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Published in final edited form as: Discov Med. 2012 Oct;14(77):229–236.

T Cell Coinhibition and Immunotherapy in Human Breast Cancer

Murali Janakiram 1, Yael M Abadi 1, Joseph A Sparano 1, Xingxing Zang 1
PMCID: PMC4107447  NIHMSID: NIHMS606103  PMID: 23114578

Abstract

Costimulation and coinhibition generated by the B7 family and their receptor CD28 family have key roles in regulating T lymphocyte activation and tolerance. These pathways are very attractive therapeutic targets for human cancers including breast cancer. Gene polymorphisms of B7x (B7-H4/B7S1), PD-1 (CD279), and CTLA-4 (CD152) are associated with increased risk of developing breast cancer although the underlying mechanisms are unclear. In human breast cancer microenvironment, up-regulation of coinhibitory B7/CD28 members B7x, B7-H3 (CD276), and PD-L1 (B7-H1/CD274) on tumor cells as well as PD-1 and PD-L1 on tumor-infiltrating immune cells are emerging as immune evasion pathways. Chemotherapy can affect the expression of these molecules, and therefore may dampen the immune response against breast cancer. Immunotherapy targeting T cell coinhibition as monotherapy or combined with standard therapies are in early stages of clinical development, but hold great promise for treatment of human breast cancer.

Introduction

Despite the development of more effective cytotoxic, hormonal, and anti-HER2 directed therapies for treatment of breast cancer, metastatic disease remains incurable, and one third of women with localized disease will develop metastases and die of the disease (Newman, 2009). Treatment options are particularly limited for patients with “triple negative” breast cancer (TNBC), defined as tumors lacking the expression of the estrogen receptor (ER) and progesterone receptor (PR) and the over-expression of human epidermal growth factor receptor 2 (HER2/neu) protein or HER2/neu gene amplification. TNBC accounts for about 15% of all breast cancers in the United States, and occurs more commonly in younger women and women of black race or Hispanic ethnicity (Brown et al., 2008). TNBC is associated with a higher risk of distant recurrence, earlier time to recurrence, and worse prognosis after recurrence (Liedtke et al., 2008). New therapeutic strategies are therefore needed for breast cancer in general and TNBC in particular. Immunotherapy approaches offer the hope of activating a patient’s immune system to specifically target and eliminate cancer cells even once they have metastasized to distal organs. However, this hope is not reflected by the current reality. The slow pace of progress is likely due to incomplete understanding of immune regulation in the context of cancer, but this understanding is now advancing and being translated into improved anti-tumor immunotherapy. In this review we discuss some of the developing T cell-based immunotherapy strategies in breast cancer, the role of T cell coinhibition in breast cancer outcomes and efficacy of immunological intervention, and current progress in coinhibition blockade as T cell immunotherapy.

T Cell-based Immunotherapy for Breast Cancer

Various immunotherapeutic strategies have been tested in preclinical and clinical settings for the treatment of breast cancer. Among these are vaccines, adoptive T cell transfer, cytokine therapy, and others. Perhaps most successful are “passive immunotherapy” antibodies such as the HER-2/neu blocking antibody trastuzumab (Herceptin, Genentech) which was FDA approved in 1998 and became a standard treatment for HER-2/neu-positive tumors, and which may mediate its antitumor effects in part via antibody dependent cellular cytotoxicity (Goldenberg, 1999). While all treatments likely interact with the T cell arm of the immune system, we focus here specifically on T cell-based immunotherapy and ultimately the role of coinhibition of T cells in disease progression and treatment.

