Manganese is critical for antitumor immune responses via cGAS-STING and improves the efficacy of clinical immunotherapy

CD8⁺ T cell-mediated cancer clearance is often suppressed by the interaction between inhibitory molecules like PD-1 and PD-L1, an interaction acts like brakes to prevent T cell overreaction under normal conditions but is exploited by tumor cells to escape the immune surveillance. Immune checkpoint inhibitors have revolutionized cancer therapeutics by removing such brakes. Unfortunately, only a minority of cancer patients respond to immunotherapies presumably due to inadequate immunity. Antitumor immunity depends on the activation of the cGAS-STING pathway, as STING-deficient mice fail to stimulate tumor-infiltrating dendritic cells (DCs) to activate CD8⁺ T cells. STING agonists also enhance natural killer (NK) cells to mediate the clearance of CD8⁺ T cell-resistant tumors. Therefore STING agonists have been intensively sought after. We previously discovered that manganese (Mn) is indispensable for the host defense against cytosolic dsDNA by activating cGAS-STING. Here we report that Mn is also essential in innate immune sensing of tumors and enhances adaptive immune responses against tumors. Mn-insufficient mice had significantly enhanced tumor growth and metastasis, with greatly reduced tumor-infiltrating CD8⁺ T cells. Mechanically, Mn²⁺ promoted DC and macrophage maturation and tumor-specific antigen presentation, augmented CD8⁺ T cell differentiation, activation and NK cell activation, and increased memory CD8⁺ T cells. Combining Mn²⁺ with immune checkpoint inhibition synergistically boosted antitumor efficacies and reduced the anti-PD-1 antibody dosage required in mice. Importantly, a completed phase 1 clinical trial with the combined regimen of Mn²⁺ and anti-PD-1 antibody showed promising efficacy, exhibiting type I IFN induction, manageable safety and revived responses to immunotherapy in most patients with advanced metastatic solid tumors. We propose that this combination strategy warrants further clinical translation.Subject terms: Pattern recognition receptors, Immunosurveillance     

The presence of LC3B puncta and HMGB1 expression in malignant cells correlate with the immune infiltrate in breast cancer

Several cell-intrinsic alterations have poor prognostic features in human breast cancer, as exemplified by the absence of MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 β)-positive puncta in the cytoplasm (which indicates reduced autophagic flux) or the loss of nuclear HMGB1 expression by malignant cells. It is well established that breast cancer is under strong immunosurveillance, as reflected by the fact that scarce infiltration of the malignant lesion by CD8⁺ cytotoxic T lymphocytes or comparatively dense infiltration by immunosuppressive cell types (such as FOXP3⁺ regulatory T cells or CD68⁺ tumor-associated macrophages), resulting in low CD8⁺:FOXP3⁺ or CD8⁺:CD68⁺ ratios, has a negative prognostic impact. Here, we reveal the surprising finding that cell-intrinsic features may influence the composition of the immune infiltrate in human breast cancer. Thus, the absence of LC3B puncta is correlated with intratumoral (but not peritumoral) infiltration by fewer CD8⁺ cells and more FOXP3⁺ or CD68⁺ cells, resulting in a major drop in the CD8⁺:FOXP3⁺ or CD8⁺:CD68⁺ ratios. Moreover, absence of HMGB1 expression in nuclei correlated with a general drop in all immune effectors, in particular FOXP3⁺ and CD68⁺ cells, both within the tumor and close to it. Combined analysis of LC3B puncta and HMGB1 expression allowed for improved stratification of patients with respect to the characteristics of their immune infiltrate as well as overall and metastasis-free survival. It can be speculated that blocked autophagy in, or HMGB1 loss from, cancer cells may favor tumor progression due to their negative impact on anticancer immunosurveillance.     

