increases the MICA mRNA levels in tumors, which correlate with increased sensitivity to cytolysis [3]. The interaction between NKG2D and MICA/B is just an example, and interactions between other NK cell-activating or inhibitory receptors and ligands are also important in enhancing the cytotoxicity of NK cells against cancer cells [5]. Hyperthermia enhances the anti-cancer activity of NK cells through these molecular events.

12.3 Activating DC and CTL by Hyperthermia

DCs are key players in antigen-specific CTL induction for acquired immunity against cancer cells. DCs capture and process cancer antigens originating from damaged cancer cells before presenting them on the cell surface along with MHC antigens. The complex made up of cancer antigens and MHC class II antigens stimulates helper T cells, while the complex made up of cancer antigens and MHC class I antigens induces CTLs via cross-presentation. The resulting CTLs recognize cancer cells through the cancer antigen presented on the MHC class I antigen, and thus attacks them.

Hyperthermia causes the generation of heat shock proteins (HSPs), which serve as molecular chaperones, in cancer cells. HSPs enhance the intracellular transport of cancer antigens generated in proteasomes and are presented on the cell surface along with the MHC class I antigen to induce antigen-specific CTLs [6, 7, 8].

The complex of HSPs and cancer antigens can also be released to the extracellular space when cancer cells are destroyed. Such complexes are captured by DCs, which express HSP receptors, and are processed for cross-presentation to efficiently induce antigen-specific CTLs [7].

DCs and CTLs are activated further by cytokines released from T lymphocytes, which are also activated by hyperthermia [9].

In order to make T cells interact with DCs for CTL induction in lymphoid organs, it is crucial to recruit T cells into lymphoid organs. Thermal stress up-regulates the expression of adhesion molecules such as L-selectin and α4β7 integrin on lymphocytes, and CCL21+ and ICAM-1 on high endothelial venules, which serve as gatekeepers for lymphocyte recruitment into lymphoid organs [10]. Interestingly, CD3-negative CD56-bright NK cells also highly express L-selectin, and these immunomodulatory NK cells may activate DCs to induce CTLs.

Collectively, hyperthermia potentiates the anti-cancer acquired immunity [11, 12, 13].

12.4 Release from Immune Suppression and Hyperthermia

The balance between immunity and tolerance has to be coordinated in a finely tuned manner within the body. Tolerance is critically important in protecting oneself from autoimmune response. Cancer cells, however, take advantage of the tolerance mechanisms to escape immune surveillance; e.g., by utilizing Tregs, which negatively regulate both innate and acquired immunity at least in vitro by suppressing the function of immune cells including T lymphocytes, B lymphocytes, DCs, and NK cells [14, 15].

The number of Tregs within the peripheral blood as well as in cancer tissues is higher in cancer patients compared to that of non-cancer individuals. Moreover, cancer patients with increased numbers of Tregs in cancer tissues have poorer prognoses (cervical, renal, hepatocellular, gastric, and breast cancers and malignant melanoma) and higher risks of recurrence (lung, gastric, and hepatocellular carcinomas and malignant melanoma) [16]. These observations imply that cancer patients are prone to cancer growth due to the immune suppression caused by Tregs.

Malignant melanoma patients who were treated with a combination of hyperthermia and intratumoral injection of immature DCs exhibited lower numbers of Tregs and higher numbers of CTLs compared to patients treated without hyperthermia [17]. Furthermore, patients with hyperthermia showed significant delays in cancer growth. These results suggest that hyperthermia exerts therapeutic benefits at least partly by decreasing the number of Tregs in cancer tissues. We also observed a decrease in the number of Tregs within the peripheral blood after applying fever-range hyperthermia to the upper abdomen of healthy individuals [18]. Hyperthermia could be reducing the number of Tregs by inducing the apoptosis of Tregs, enhancing NK cytotoxicity against Tregs, or inhibiting the induction of Tregs. Further studies are needed to clarify the precise mechanisms of the hyperthermia-induced decrease of Tregs.

Additionally, immune checkpoint modulation is a recent breakthrough in cancer therapy. Therapeutic use of the anti-PD-1 antibody is one of them. We have studied the effects of hyperthermia on the PD-1/PD-L1 immune checkpoint, and observed that the expression of PD-L1 in some cancer cell lines is reduced by exposure to temperatures between 40 and 43 °C (Fig. 12.3). This observation implies the potential of hyperthermia in inhibiting immune checkpoints to sensitize cancer cells to anti-cancer immune reactions. Further studies on this matter shall establish yet another strategy of cancer therapy using hyperthermia.
Fig. 12.3

Thermal stress reduces the PD-L1 expression in cancer cell lines

12.5 Summary

Hyperthermia activates the host immune system against cancer whereas the overall mechanisms are yet to be fully elucidated. So far, several mechanisms of anti-cancer activity by hyperthermia have been postulated (Fig. 12.4): increasing cytotoxicity of effector cells such as NK cells against cancer [18, 19]; enhancing maturation of immature DCs via the production of the HSP-tumor antigen complex in tumors [11, 12, 13]; facilitating antigen presentation to CD8+ T cells for CTL induction [6]; decreasing the numbers of Tregs in tumor tissues and blood [18, 20]; increasing the expression levels of adhesion molecules in endothelial venules [10]; enhancing the trafficking of lymphocytes, such as T cells and cytokine-producing NK cells, to secondary lymphoid tissues [10, 21]; and sensitizing cancer cells to immune effector cells such as NK cells via MICA/B molecules [3, 4].
Fig. 12.4

Regional hyperthermia induces the activation of immune system against cancer. NK cells: natural killer cells, iDC: immature dendritic cells, mDC: mature dendritic cells, CTL: cytotoxic T lymphocytes, Treg: regulatory T cells, HEV: high endothelial venules, HSP: heat shock protein, Ag: antigen, MHC: major histocompatibility complex, MICA/B: major histocompatibility complex class I-related chain A/B, IFN: interferon, ICAM-1: intercellular adhesion molecule 1, CCL: chemokine (C-C motif) ligand, CD: cluster of differentiation. Based on [4]

Better uses of hyperthermia will be achieved by further understanding of its effects on the immune system. Additionally, clinical data on cancer treatments using hyperthermia in combination with other therapies need to be accumulated and evaluated for their respective clinical efficacies. These efforts will lead to the development of efficient combinatory therapies using immune therapy, chemotherapy, radiation, and hyperthermia.


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Copyright information

© Springer Science+Business Media Singapore 2016

Authors and Affiliations

  1. 1.Tokyo ClinicTokyoJapan
  2. 2.Biotherapy Institute of JapanTokyoJapan

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