One avenue of T cell immunotherapy in breast cancer is the adoptive transfer of lymphocytes into patients. Such approaches include the transfer of immune cells not specific for breast cancer and transfer of tumor-specific T cells. In a number of non-specific transfer studies, breast cancer patients were treated with allogeneic stem cell transplants in addition to high dose chemotherapy with the goal of inducing graft versus tumor (GVT) responses through the induction of graft versus host disease (GVHD) (reviewed in Wright, 2012). Some patients achieved complete or partial remissions associated with GVHD, including, in some cases, long-term survival (Carella et al., 2005; de Souza et al., 2009; Ueno et al., 1998). However, treatment was also associated with relatively high morbidity and mortality due to GVHD (Bishop et al., 2004; de Souza et al., 2009; Ueno et al., 1998). Adoptive transfer of autologous lymphocytes conditioned in vitro has been better tolerated by patients. Prior studies have shown that administration of high-dose interleukin-2 in combination with lymphocyte-activated killer cells were ineffective in breast cancer (Sparano et al., 1994). Currently in clinical trials, these approaches enrich the patient’s own T cells for specificity to particular breast cancer associated antigens and stimulate them before returning them to the patient. In the first reported study of such, T cells from a HER-2+ breast cancer patient were isolated and stimulated with HER-2 peptide-loaded dendritic cells in vitro and then reinfused to the patient. These T cells successfully eliminated cancer cells disseminated in the bone marrow, but could not penetrate solid metastases (Bernhard et al., 2008). In another study, cytotoxic T lymphocytes (CTLs) isolated from peripheral blood mononuclear cells (PMBC) of four breast cancer patients were stimulated with MUC1 peptides before return to the patients. The therapy was safe but there were no tumor regressions among the two patients that had macroscopic disease. However, in the two patients that were in remission, CTLs isolated after stimulation and reinfusion displayed higher tumor-specific lysis ex vivo than those isolated originally before stimulation (Wright et al., 2009). Attempts to improve the efficacy of adoptive T cell treatments through different in vitro activation protocols and combination with chemotherapy or vaccines are in active or planned clinical trials (clinicaltrials.gov: NCT01147016, NCT01219907, and NCT01022138).

In addition to adoptive T cell therapy, breast cancer vaccines aiming to generate effective anti-tumor CTL responses are in development and clinical trials. These vaccines utilize antigens aberrantly expressed in breast cancer including HER-2/neu, MUC1, carcinoembryonic antigen (CEA), and alpha-lactalbumin (Florescu et al., 2011), administered with adjuvant or via viral vectors, tumor cells, dendritic cells, or DNA. Vaccines have demonstrated safety and limited success in small, early trials but there is still no definitive evidence of survival advantage in larger trials. In a phase I trial that included some breast cancer patients, a pox viral vaccine combining CEA with 3 costimulatory molecules (TRICOM) had modest results including stabilization of disease among 40% of patients (Marshall et al., 2005). A pilot study vaccinating breast cancer patients with a similar TRICOM vaccine including both CEA and MUC1 peptides and involving alternating vaccinia-based and fowlpox-based booster vaccines (PANVACVF, Therion Biologics) was associated with both CD8 and CD4 T cell responses and one breast cancer patient had a greater than 20% reduction in size of a liver metastasis (Gulley et al., 2008). A phase II trial showed an overall survival advantage in patients receiving a MUC1 vaccine (Miles and Papazisis, 2003), but no overall survival advantage was found in a larger phase III trial (Ibrahim et al., 2004). Phase I trials for dendritic cells-based HER-2/neu vaccine Lapuleucel-T were safe and showed T cell proliferative and IFN-γ responses, some instances of stable disease, and one partial response (Park et al., 2007; Peethambaram et al., 2009). Increased survival and reduced recurrence were observed in an ongoing phase I/II clinical trial for a HER-2/neu vaccine composed of peptide and GM-CSF [(Peoples et al., 2005), clinicaltrials.gov: NCT00854789].

While many studies seeking to develop effective adoptive T cell therapy and vaccines are underway, clinical success has been limited and the immunotherapy endeavor has overall been somewhat frustrating. This is probably due to a variety of tumor-induced T cell suppressive mechanisms that operate in the context of breast cancer and can undermine even well-designed immunotherapy approaches.