DEAD/H (Asp–Glu–Ala–Asp/His) box polypeptide 3, X-linked is an immunogenic target of cancer stem cells

Accumulating evidence suggests that most solid malignancies consist of heterogeneous tumor cells and that a relatively small subpopulation, which shares biological features with stem cells, survives through potentially lethal stresses such as chemotherapy and radiation treatment. Since the survival of this subpopulation of cancer stem cells (CSC) plays a critical role in recurrence, it must be eradicated in order to cure cancer. We previously reported that vaccination with CD133⁺ murine melanoma cells exhibiting biological CSC features induced CSC-specific effector T cells. These were capable of eradicating CD133⁺ tumor cells in vivo, thereby curing the parental tumor. In the current study, we indicated that DEAD/H (Asp–Glu–Ala–Asp/His) box polypeptide 3, X-linked (DDX3X) is an immunogenic protein preferentially expressed in CD133⁺ tumor cells. Vaccination with DDX3X primed specific T cells, resulting in protective and therapeutic antitumor immunity. The DDX3X-primed CD4⁺ T cells produced CD133⁺ tumor-specific IFNγ and IL-17 and mediated potent antitumor therapeutic efficacy. DDX3X is expressed in various human cancer cells, including lung, colon, and breast cancer cells. These results suggest that anti-DDX3X immunotherapy is a promising treatment option in efforts to eradicate CSC in the clinical setting.     

The capacity of CD4+ Vγ9Vδ2 T cells to kill cancer cells correlates with co-expression of CD56

Human Vγ9Vδ2 T cells are a unique T-cell type, and data from recent studies of Vγ9Vδ2 T cells emphasize their potential relevance to cancer immunotherapy. Vγ9Vδ2 T cells exhibit dual properties since they are both antigen-presenting cells and cytotoxic toward cancer cells. The majority of Vγ9Vδ2 T cells are double-negative for the co-receptors CD4 and CD8, and only 20–30% express CD8. Even though they are mostly neglected, a small fraction of Vγ9Vδ2 T cells also express the co-receptor CD4. Here the authors show that CD4⁺ Vγ9Vδ2 T cells comprise 0.1–7% of peripheral blood Vγ9Vδ2 T cells. These cells can be expanded in vitro using zoledronic acid, pamidronic acid or CD3 antibodies combined with IL-2 and feeder cells. Unlike most conventional CD4⁺ αβ T cells, CD4⁺ Vγ9Vδ2 T cells are potently cytotoxic and can kill cancer cells, which is here shown by the killing of cancer cell lines of different histological origins, including breast cancer, prostate cancer and melanoma cell lines, upon treatment with zoledronic acid. Notably, the killing capacity of CD4⁺ Vγ9Vδ2 T cells correlates with co-expression of CD56.     

Homeostasis of peripheral FoxP3+ CD4+ regulatory T cells in patients with early and late stage breast cancer

FoxP3 ⁺ CD4 ⁺ regulatory T cells (Tregs) are important mediators of peripheral immune tolerance, acting via multiple mechanisms to suppress cellular immunity including antitumor responses. Although therapeutic strategies have been proposed to deplete Tregs in patients with breast cancer and other malignancies, dynamic changes in the Treg compartment as a function of stage and treatment of breast cancer remain poorly understood. Here, we evaluated peripheral blood CD4⁺ T cells and FoxP3⁺ CD4⁺ T cells from 45 patients with early or late stage breast cancer and compared percentages, absolute counts, and Treg function to those from healthy volunteers (HV) of comparable age. Patients having completed adjuvant chemotherapy and patients with metastatic cancer exhibited significantly lower absolute CD4 counts and significantly higher percentages of FoxP3⁺ CD4⁺ T cells. In contrast, the absolute counts of circulating FoxP3⁺ CD4⁺ T cells did not differ significantly among early stage patients, late stage patients, or HV. Functionally, FoxP3⁺ CD4⁺ T cells from all donor groups similarly expressed CTLA-4 and failed to secrete IFN-γ in response to stimulation. Thus, although Tregs comprise an increased percentage of circulating CD4⁺ T cells in patients with metastatic breast cancer and patients in remission after completing the adjuvant chemotherapy, the systemic Treg pool, as measured by absolute counts, appears relatively constant regardless of disease stage or treatment status. Total CD4⁺ T cell counts are not constant, however, suggesting that homeostatic mechanisms, or susceptibility to cytotoxic or malignant insults, fundamentally differ for regulatory and non-regulatory CD4⁺ T cells.     

