De Duve Institute

2. A mechanism causing anergy of CD8 and CD4 T lymphocytes


The identification of specific tumor antigens recognized by T lymphocytes on human cancer cells has elicited numerous clinical trials involving vaccination of tumor-bearing cancer patients with defined tumor antigens. These treatments have shown a low clinical efficacy. Among metastatic melanoma patients, about 5% show a complete or partial clinical response following vaccination, whereas an additional 10% show some evidence of tumor regression without clear clinical benefit. We believe that progress depends on unraveling the different blockages for efficient tumor destruction.

The tumors of the patients about to receive the vaccine, already contain T cells directed against tumor antigens. Presumably these T cells are exhausted and this impaired function is maintained by immunosuppressive factors present in the tumor. The T cell response observed in some vaccinated patients reinforce an hypothesis proposed by Thierry Boon and Pierre Coulie: anti-vaccine CTL are not the effectors that kill the tumor cells but their arrival at the tumor site containing exhausted anti-tumor CTL, generates conditions allowing the reawakening of the exhausted CTL and/or activation of new anti-tumor CTL clones, some of them contributing directly to tumor destruction. Accordingly, the difference between the responding and the non-responding vaccinated patients is not the intensity of their direct T cell response to the vaccine but the intensity of the immunosuppression inside the tumor. It is therefore important to know which immunosuppressive mechanisms operate in human tumors.

Human tumor-infiltrating T lymphocytes show impaired IFN-ϒ secretion
Both human CD8 and CD4 tumor-infiltrating T lymphocytes (TIL) were isolated from tumor ascites or solid tumors and compared with T lymphocytes from blood donors. TIL secrete low levels of INF-ϒ and other cytokines upon non-specific stimulation with anti-CD3 and anti-CD28 antibodies. TCR were observed to be distant from the co-receptors on the cell surface of TIL, either CD8 or CD4, whereas TCR and the co-receptors co-localized on blood T lymphocytes.


figureTCR-CD8

TCR and CD8 do not co-localize on CD8 T cells with impaired functions

 

Reversing the anergy of tumor-infiltrating T lymphocytes with galectin ligands
We have attributed the decreased IFN-ϒ secretion to a reduced mobility of T cell receptors upon trapping in a lattice of glycoproteins clustered by extracellular galectin-3. Indeed, we have shown that treatment of TIL with N-acetyllactosamine (LacNAc), a galectin ligand, restored this secretion.


Figure Treatment of TIL Treatment of tumor-infitrating lymphocytes with a galectin ligand reverses anergy

 

Our working hypothesis is that TIL have been stimulated by antigen chronically, and that the resulting activation of T cells could modify the expression of enzymes of the N-glycosylation pathway, as shown for murine T cells. The chronically activated TIL, compared to resting T cells, could thus express surface glycoproteins decorated with a set of glycans that are either more numerous or better ligands for galectin-3. Galectin-3 is an abundant lectin in many solid tumors and carcinomatous ascites, and can thus bind to surface glycoproteins of TIL and form lattices that would thereby reduce TCR mobility. This could explain the impaired function of TIL. The release of galectin-3 by soluble competitor ligands would restore TCR mobility and boost IFN-ϒ secretion by TIL. We recently strengthened this hypothesis by showing that both CD4 and CD8 TIL that were treated with an anti-galectin-3 antibody, which could disorganize lattice formation, had an increased IFN-ϒ secretion compared to untreated cells.

Towards a clinical trial combining vaccination and galectin-binding polysaccharides
Galectin competitor ligands, e.g. disaccharides LacNAc, are rapidly eliminated in urine, preventing their use in vivo. We recently found that a plant-derived polysaccharide, currently in clinical development, detached galectin-3 from TIL and boosted their IFN-ϒ secretion. Importantly, we observed that not only CD8+ TIL but also CD4+ TIL that were treated with this polysaccharide secreted more IFN-ϒ upon ex vivo re-stimulation. In tumor-bearing mice vaccinated with a tumor antigen, injections of this polysaccharide led to tumor rejection in half of the mice, whereas all control mice died. In non-vaccinated mice, the polysaccharide had no effect by itself. These results suggest that a combination of galectin-3 ligands and therapeutic vaccination may induce more tumor regressions in cancer patients than vaccination alone. Translation of these results to the clinic was unfortunately impossible because the company producing this polysaccharide got bankrupted. We recently identified another plant-derived polysaccharide that binds to galectins and was already used in combination with chemotherapy in phase II clinical trials in colorectal cancer patients. This compound was as effective as LacNAc in boosting the secretion of IFN-ϒ by treated TIL. A clinical trial with this new compound, in combination with anti-tumoral vaccination, will hopefully be launched in 2011 in clinical centers close to the Brussels Branch. We are currently trying to understand the very early activation events that are defective in TIL.

Further reading

Demotte N, Wieërs G, Van Der Smissen P, Moser M, Schmidt CW, Thielemans K, Squifflet J-L, Weynand B, Carrasco J, Lurquin C, Courtoy PJ, van der Bruggen P.
A galectin-3 ligand corrects the impaired function of human CD4 and CD8 tumor-infiltrating lymphocytes and favors tumor rejection in mice.
Cancer Research. 2010 Oct 1;70(19): 7476-7488

Demotte N, Stroobant V, Courtoy PJ, Van der Smissen P, Colau D, Luescher IF, Hivroz C, Nicaise J, Squifflet J-L, Mourad M, Godelaine D, Boon T, van der Bruggen P.
Restoring the association of the T cell receptor with CD8 reverses anergy in human tumor-infiltrating lymphocytes.
Immunity. 2008 Mar;28(3): 414-424

Demotte N, Colau D, Ottaviani S, Godelaine D, Van Pel A, Boon T, van der Bruggen P.
A reversible functional defect of CD8+ T lymphocytes involving loss of tetramer labeling.
European Journal of Immunology. 200 Jun;32(6):1688-1697



To know more... (pdf chapter of the last de Duve Institute report)


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