T Cell Coinhibition in Breast Cancer: Immune Evasion Mechanisms

Breast cancers are very ineffective in stimulating effective immune responses despite expressing antigens recognizable by their host’s immune system. Tumors escape immune surveillance by several mechanisms including self-tolerance, loss of tumor specific antigens due to epigenetic changes, tumor induced immune suppression, and other mechanisms (Manjili and Payne, 2012). In this section we will discuss breast tumor-induced immune suppression and specifically, the ability of tumors to suppress the T cell response through T cell coinhibition.

Activation of a T cell requires two signals: the presentation of specific antigen on the major histocompatibility complex (MHC) of an antigen presenting cell (APC) (signal 1), together with a costimulatory signal delivered by the APC to the T cell (signal 2). Costimulation is mainly generated by signaling between members of the B7/CD28 families, classically, B7-1 or B7-2 ligands on the APC binding to CD28 on the T cell (Scandiuzzi et al., 2011). Upon initial T cell activation, additional costimulatory as well as coinhibitory signals follow, and the integration of these signals determine the outcome of the T cell response. The first coinhibitory member of the B7/CD28 families discovered was cytotoxic T lymphocyte antigen 4 (CTLA-4/CD152), a homolog of CD28 which is upregulated in activated T cells and strongly binds B7-1 and B7-2, serving to reduce T cell activation through direct inhibitory signaling and opposition of CD28 binding (Krummel and Allison, 1995; Linsley et al., 1991). Over the past number of years, additional members of the B7 family have been identified, including costimulatory B7h (ICOS-L/CD275) which binds ICOS (Yoshinaga et al., 1999), coinhibitory PD-L1 (B7-H1/CD274) (Freeman et al., 2000) and PD-L2 (B7-DC/CD273) which bind PD-1 (Latchman et al., 2001), B7-H3 (CD276) (Chapoval et al., 2001) which can be both costimulatory and coinhibitory, and inhibitory B7x (B7-H4/B7S1) (Prasad et al., 2003; Sica et al., 2003; Zang et al., 2003). In contrast to B7-1 and B7-2, whose expression is limited to lymphoid organs, PD-L1, B7-H3, and B7x can be expressed in non-lymphoid organs as well as on tumor cells in various cancers, where they are thought to contribute to tumor immune evasion. The balance of costimulation and coinhibition is an important checkpoint in T cell function. In breast cancer, the balance of costimulation and coinhibition appears skewed towards coinhibition due to dysregulation of the expression of several B7 and CD28 family members.

CTLA-4 gene polymorphisms in human breast cancer

CTLA-4 expression on T cells is induced by antigenic stimulation while regulatory T cells constitutively express CTLA-4. CTLA-4 gene polymorphisms were found to be associated with breast cancer risk in a comprehensive meta-analysis (Zhang et al., 2011) and in the Chinese Han population (Li et al., 2012). Studies have revealed increased soluble CTLA-4 in plasma and increased FoxP3 and CTLA-4 transcripts in peripheral blood mononuclear cells of breast cancer patients as compared to normal controls (Jaberipour et al., 2010). All of these studies indicate that there is a certain degree of immune dysregulation in breast cancer patients involving CTLA-4, not only in the tumor microenvironment but also possibly extending to the systemic immune system. Still, the extent to which CTLA-4 contributes to tumor immunity in breast cancer is currently uncertain.