Knockdown of CDK5 down-regulates PD-L1 via the ubiquitination-proteasome pathway and improves antitumor immunity in lung adenocarcinoma

Although immunotherapy (anti-PD-1/PD-L1 antibodies) has been approved for clinical treatment of lung cancer, only a small proportion of patients respond to monotherapy. Hence, understanding the regulatory mechanism of PD-L1 is particularly important to identify optimal combinations. In this study, we found that inhibition of CDK5 induced by shRNA or CDK5 inhibitor leads to reduced expression of PD-L1 protein in human lung adenocarcinoma cells, while the mRNA level is not substantially altered. The PD-L1 protein degradation is mediated by E3 ligase TRIM21 via ubiquitination-proteasome pathway. Subsequently, we studied the function of CDK5/PD-L1 axis in LUAD. In vitro, the absence of CDK5 in mouse Lewis lung cancer cell (LLC) has no effect on cell proliferation. However, the attenuation of CDK5 or combined with anti-PD-L1 greatly suppresses tumor growth in LLC implanted mouse models in vivo. Disruption of CDK5 elicits a higher level of CD3⁺, CD4⁺ and CD8⁺ T cells in spleens and lower PD-1 expression in CD4⁺ and CD8⁺ T cells. Our findings highlight a role for CDK5 in promoting antitumor immunity, which provide a potential therapeutic target for combined immunotherapy in LUAD.     

Signaling through CD40 enhances cytotoxic T lymphocyte generation by CD8+ T cells from mice bearing large tumors

Recent studies have demonstrated the importance of CD40/CD154 (CD40L) interactions for the generation of cell-mediated antitumor immune responses. Here we show that signaling via CD40 (through the use of the activating anti-CD40 mAb, 1C10) can actually promote the in vitro generation of CTL activity by CD8⁺ splenic T cells from mice bearing a large MOPC-315 tumor. Anti-CD40 mAb had to be added at the initiation of the stimulation cultures of tumor-bearing splenic cells in order to realize fully its potentiating activity for cytotoxic T lymphocyte (CTL) generation, suggesting that signaling through CD40 is important at the inductive stage of antitumor cytotoxicity. Moreover, anti-CD40 mAb was found to enhance the expression of the B7-2 (CD86) and, to a lesser extent, the B7-1 (CD80) costimulatory molecules on B220⁺ cells (i.e., B cells), and B7-2 and, to a lesser extent, B7-1 molecules played an important role in the potentiating effect of anti-CD40 mAb for CTL generation by tumor-bearer splenic cells. Furthermore, B220⁺ cells were found to be essential for the potentiating effect of anti-CD40 mAb, as depletion of B220⁺ cells at the inductive stage completely abrogated the ability of anti-CD40 mAb to enhance CTL generation. Thus, signaling through CD40 enhances CTL generation by CD8⁺ T cells from tumor-bearing mice by a mechanism that involves the up-regulation of B7-2 and, to a lesser extent, B7-1 expression on B220⁺ cells. Received: 23 December 1998 / Accepted: 22 February 1999     

PD-1+ immune cell infiltration inversely correlates with survival of operable breast cancer patients

The programmed death-1 (PD-1) molecule is mainly expressed on functionally “exhausted” CD8⁺ T cells, dampening the host antitumor immune response. We evaluated the ratio between effective and regulatory T cells (Tregs) and PD-1 expression as a prognostic factor for operable breast cancer patients. A series of 218 newly diagnosed invasive breast cancer patients who had undergone primary surgery at Ruijin Hospital were identified. The influence of CD8⁺ cytotoxic T lymphocytes, FOXP3⁺ (Treg cell marker), and PD-1⁺ immune cell counts on prognosis was analyzed utilizing immunohistochemistry. Both PD-1⁺ immune cells and FOXP3⁺ Tregs counts were significantly associated with unfavorable prognostic factors. In bivariate, but not multivariate analysis, high tumor infiltrating PD-1⁺ cell counts correlated with significantly shorter patient survival. Our results suggest a prognostic value of the PD-1⁺ immune cell population in such breast cancer patients. Targeting the PD-1 pathway may be a feasible approach to treating patients with breast cancer.     

Adoptive cellular immunotherapy for the treatment of patients with breast cancer: A meta-analysis

