Intratumoral injection of alpha-gal glycolipids induces a protective anti-tumor T cell response which overcomes Treg activity

Abstract

alpha-Gal glycolipids capable of converting tumors into endogenous vaccines, have alpha-gal epitopes (Gal alpha 1-3 Gal beta 1-4GlcNAc-R) and are extracted from rabbit RBC membranes. alpha-Gal epitopes bind anti-Gal, the most abundant natural antibody in humans constituting 1% of immunoglobulins. alpha-Gal glycolipids insert into tumor cell membranes, bind anti-Gal and activate complement. The complement cleavage peptides C5a and C3a recruit inflammatory cells and APC into the treated lesion. Anti-Gal further opsonizes the tumor cells and targets them for effective uptake by recruited APC, via Fc gamma receptors. These APC transport internalized tumor cells to draining lymph nodes, and present immunogenic tumor antigen peptides for activation of tumor specific T cells. The present study demonstrates the ability of alpha-gal glycolipids treatment to prevent development of metastases at distant sites and to protect against tumor challenge in the treated mice. Adoptive transfer studies indicate that this protective immune response is mediated by CD8+ T cells, activated by tumor lesions turned vaccine. This T cell activation is potent enough to overcome the suppressive activity of Treg cells present in tumor bearing mice, however it does not elicit an autoimmune response against antigens on normal cells. Insertion of alpha-gal glycolipids and subsequent binding of anti-Gal are further demonstrated with human melanoma cells, suggesting that intratumoral injection of alpha-gal glycolipids is likely to elicit a protective immune response against micrometastases also in cancer patients.

Fig. 1

Induction of a protective immune response against tumor challenge by intratumoral injection of α-gal glycolipids. Primary B16 tumors received three weekly injections of 1 mg α-gal glycolipids (a), or one injection of ethanol (b). One day after last injection of α-gal glycolipids, or 3 weeks after ethanol injection, mice were challenged with 0.5 × 10⁶ B16 cells in the contra-lateral left flank and tumor growth in the left flank monitored. Note that ten of 15 mice injected with α-gal glycolipids (a) displayed no tumor development in the left flank (one curve labeled with filled circle for all ten mice). Tumors in the remaining five mice are presented as individual curves each labeled with open circle. Mice with tumors injected with ethanol (b), all developed tumors in the left flank (n = 5)

Fig. 2

Prevention of distant tumor growth by intratumoral injection of α-gal glycolipids. The figure describes development of tumors in the left flank from inoculates of 10⁴ B16 cells, administered subcutaneously at the same time as the 10⁶ B16 cells were administered subcutaneously in the right flank. (a) Development of the distant tumor in mice receiving two α-gal glycolipids injections into the right flank “primary” tumor (n = 8). b Development of the distant tumor in mice with “primary” tumor injected twice with PBS (n = 8)

Fig. 3

ELISPOT data from mice with B16 tumors injected with PBS, or with α-gal glycolipids (GSL-glycosphingolipids), and which are stimulated in vitro with various immunodominant MAA peptides (n = 6 mice per group). a Data with spleen lymphocytes from individual mice stimulated with the following MAA peptides: TRP2_180–188 (closed columns); TRP2_180–188 without S₁₈₀ and V₁₈₇ (TRP2_181–188 open columns); mouse gp100_25–33 (diagonal hatched columns); human gp100_25–33 (horizontal hatched columns). Data presented as number of IFNγ spots per million lymphocytes (i.e., T cells secreting IFNγ/10⁶ cells). b Data presented as mean + standard deviation of ELISPOT response to the various MAA peptides in mice with tumors treated with PBS (open columns), or with α-gal glycolipids (closed columns)

Fig. 4

Tumor growth following adoptive transfer of lymphocytes from donor mice with tumors injected with α-gal glycolipids. Naïve recipients were challenged subcutaneously with 0.5 × 10⁶ live B16 cells, 24 h prior to transfer of lymphocytes. Adoptive transfer of lymphocytes was performed in pairs of recipients, each receiving 40 × 10⁶ cells. Open circle total lymphocytes, or filled circle lymphocytes depleted of CD8+ T cells. Data on tumor growth for 30 days post adoptive transfer in pairs receiving total, or CD8+ depleted lymphocytes from eight donors

Fig. 5

Tumor growth following adoptive transfer of lymphocytes from donor mice with tumors injected with PBS. Adoptive transfer performed as in Fig. 4. Open circle total lymphocytes; filled circle lymphocytes depleted of CD4+ T cells. Data are presented in pairs receiving total, or CD4+ depleted lymphocytes from eight donors

Fig. 6

Immunostaining of Treg cells from spleens of mice with tumors injected with PBS (a, b), or injected with α-gal glycolipids (c, d). Spleen lymphocytes were obtained 2 weeks after second injection and subjected to Foxp3 and CD4 double staining. The Treg cells are located in the upper right quadrant. Presented data are of two representatives out of five mice in each group

Fig. 7

Insertion of α-gal glycolipids into the cell membranes of human melanoma cells (SK-N-MC pLXSN cell line). a Binding of the monoclonal anti-Gal Ab M86 to untreated melanoma cells (thin line histogram), or cells incubated for 2 h at 37°C with 0.1 or 1 mg/ml α-gal glycolipids (dashed line and thick line histograms, respectively). b Complement dependent cytolysis of the human melanoma cells with inserted α-gal glycolipids following 30 min incubation at 37°C with human serum. Open circle cells incubated with 1 mg/ml α-gal glycolipids; open square 0.1 mg/ml; open triangle no α-gal glycolipids