PD-L1 expression in human breast cancer

PD-L1 is not expressed in normal breast tissue but is highly expressed in breast cancer tissue samples and breast cancer cell lines. PD-L1 polymorphisms are associated with sporadic breast cancer in the Chinese Han population (Hua et al., 2011), but the significance of this association is not yet known. The expression of PD-L1 was studied in 44 patients with breast cancer and was correlated to clinicopathological parameters (Ghebeh et al., 2006). Tumor expression of PD-L1 is associated with Grade III, estrogen (ER)/progesterone receptor (PR) negative tumors. When PD-L1 expression was evaluated in tumor infiltrating lymphocytes, a higher expression was associated with larger size, Grade III and HER2/neu positive tumors. It was also reported that tumor PD-L1 expression and Foxp3+ regulatory T cell (Treg) infiltration were highly correlated in breast cancer tissue specimens (Ghebeh et al., 2008). Moreover the presence of Foxp3+ Tregs, tumor PD-L1 expression, and PD-1+ tumor infiltrating lymphocytes was correlated with a high histological grade, ER negative status, and severe lymphocyte infiltration. Taken together, these observations suggest that the expression of PD-L1 by the tumor or PD-1/PD-L1 by the tumor infiltrating lymphocytes may have a role in modulating the immune response. Furthermore, PD-L1 expression is increased in tumors that have a higher proliferation index as measured by Ki-67 and in cell cultures PD-L1 expression was down-regulated in cells in a quiescent state (Ghebeh et al., 2007). These observations suggest that the PD-L1/PD-1 pathway may be more important in certain breast cancer subtypes like triple negative breast cancers which have a higher proliferation rate, higher grade, and have a high lymphocytic response while in low grade tumors these molecules may play a role in the later stages contributing to their invasiveness.

Loss of the tumor suppressor gene phosphatase and tensin homolog (PTEN) in gliomas results in activation of the phosphatidylinositol 3-kinase (PI3k) pathway and increased PD-L1 expression (Parsa et al., 2007), and activation of the PI3k pathway mediated via PI3k mutations or PTEN loss is also known to be common in breast cancer. In breast and prostate cancer cell lines, treatment with PI3/Akt inhibitors resulted in down-regulation of PD-L1 expression and a reversal of their immunoresistant phenotype (Crane et al., 2009). In anaplastic lymphoma kinase (ALK)+ T cell lymphoma, the chimeric tyrosine kinase from the nucleophosmin/ALK translocation results in increased expression of PD-L1 by activating the transcription activator STAT3 (Marzec et al., 2008). Thus, oncogenic pathways may be directly involved in the up-regulation of PD-L1, contributing to immune resistance, and blockade of the PD-1 signal transduction pathway can reverse immune resistance. The effects of chemotherapeutic agents on PD-L1 expression were studied and it was shown that doxorubicin down-regulates PD-L1 expression on the cell surface but up-regulates its expression in the nucleus, and this up-regulation of PDL1 was inhibited by blocking the PI3/AKT pathway (Ghebeh et al., 2010). In contrast, other chemotherapeutic agents like paclitaxel, etoposide, and fluorouracil have been shown to up-regulate PD-L1 expression which contributes to immune resistance (Hasan et al., 2011; Zhang et al., 2008). These observations show that chemotherapy can affect immune resistance and with the advent of neoadjuvant chemotherapy as a standard treatment, this provides us a window of opportunity to study the effects of chemotherapy on these molecules in pre- and post-treatment. Moreover this principle is also important when evaluating monoclonal antibodies against PD-L1 in clinical trials, as these might have differential effects depending on whether these are evaluated in patients who have received anthracycline or taxane based chemotherapy, both of which are used in breast cancer.

B7-H3 expression in human breast cancer

B7- H3 is expressed in breast cancer cell lines and tissue samples but not in normal breast tissue (Arigami et al., 2010). The receptor for this ligand is still unknown. Currently, the majority of evidence in humans suggests a predominantly coinhibitory role for B7-H3, and in cancers its expression has been associated with worse outcomes (Crispen et al., 2008; Zang et al., 2007). B7-H3 expression was evaluated in breast tumors of 82 breast cancer patients using quantitative real-time reverse transcription-polymerase chain reaction and immunohistochemistry. Expression of B7-H3 correlated with increased tumor size, lymphovascular invasion, and sentinel lymph node and overall lymph node metastasis, but was not significantly associated with tumor grade, ER/PR, or Her2 status. These observations suggest that B7-H3 may play a more important role in tumor metastases, rather than the early phases of the tumor immune response. In vitro silencing of B7-H3 by siRNA resulted in increased sensitivity of breast cancer cell lines to paclitaxel (Liu et al., 2011) by interfering with the Jak/Stat3 pathway. Overall the pattern of B7-H3 expression in breast cancer tissue samples appears to be different from PD-L1 and whether these molecules play important roles in different stages of the disease needs to be determined.