BackgroundTo evaluate the therapeutic efficacy of dendritic cells (DC) alone, cytokine-induced killer (CIK) cells alone and the combination of DC and CIK cells in the treatment of breast cancer, we performed a systemic review of the relevant published clinical studies, collectively referred to as DC-CIK cell therapy.MethodsSix hundred thirty-three patients with breast cancer were assigned to cohorts, and a meta-analysis was conducted.ResultsThe treatment of breast cancer with DC-CIK cells was associated with a significantly improved 1-year survival (P = 0.0001). The Karnofsky performance status scale of the patients treated with DC-CIK cells was significantly improved compared with that of the non-DC-CIK group (P < 0.0001). The percentage of T cells (CD3⁺, CD4⁺ and CD4⁺CD8⁺), CD16⁺ monocytes, and CD3⁺CD56⁺ natural killer T cells in the peripheral blood of cancer patients was significantly increased (P ≤ 0.05), whereas the percentage of CD4⁺CD25⁺ regulatory T cells was not significantly decreased (P = 0.32) in the DC-CIK treatment group compared with the non-DC-CIK group. The levels of interleukin-2, interleukin-12, tumor necrosis factor-α, interferon-γ, and nucleolar organizer region protein in the peripheral blood of cancer patients, which reflect immune function, were significantly increased (P < 0.001) after DC-CIK cell treatment. Furthermore, after DC-CIK treatment, the average levels of the alpha-fetoprotein, cancer antigen embryonic antigen and carbohydrate antigen tumor markers were decreased (P < 0.00001).ConclusionsDC-CIK cell therapy markedly prolongs survival time, enhances immune function, and improves the efficacy of the treatment of breast cancer patients.     

小鼠乳腺表达抗PD-1抗体对其脾脏T细胞的影响

为从细胞水平研究小鼠乳腺表达抗PD-1抗体对转基因鼠脾脏T细胞表面抗原蛋白、细胞因子表达、脾脏CD4⁺T细胞增殖以及增殖相关通路的影响,将8周龄未经历过怀孕的和18周龄经历过哺乳的表达抗人PD-1抗体的转基因小鼠分成两组,每组以转基因阴性鼠为对照,提取脾脏淋巴细胞,检测脾脏淋巴细胞的变化。与转基因阴性小鼠相比,乳腺表达抗PD-1抗体的转基因小鼠的免疫系统中的脾脏T细胞的效应T细胞比例上升,Treg细胞比例下降,CD4⁺T细胞表达的IFN-γ、IL-17以及IL-2有不同程度的增加。IL-4、IL-10以及TGF-β都没有发生变化。与T细胞刺激相关的一些细胞表面的蛋白分子也没有引起变化。转基因阳性鼠和转基因阴性鼠中T细胞增殖没有显著性差异,转基因阳性鼠中PI3K/Akt/mTOR和Ras/MEK/ERK这两条通路上的磷酸化蛋白只有部分表达上调,整个通路没有完全上调。结果表明,转基因小鼠作为表达抗PD-1抗体这类免疫系统相关单克隆抗体的宿主是可行的。     

Blocking of the PD-1/PD-L1 interaction by a novel cyclic peptide inhibitor for cancer immunotherapy

The interaction of PD-1/PD-L1 allows tumor cells to escape from immune surveillance. Clinical success of the antibody drugs has proven that blockade of PD-1/PD-L1 pathway is a promising strategy for cancer immunotherapy. Here, we developed a cyclic peptide C8 by using Ph.D.-C7 C phage display technology. C8 showed high binding affinity with h PD-1 and could effectively interfere the interaction of PD-1/PD-L1. Furthermore, C8 could stimulate CD8+T cell activation in human peripheral blood mononuclear cells(PBMCs). We also observed that C8 could suppress tumor growth in CT26 and B16-OVA, as well as anti-PD-1 antibody resistant B16 mouse model. CD8+T cells infiltration significantly increased in tumor microenvironment, and IFN-γ secretion by CD8+T cells in draining lymph nodes also increased. Simultaneously, we exploited T cells depletion models and confirmed that C8 exerted anti-tumor effects via activating CD8+T cells dependent manner. The interaction model of C8 with h PD-1 was simulated and confirmed by alanine scanning. In conclusion, C8 shows anti-tumor capability by blockade of PD-1/PD-L1 interaction, and C8 may provide an alternative candidate for cancer immunotherapy.     

T-Cell Autophagy Deficiency Increases Mortality and Suppresses Immune Responses after Sepsis

Background

Although the role of autophagy in sepsis has been characterized in several organs, its role in the adaptive immune system remains to be ascertained. This study aimed to investigate the role of autophagy in sepsis-induced T cell apoptosis and immunosuppression, using knockout mice with T cell specific deletion of autophagy essential gene Atg7.

