Active tumor immunotherapy shows great promise in animal models but has yet to achieve widespread success in the clinic. Vaccines have been extensively tested in clinical trials for the treatment of glioma. Glioma patients have been vaccinated in multiple sites including the scapula draining into the axilla (1
), the anterior upper thigh (2
), the upper arm (3
), and the cervical regions (4
). Data are insufficient to correlate response rates with vaccination site. There have been few basic studies examining priming following vaccination as it relates to anatomic location.
The sentinel lymph nodes (draining lymph node, DLN, nearest to the tumor) are in direct lymphatic drainage from the primary tumor (6
) and are the DLN most prone to immune suppression (8
). In breast cancer and melanoma patients, T cells isolated from the sentinel lymph nodes have suppressed activation and proliferation in response to various mitogens compared to T cells isolated from the more distal lymph nodes (11
). Multiple mechanisms contribute to this local suppression. Tumor-elaborated soluble factors, such as TGFγ and prostaglandin E2 (15
), can act at the tumor site or DLN to dampen T cell reactivity. In experimental systems, additional documented mechanisms of local immune suppression at the DLN include regulatory T cell-mediated killing of tumor antigen presenting DCs (19
) and T cell receptor nitration by myeloid derived suppressor cells (20
With respect to brain tumors, the immunologically specialized nature of the brain and its draining cervical lymph nodes must also be considered. Initial experiments revealed that vaccination with antigen into the brain can trigger higher antibody titers compared to vaccination in the periphery (21
). In contrast, Th1-mediated delayed type hypersensitivity responses are absent or blunted when the same antigen is delivered to the brain (reviewed in (22
)). These findings support a model whereby the cervical lymph nodes have an intrinsic Th2 bias in steady state conditions. Additional experiments showed that CD8 T cells undergo initial expansion following intracerebral tumor cell challenge, but fail to differentiate into cytotoxic T lymphocytes (CTLs) (23
). However, it was unclear if this was due to tumor-induced immune suppression, a lack of co-stimulation, or an intrinsic bias against CTL development in the cervical lymph nodes. Normal mouse cerebral spinal fluid can suppress CD8 T cell activation in ex vivo
assays, which is restored by a TGFγ blocking antibody (24
), implicating brain-derived TGFγ as one soluble mediator of CTL suppression in the cervical lymph nodes.
Despite support for Th2 immune deviation in the brain DLN, there is evidence that CD8 T cell responses play a tumoricidal role in human gliomas. Infiltration of CD8 T cells is a positive prognostic factor in glioma patients (25
). Furthermore, immunological synapses between CD8 T cells and glioma cells have been documented in humans (26
). Interestingly, glioblastoma patients receiving autologous tumor lysate-pulsed dendritic cell vaccines had superior survival when their gene expression was of mesenchymal rather than the proneural molecular signature; the mesenchymal signature is inflammatory and was correlated with significantly more infiltrating CD8 T cells at the tumor site compared to proneural tumors (27
). Regardless of these spontaneous or vaccine-induced T cell responses, global immune suppression has been widely accepted to occur in glioma patients. Many studies done to establish this conclusion were conducted with leukocytes harvested after treatment with glucocorticoids or chemotherapy, clouding the contribution of the tumor versus the treatment on dampened immunity. More recent data suggest that treatment with glucocorticoids and alkylating chemotherapy plays a significant role in inducing global immune suppression, because both drugs are associated with rapid post-treatment lymphopenia, and elevation in regulatory T cell or myeloid derived suppressor cell frequency (28
). The severity of lymphopenia following the standard of care (steroids, chemotherapy, and radiation) negatively correlates with overall survival in glioblastoma patients (30
There is stronger experimental evidence for profound local immune suppression at the tumor site in gliomas. Studies in spontaneous murine models suggest that gliomas accrue immune suppressive cell populations even at early, asymptomatic stages (19
). As gliomas develop, recruitment of myeloid derived suppressor cells is mediated in part through a cyclooxygenase-2 and prostaglandin E2 axis (31
), decreasing CTL recruitment to the tumor bed. Local immune suppression is further enforced by hypoxia, leading to reduced effector function of T cells via increased production of immunosuppressive factors (32
). Moreover, glioma cells express inhibitory ligands that can trigger T cell tolerance including HLA-E and B7-H1 (33
). However, little is known about how these mechanisms of suppression at the tumor site impact priming responses in the cervical lymph nodes. One purpose of this study was to shed insight on this poorly understood area.
Based on evidence that much of the tumor-derived immune suppression in other malignancies is anatomically graded rather than global (8
), we reasoned that vaccination distal to gliomas could be exploited to improve active immunotherapy. We focused our investigation on the relationship between vaccination sites and the ensuing CD8 T cell response. Vaccination in close proximity to the tumor DLN (cervical or axillary regions) resulted in no survival benefit. In contrast, vaccinations in DLN further from the brain (in the inguinal region) increased survival of glioma-bearing mice. Mice vaccinated in the cervical region had CD8 T cells with lower TCR affinity and weakened effector functions compared to priming at the inguinal region. Considering that previous clinical trials have used a variety of injection sites, these data reveal a neglected yet potentially critical variable for trial design. Injection site is a crucial aspect of active tumor immunotherapy in this murine model that demands investigation in glioma patients.