B7x gene polymorphisms and protein expression in human breast cancer

B7x [B7-H4/B7-S1] is a protein of the B7 family with a yet-unidentified receptor expressed on activated T cells (Prasad et al., 2003; Sica et al., 2003; Zang et al., 2003). B7x inhibits CD4 and CD8 T cell proliferation and cytokine production (Zang et al., 2003). A study identified three single nucleotide polymorphisms (SNPs) in untranslated regions of the B7x gene which were associated with rates of sporadic breast cancer in a Chinese Han population (Zhang et al., 2009). These SNPs may affect the efficiency of cellular production of B7x rather than the function of the protein itself. Two SNPs were associated with a decreased risk of cancer, while one SNP was linked to a greater incidence of cancer, increased lymph node metastasis, and altered steroid hormone receptor status. It was reported that knockdown of B7x expression in breast cancer cell lines in vitro by siRNA leads to increased caspase activity and apoptosis of tumor cells (Salceda et al., 2005). Higher expression of B7x was found in breast and ovarian cancers as compared to their corresponding normal tissues (Qian et al., 2011), indicating that B7x may be more important in tumor biology in these cancers. In contrast to other coinhibitory molecules, B7x can be expressed at low levels in normal breast tissue epithelium (Tringler et al., 2005). In a study of 419 cases of human breast cancer, more than 95% of tissue samples expressed B7x (Tringler et al., 2005). Increased staining intensity correlated with PR negative status and neoadjuvant chemotherapy while the proportion of B7x positive cells was correlated with PR negative and Her2 negative tumors. These observations suggest that B7x expression is highly up-regulated in breast cancer and that B7x may be a useful diagnostic marker and potential therapeutic target.

Blockade of T Cell Coinhibition for Breast Cancer Therapy

After decades of evaluation of various immune therapies, researchers have finally achieved some therapeutic success with blockade of T cell coinhibitory molecules in the past few years. The proof of principle for blockade of coinhibitory therapy in humans came from preclinical mouse models (Leach et al., 1996) and clinical studies in metastatic melanoma showing tumor regression and an improvement in overall survival after treatment with the anti-CTLA-4 antibody (Downey et al., 2007; Hodi, 2007; Hodi et al., 2003; Phan et al., 2003; Robert et al., 2011). CTLA-4 and PD-1 antibodies have produced some impressive results in the few cases of durable remission seen in select patients treated with these antibodies, proving that stimulating the immune system can effectively induce long standing anti-tumor immunity even in advanced cancers. These drugs can also have adverse autoimmune side effects including pneumonitis, vitiligo, colitis, hepatitis, hypophysitis, sarcoidosis, endophthalmitis, diabetes mellitus, and myasthenia gravis. Currently there are no proven biomarkers available to predict response to immune therapy, although in a recent trial, expression of PD-L1 ligand in tumor specimens shows promise as a biomarker (Topalian et al., 2012). The results of some T cell coinhibitory blockade clinical trials are discussed below.

Blockade of CTLA-4 in human breast cancer

Initial enthusiasm for CTLA-4 antibody based therapy in breast cancer was generated from mouse models in which mice treated with a GM-CSF-expressing vaccine followed by anti-CTLA-4 antibody rejected SM1 cell line-induced mammary carcinoma and were also immune to subsequent re-challenge with the same cell line (Hurwitz et al., 1998). Following this, Tremelimumab (CP-675,206 or ticilimumab; Pfizer) a fully human IgG2 anti-CTLA-4 monoclonal antibody that blocks the binding of CTLA-4 to B7-1 and B7-2 was tested in combination with exemestane, an aromatase inhibitor that blocks the production of estrogen (Vonderheide et al., 2010). In this phase 1 study, 26 patients with advanced hormone receptor positive breast cancer were treated with tremelimumab (3–10 mg/kg) every 28 days or every 90 days along with exemestane 25 mg orally daily. Since other studies suggested that there was an increase in diarrhea with the 28 day dosing schedule, this was discontinued later in the trial. Dose limiting toxicities were liver dysfunction and diarrhea and the maximum tolerated dose was 6 mg/kg every 90 days. The overall response rate (ORR) was stable disease in 11 patients (42%). Treatment was associated with increase in the ratio of ICOS+ CD4 and CD8 T cells when compared with Foxp3+ Treg cells, suggesting enhanced cellular immune function. In another phase II trial investigating Ipilimumab (MDX-010, Yervoy; Bristol-Myers Squibb) at 3 mg/kg in subjects with metastatic breast cancer (clinicaltrials.gov: NCT0083278), enrollment was stopped early as there were no objective responses as defined by the RECIST criteria in 31 subjects who were treated. Trials which combine ipilimumab with cryoablation to prevent recurrence before mastectomy are in progress (clinical-trials. gov: NCT01502592). These clinical trials show that CTLA-4 inhibition alone is not an effective approach in breast cancer.