Methods and Results

Sepsis was induced in a cecal ligation and puncture (CLP) model, with T-cell-specific Atg7-knockout mice compared to control mice. Autophagic vacuoles examined by electron microscopy were decreased in the spleen after CLP. Autophagy proteins LC3-II and ATG7, and autophagosomes and autolysosomes stained by Cyto-ID Green and acridine orange were decreased in CD4⁺ and CD8⁺ splenocytes at 18 h and 24 h after CLP. This decrease in autophagy was associated with increased apoptosis of CD4⁺ and CD8⁺ after CLP. Moreover, mice lacking Atg7 in T lymphocytes showed an increase in sepsis-induced mortality, T cell apoptosis and loss of CD4⁺ and CD8⁺ T cells, in comparison to control mice. This was accompanied by suppressed cytokine production of Th1/Th2/Th17 by CD4⁺ T cells, reduced phagocytosis in macrophages and decreased bacterial clearance in the spleen after sepsis.

Conclusion

These results indicated that sepsis led to down-regulation of autophagy in T lymphocytes, which may result in enhanced apoptosis induction and decreased survival in sepsis. Autophagy may therefore play a protective role against sepsis-induced T lymphocyte apoptosis and immunosuppression.     

Major Histocompatibility Complex Class II+ Invariant Chain Negative Breast Cancer Cells Present Unique Peptides that Activate Tumor-specific T Cells from Breast Cancer Patients

The major histocompatibility complex (MHC) class II-associated Invariant chain (Ii) is present in professional antigen presenting cells where it regulates peptide loading onto MHC class II molecules and the peptidome presented to CD4⁺ T lymphocytes. Because Ii prevents peptide loading in neutral subcellular compartments, we reasoned that Ii⁻ cells may present peptides not presented by Ii⁺ cells. Based on the hypothesis that patients are tolerant to MHC II-restricted tumor peptides presented by Ii⁺ cells, but will not be tolerant to novel peptides presented by Ii⁻ cells, we generated MHC II vaccines to activate cancer patients'' T cells. The vaccines are Ii⁻ tumor cells expressing syngeneic HLA-DR and the costimulatory molecule CD80. We used liquid chromatography coupled with mass spectrometry to sequence MHC II-restricted peptides from Ii⁺ and Ii⁻ MCF10 human breast cancer cells transfected with HLA-DR7 or the MHC Class II transactivator CIITA to determine if Ii⁻ cells present novel peptides. Ii expression was induced in the HLA-DR7 transfectants by transfection of Ii, and inhibited in the CIITA transfectants by RNA interference. Peptides were analyzed and binding affinity predicted by artificial neural net analysis. HLA-DR7-restricted peptides from Ii⁻ and Ii⁺ cells do not differ in size or in subcellular location of their source proteins; however, a subset of HLA-DR7-restricted peptides of Ii⁻ cells are not presented by Ii⁺ cells, and are derived from source proteins not used by Ii⁺ cells. Peptides from Ii⁻ cells with the highest predicted HLA-DR7 binding affinity were synthesized, and activated tumor-specific HLA-DR7⁺ human T cells from healthy donors and breast cancer patients, demonstrating that the MS-identified peptides are bonafide tumor antigens. These results demonstrate that Ii regulates the repertoire of tumor peptides presented by MHC class II⁺ breast cancer cells and identify novel immunogenic MHC II-restricted peptides that are potential therapeutic reagents for cancer patients.Cancer vaccines are a promising tool for cancer treatment and prevention because of their potential for inducing tumor-specific responses in conjunction with minimal toxicity for healthy cells. Cancer vaccines are based on the concept that tumor cells synthesize multiple peptides that are potential immunogens, and that with the appropriate vaccine protocol, these peptides will activate an efficacious antitumor response in the patient. Much effort has been invested in identifying and testing tumor-encoded peptides, particularly peptides presented by major histocompatibility complex (MHC)¹ class I, molecules capable of activating CD8⁺ T-cells that directly kill tumor cells (1, 2). Fewer studies have been devoted to identifying MHC class II-restricted peptides for the activation of tumor-reactive CD4⁺ T-cells despite compelling evidence that Type 1 CD4⁺ T helper cells facilitate the optimal activation of CD8⁺ T-cells and the generation of immune memory, which is likely to be essential for protection from metastatic disease.Activation of CD4⁺ T cells requires delivery of a costimulatory signal plus an antigen-specific signal consisting of peptide bound to an MHC II molecule. Most cells do not express MHC II or costimulatory molecules, so CD4⁺ T cells are typically activated by professional antigen presenting cells (APC), which endocytose exogenously synthesized antigen and process and present it in the context of their own MHC II molecules. This processing and presentation process requires Invariant chain (Ii), a molecule that is coordinately synthesized with MHC II molecules and prevents the binding and presentation of APC-encoded endogenous peptides (3, 4). As a result, tumor-reactive CD4⁺ T cells are activated to tumor peptides generated by the antigen processing machinery of professional APC, rather than peptides generated by the tumor cells. Because of the potential discrepancy in peptide generation between professional APC and tumor cells, and the critical role of Ii in preventing the presentation of endogenous peptides, we have generated “MHC II cancer vaccines” that consist of Ii⁻ tumor cells transfected with syngeneic MHC class II and CD80 genes. We reasoned that MHC II⁺Ii⁻CD80⁺ tumor cells may present a novel repertoire of MHC II-restricted tumor peptides that are not presented by professional APC, and therefore may be highly immunogenic. Once activated, CD4⁺ T cells produce IFNγ and provide help to CD8⁺ T cells and do not need to react with native tumor cells. Therefore, the MHC II vaccines have the potential to activate CD4⁺ Th1 cells that facilitate antitumor immunity. In vitro (5) and in vivo (5–7) studies with mice support this conclusion. In vitro studies with human MHC II vaccines further demonstrate that the absence of Ii facilitates the activation of MHC II-restricted tumor-specific CD4⁺ type 1 T cells of HLA-DR-syngeneic healthy donors and cancer patients, and that the vaccines activate CD4⁺ T cells with a distinct repertoire of T cell receptors (8–12). A critical negative role for Ii is also supported by studies of human acute myelogenous leukemia (AML). High levels of class II-associated invariant chain peptide (CLIP), a degradation product of Ii, by leukemic blasts is associated with poor patient prognosis (13, 14), whereas down-modulation of CLIP on AML cells increases the activation of tumor-reactive human CD4⁺ T cells (14, 15).We have now used mass spectrometry to identify MHC II-restricted peptides from MHC II⁺Ii⁻ and MHC II⁺Ii⁺ human breast cancer cells to test the concept that the absence of Ii facilitates the presentation of unique immunogenic MHC II-restricted peptides. We report here that a subset of MHC II-restricted peptides from HLA-DR7⁺ breast cancer cells are unique to Ii⁻ cells and are derived from source proteins not used by Ii⁺ cells. Ii⁻ peptides have high binding affinity for HLA-DR7 and activate tumor-specific T-cells from the peripheral blood of healthy donors and breast cancer patients. This is the first study to compare the human tumor cell MHC II peptidome in the absence or presence of Ii and to demonstrate that MHC II⁺Ii⁻ tumor cells present novel immunogenic MHC II-restricted peptides that are potential therapeutic reagents for cancer patients.     

