Retargeting of regulatory T cells to surface-inducible autoantigen La/SS-B
Highlights
► La autoantigen is not limited to the surface of dying cells. ► La binds to the surface of intact cells. ► Surface bound La on intact cells is accessible for abs. ► A novel bispecific ab to La and CD3 was developed. ► Surface bound La can be used for retargeting of Tregs via this bsAb.
Introduction
Non-physiological cell death poses a potential threat to the organism and therefore should alert the immune system of danger [1], [2]. The concept of a proinflammatory activity of dead cells was already postulated in 1994 by Matzinger [3]. She hypothesized that our immune system can not only discriminate between infectious and non-infectious agents but can also sense and respond to intracellular alarming signals. These endogenous danger signals, designated as damage associated molecular patterns (DAMPs) or alarmins are hidden inside the cells under physiological conditions. However, upon cellular stress, damage or necrotic cell death these molecules are released due to a loss of cell membrane integrity [4]. In the extracellular space, DAMPs can act like conventional cytokines or chemokines through binding to different activating receptors on immune cells and trigger both innate and adaptive immune responses [5], [6], [7], [8]. Many pathological immune-mediated conditions, e.g. autoinflammatory diseases, autoimmune diseases or graft rejection are associated with massive tissue damage. DAMPs released in this context may not only be useful biomarkers of inflammation but also valuable novel target structures for therapeutic purposes.
In order to downregulate infiltrating proinflammatory T cells one could for example try to recruit Tregs to sites of massive tissue damage and DAMP release. Tregs are indispensable players in immune homeostasis for both maintenance of self-tolerance as well as for regulating immune responses against foreign antigens [9], [10], [11]. In recent years, a plethora of in vitro and in vivo studies highlighted the tremendous potential of Treg cells to ameliorate or even prevent various immune-mediated pathologies including autoimmunity, graft versus host disease (GvHD) and graft rejection (e.g. [12], [13], [14], [15], [16]). Nevertheless, in order to exert their suppressive effect locally in inflamed tissues Tregs have to be recruited from the circulation and enriched at the appropriate sites of ongoing immune responses [17], [18], [19].
Already in 1987 we reported a stress-dependent shuttling of the nuclear autoantigen La/SS-B [20]. Since then we and others reported not only a shuttling of the La protein between the nucleus and the cytoplasm but also a staining of the cell surface including for example after virus-infection or UV irradiation or exposure to inflammatory cytokines such as TNF [21], [22], [23], [24], [25], [26], [27], [28]. In general La protein was found on the cell surface of cells exposed to oxidative stress where it remained detectable for more than 48 h [25]. As all these conditions can also cause cell death, it was not surprising that La protein was also found on the surface of apoptotic cells [29]. Due to the limitations of the experimental settings in all of these studies it remains, however, unclear even until today whether or not cell surface detectable La protein is (i) coming from inside of the cell and (ii) limited to the surface of dying or already dead cells.
Here we present experimental data that La protein is not restricted to the surface of dying cells. La protein is released and binds tightly to neighboring intact cells. In order to show first proof of concept for the idea that such cell surface bound La protein can be used as target for redirection of Tregs to inflamed tissues we constructed a novel bsAb which is directed on the one hand to the La protein and on the other hand to the CD3 complex of T cells. The general potential of such cross-linking bsAbs to activate immune effector T cells (Teff cell) against tumor cells has already been successfully substantiated in various in vitro and animal studies as well as in first clinical trials [30], [31], [32], [33], [34], [35], [36]. Moreover, our group demonstrated recently that bsAbs targeting a cell surface receptor can activate Tregs in a similar way as their Teff cell counterparts [37]. Here we show that a cross-linkage of Tregs with La-decorated target cells mediated by our novel, fully humanized bsAb to the La protein and the CD3 complex leads to an activation of freshly isolated human Tregs. Moreover, such bsAb activated Tregs displayed a potent suppressive capacity and negatively influenced proliferation, expansion and cytokine production of autologous CD4+ and CD8+ Teff cells.
Section snippets
Monoclonal antibodies
In the current study we used three monoclonal anti-La abs including the previously described and well characterized anti-La mabs La-SW5 [38], [39] and La7B6 [40]. The anti-La mAb SW5 recognizes a split epitope in the N-terminal domain of human La protein. The anti-La mAb La7B6 is directed to the amino acids (aa) 311-329 (EKEALKKIIEDQQESLNKW) of human La protein which are part of a helical region in the C-domain. This epitope is unique for human La protein: In the corresponding murine La
The human La protein is released from apoptotic cells and binds to the membrane of adjacent living cells
Recruiting Tregs locally to sites of ongoing immune reactions and tissue damage requires the identification of target antigens, which are specifically expressed or released in the affected infiltrated tissues. Three decades of intensive research on the nuclear autoantigen La/SS-B revealed that the protein shuttles from the nucleus to the cytoplasm in response to cellular stress conditions and/or apoptotic signals (e.g. [20], [21]). Besides it was also demonstrated, that the protein translocates
Discussion
Infiltrating inflammatory immune effector T cells are involved in tissue destruction in autoimmune patients, rejection of transplanted organs and also during GvHD. Thus, redirecting of Tregs into damaged tissue may result in an attenuation of the local immune response and perhaps even in induction of peripheral tolerance. One consequence of tissue damage is the release of the cell content including nuclear material. To this end it has been reported that in eukaryotic cells the nuclear
Contributions
S.K., M.C., C.A., C.C.B., A.F., I.M., K. W., S.S. performed experiments, collected and analyzed data. M.B., G.E. provided critical reagents. S.K., M.C., M.S., M.B. designed the experiments and wrote the manuscript.
Disclosures/conflict of interests
M.B. and G.E. have filed provisional patent applications related to the abs directed to the La antigen.
Acknowledgments
We thank Livia Schulze, Barbara Uteβ, and Christine Gräfe for excellent technical assistance.
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- 1
Contributed equally to this work.
- 2
Present address: Department of Biomedicine, University Hospital Basel, Hebelstr. 20, 4031 Basel, Switzerland.