Blockade of PD-1/PD-L1 pathway

In a phase 1 study, the anti-PD-1 antibody (BMS-936558, Bristol-Myers Squibb) was evaluated in 296 patients with advanced refractory solid tumors — non-small cell lung, renal, melanoma, colon, and castration resistant prostate cancer — and was studied at different doses of 1.0, 3.0, or 10.0 mg per kilogram of body weight (Topalian et al., 2012). The maximum tolerated dose (MTD) was not reached and the common side effects were skin rash, diarrhea, and pruritus. Grade 3 or 4 adverse events included diarrhea, pneumonitis, and liver dysfunction. The ORR was 18% in patients with non-small cell lung cancer, 28% in melanoma, and 27% in patients with renal cell cancer with some durable responses. Of note, 36% of patients whose tumors expressed PD-L1 (defined as >5%) demonstrated an objective response while none of the patients whose tumors did not express PD-L1 demonstrated a response. In another phase 1 study the anti-PD-L1 antibody (BMS-936559, Bristol-Myers Squibb) was evaluated to assess the safety and efficacy in advanced cancers (Brahmer et al., 2012). A total of 207 patients were treated at increasing dose levels from 0.3 to 10 mg/kg every 14 days in 6-week cycles for up to 16 cycles. The ORR was 6 to 17% in various tumors with median response duration equal to or greater than 24 weeks, but none of the four breast cancer patients treated had a response. With respect to breast cancer patients, the absence of response noted may be due to the small number of patients treated in this study (n=4) or they might have had PD-L1 negative breast cancer as we know not all breast cancers express PD-L1. Another PD -L1 antibody (MPDL3280A) is currently being evaluated in advanced solid cancers along with bevacizumab (clinicaltrials.gov: NCT01633970).

Blockade of the B7-H3 pathway

A phase 1 trial of anti B7-H3 antibody (MGA 271) is underway in the treatment of refractory solid tumors (clinicaltrials.gov: NCT01391143) (Seliger and Quandt, 2012).

Concluding Remarks

The past few years have witnessed a new era in the understanding of tumor immunity with the discovery of the B7 family of coinhibitory molecules and their functions. But we are still in the early stages of understanding B7 coinhibitory molecule expression and co-expression in various tumors, mechanisms of signal transduction, and the interaction of these pathways with other oncogenic pathways contributing to tumor immunity. Even though breast cancer therapies have improved in the past decade, treatment of certain breast cancer subtypes like TNBC remains challenging and currently the cure rates for metastatic disease are dismal. T cell immunotherapy has shown some success in metastatic disease treatment, but progress is likely hindered by cancer-induced immunosuppression including T cell coinhibition. While still in development, coinhibition blockade, likely in combination with other T cell immunotherapies as well as chemotherapies, may prove to be an effective approach in breast cancer therapy.

Acknowledgements

Y.M.A. was supported by NIH training grant T32GM007491. Research in the Zang lab is supported in part by NIH DP2DK083076 and DOD PC094137. The Albert Einstein Cancer Center is supported by NIH P30CA013330.

Footnotes

Disclosure

The authors report no conflicts of interest.

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