Local injections of OK432 can help the infiltration of adoptively transferred CD8+ T cells into the tumor sites and synergistically induce the local production of Th1-type cytokines and CXC3 chemokines

The effect of local injections with streptococcal preparation OK432 on the antitumor effect induced by adoptive immunotherapy (AIT) was investigated. Draining lymph node cells on day 14 after B7-P815 inoculation were used for AIT after in vitro stimulation. AIT on days 7 and 10 showed no effect on the growth of s.c. established P815 mastocytoma, but local injections with OK432 into the tumor sites on days 3, 6 and 9 resulted in a moderate antitumor effect. On the other hand, the combination therapy significantly suppressed tumor growth, and the tumor-bearing mice survived longer than those receiving only one of the treatment modalities. The significant infiltration of CD4⁺ or CD8⁺ T cells and multiple necrosis in the tumor sites were observed only when the tumor-bearing mice were treated with the combination therapy. In addition, a transfer experiment using labeled effector cells revealed these infiltrated CD8⁺ T cells and CD4⁺ T cells to be derived from the donor and the host respectively. More importantly, the combination therapy clearly led to higher expression of the mRNA for Th1-type cytokines and CXC3 chemokines in the tumor sites than resulted from each of the treatment modalities alone. Collectively, these results indicate that local injections with OK432 can help the infiltration of adoptively transferred CD8⁺ T cells into the tumor sites and synergistically induce the local production of the Th1-type cytokines and CXC3 chemokines. Received: 3 April 2000 / Accepted: 5 May 2000     

Inhibition of breast cancer growth via miR-7 suppressing ALDH1A3 activity concomitant with decreasing breast cancer stem cell subpopulation

Breast cancer patients with high expression of aldehyde dehydrogenases (ALDHs) cell population have higher tolerability to chemotherapy since the cells posses a characteristic of breast cancer stem cells (BCSCs) that are resistant to conventional chemotherapy. In this study, we found that the ALDH-positive cells were higher in CD44⁺CD24⁻ and CD44⁺CD24⁻ESA⁺BCSCs than that in both BT549 and MDA-MB-231 cell lines but microRNA-7 (miR-7) level was lower in CD44⁺CD24⁻ and CD44⁺CD24⁻ESA⁺BCSCs than that in MDA-MB-231 cells. Moreover, miR-7 overexpression in MDA-MB-231 cells decreased ALDH1A3 activity by miR-7 directly binding to the 3′-untranslated region of ALDH1A3; while the ALDH1A3 expression was downregulated in MDA-MB-231 cells, the expressions of CD44 and Epithelium Specific Antigen (ESA) were reduced along with decreasing the BCSC subpopulation. Significantly, enforced expression of miR-7 in CD44⁺CD24⁻ESA⁺BCSC markedly inhibited the BCSC-driven xenograft growth in mice by decreasing an expression of ALDH1A3. Collectively, the findings demonstrate the miR-7 inhibits breast cancer growth via suppressing ALDH1A3 activity concomitant with decreasing BCSC subpopulation. This approach may be considered for an investigation on clinical treatment of breast cancers.     

Effective killing of cells expressing CD276 (B7-H3) by a bispecific T cell engager based on a new fully human antibody

The pancaner molecule CD276 (B7-H3) is an attractive target for antibody based therapy. We identified from a large (10¹¹) phage-displayed single-chain variable fragment (scFv) library, a fully human antibody, B11, which bound with high avidity (K_D=0.4 nM) to CD276. B11 specifically bound to the V1/V2 domain of CD276 and competed with the antibody 8H9 (Omburtamab). It was used to design an IgG-format bispecific T cell engager B11-BiTE, which was more effective than 8H9-BiTE in 14 different cancer cell lines. B11-BiTE also exhibited strong ADCC/ADCP. Therefore, the fully human B11-BiTE is a promising candidate for treatment of tumors expressing CD276.     

Effective killing of cells expressing CD276 (B7-H3) by a bispecific T cell engager based on a new fully human antibody

The pancaner molecule CD276 (B7-H3) is an attractive target for antibody based therapy. We identified from a large (10¹¹) phage-displayed single-chain variable fragment (scFv) library, a fully human antibody, B11, which bound with high avidity (K_D=0.4 nM) to CD276. B11 specifically bound to the V1/V2 domain of CD276 and competed with the antibody 8H9 (Omburtamab). It was used to design an IgG-format bispecific T cell engager B11-BiTE, which was more effective than 8H9-BiTE in 14 different cancer cell lines. B11-BiTE also exhibited strong ADCC/ADCP. Therefore, the fully human B11-BiTE is a promising candidate for treatment of tumors expressing CD276.     

Autophagy,citrullination and cancer

A cell needs to maintain a balance between biosynthesis and degradation of cellular components to maintain homeostasis. There are 2 pathways, the proteasome, which degrades short-lived proteins, and the autophagy/lysosomal pathway, which degrades long-lived proteins and organelles. Both of these pathways are also involved in antigen presentation or the effective delivery of peptides to MHC molecules for presentation to T cells. Autophagy (macroautophagy) is a key player in providing substantial sources of citrullinated peptides for loading onto MHC-II molecules to stimulate CD4⁺ T cell responses. Stressful conditions in the tumor microenvironment induce autophagy in cancer cells as a mechanism to promote their survival. We therefore investigated if citrullinated peptides could stimulate CD4⁺ T cell responses that would recognize these modifications produced during autophagy within tumor cells. Focusing on the intermediate filament protein VIM (vimentin), we generated citrullinated VIM peptides for immunization experiments in mice. Immunization with these peptides induced CD4⁺ T cells in response to autophagic tumor targets. Remarkably, a single immunization with modified peptide, up to 14 d after tumor implant, resulted in long-term survival in 60% to 90% of animals with no associated toxicity. These results show how CD4⁺ cells can mediate potent antitumor responses against modified self-epitopes presented on tumor cells, and they illustrate for the first time how the citrullinated peptides produced during autophagy may offer especially attractive vaccine targets for cancer therapy.