Fig. 1: Scheme of P2X7 activation by NAD released from injured cells

1. * NAD * * * * virus-infected cell 1Institute of Immunology University Medical Center Hamburg-Eppendorf, Germany 2INSERM U905, Rouen, France ENDOGENOUS NAD+ DERIVED FROM INJURED CELLS MEDIATES PS EXTERNALIZATION AND CD62L SHEDDING ON MURINE REGULATORY T CELLS Björn Rissiek1, Nicole Schwarz1, Sahil Adriouch1,2, Sandra Hubert2, Friedrich Haag1, Olivier Boyer2, Michel Seman2, Friedrich Koch-Nolte1 Introduction NAD and ATP, the universal energy metabolites, function also as extracellular signaling molecules when released from stressed or injured cells (Fig. 1). T cells are equipped with sensors for extracelluar NAD and ATP, including the P2X7 ion channel, the toxin related ADP-ribosyltransferase ART2 and the NAD-hydrolase CD38. P2X7 is gated by its soluble ligand ATPor by NAD-dependent ADP-ribosylation catalyzed by ART2 (Fig. 2) (1, 2). This results in shedding of CD62L and other membrane proteins and in externalization of phosphatidylserine (PS). By removing the ART substrate NAD, CD38 limits P2X7 activation to NAD (3). Heavy chain antibodies from llamas contain a single antigen binding domain, designated VHH (Fig. 3). The long CDR3 of VHH domains can extend into and block the active site of enzymes. We have generated recombinant ART2-blocking single domain antibodies = sdAbs from an immunized llama (Fig. 4) (4). These sdAbs efficiently block NAD-induced shedding of CD62L (Fig. 5) Summary NAD released during preparation of splenocytes or lymph node cells results in ADP-ribosylation of P2X7, even at 4°C (5). When cells are returned to 37°C, e.g. for functional studies or adoptive transfer experiments, gating of P2X7 causes externalization of PS and shedding of CD62L. Tregs are particularly sensitive to this NAD “shower”, which profoundly affects their phenotype and function. An injection of ART2-blocking sdAbs 10 minutes before sacrificing mice suffices to protect Tregs from the side effects of NAD released during cell preparation. Fig. 3: The long CDR3 of heavy chain antibodies can penetrate and block enzyme active sites Fig. 1: Scheme of P2X7 activation by NAD released from injured cells Conventional IgG CD38 ART2 Heavy Chain IgG Fig. 4: Generation of recombinant ART2-blocking sdAbs from immunized llama Matahari Fig. 4: Generation of recombinant ART2-blocking sdAbs from immunized llama Matahari P2X7 CD62L Salmi & Jalkanen Nat Rev Immunol. 2005 Fig. 2: Gating of P2X7 by NAD-dependent ADP-ribosylation or ATP induces externalization of PS myc-tag His6x-tag WT purified lmyph node T cells were incubated for 30 min in the absence or presence of NAD or ATP before FACS analyses Results Tregs express the P2X7 ion channel and the ART2 ADP-ribosyltransferase. In order to study live Tregs, a foxp3 regulated GFP-transgene was backcrossed onto ART2KO, P2X7KO, and CD38KO mice. (DEREG mice from T. Sparwasser, Hannover). FACS analyses show that Tregs express ART2 and P2X7 (Fig. 6). NAD released during the preparation of splenocytes and lymph node cells induces externalization of PS when Tregs are returned to 37°C. Incubation of freshly prepared lymph node cells at 37°C induces shedding of CD62L (not shown) and externalization of PS on a large fraction of Tregs and on a small fraction of other T cells (Figs. 2, 7), in response to NAD released during cell preparation (5). Tregs from ART2KO and P2X7KO mice, which cannot ADP-ribosylate P2X7 in response to NAD, neither shed CD62L nor expose PS upon incubation at 37°C NAD (Fig. 7). In contrast, Tregs from CD38KO mice, which lack the major NAD-hydrolase, respond stronger to released NAD. Incubation with NAD leads to PS externalization on Tregs within 1-2min after start of the incubation. CD4+ T cells were isolated from C57BL/6 DEREG wt mice and incubated at 37°C in the presence of 30µM NAD for certain time periods(0.5 – 5min) (Fig. 8). The externalization of PS on CD4+GFP+ Tregs starts after 1-2min. After 5min 90% of the Tregs show PS externalization compared to only 20% of the CD4+GFP- T cells. Intravenous injection of ART2-blocking single domain antibodies prevents activation of P2X7 on Tregs during cell preparation. Injection of ART2-blocking sdAbs 10 minutes prior to sacrificing the mice effectively prevents shedding of CD62L (not shown) and externalization of PS on Tregs from both, WT and CD38KO mice (Fig. 9). ART2-/- affinity purification of recombinant single domain antibodies sdAbs from E.coli periplasma lysates (yield 1-5 mg protein per liter of E.coli culture) P2X7-/- Fig. 5: i.v. injection of ART2-blockig sdAbs blocks NAD-induced shedding of CD62L PBS αART2 VHH 4°C 37°C 37°C Fig. 6: Tregs express P2X7 and ART2 mice were sacrified at the indicated times after injection of sdAb or PBS. The small (15kd) sdAbs reach lymph node cells within 10 min after injection and are rapidly eliminated via the kidney. The blockade of ART2 is effective for > 2h and largely reversed after 24 h. P2X7 ART2 CD4 SSC FSC GFP VHH GFP GFP purified lymph node T cells from C57BL6/J DEREG mice Fig. 7: Tregs externalize PS in responses to NAD released during cell preparation Fig. 9: ART2-blocking sdAbs protect Tregs against gating of P2X7 by NAD released during preparation purified splenic T cells from C57BL6 DEREG mice purified lymph node T cells from C57BL6/J DEREG mice Discussion and Conclusions Tregs are highly sensitive to regulation by extracelluar nucleotides. In an inflammatory setting this may provide a mechanism to facilitate the expansion of antigen-specific effector cells (Fig. 10). The latter shed ART2.2 after TCR engagement, and are rendered insensitive to NAD. Simple mechanical manipulations during cell prepareation results in the release of NAD and in ART2-catalyzed ADP-ribosylation of P2X7 on sensitive cells (e.g. T regs), even at 4°C. Gating of P2X7 occurs when cells are returned to 37°C, resulting in activation of downstream effectors including externalization of PS and shedding of CD62L or CD27. This poses a nuisance for functional and adoptive transfer studies. These unwanted side effects of cell preparation on Tregs can be prevented by blocking ART2 with inhibitory sdAbs. Fig. 8: Tregs start to expose phosphatidylserin after 1-2min of incubation onset with 30µM NAD at 37°C Fig. 10: NAD released during inflammation may prevent the unwanted activation of bystander cells naive bystander cell (Tregs, iNKT) effector CTL shed ART2 ART2 PS externailzationon Tregs starts 1-2min after onset of the 37°C incubation with 30µM NAD. Up to 90% of the Tregs expose PS after 5min of incubation • References • Seman, M., S. Adriouch, F. Scheuplein, C. Krebs, D. Freese, G. Glowacki, P. Deterre, F. Haag, and F. Koch-Nolte. 2003. NAD-induced T cell death: ADP-ribosylation of cell surface proteins by ART2 activates the cytolytic P2X7 purinoceptor. Immunity19:571-582 • Adriouch, S., P. Bannas, N. Schwarz, R. Fliegert, A. H. Guse, M. Seman, F. Haag, and F. Koch-Nolte. 2008. ADP-ribosylation at R125 gates the P2X7 ion channel by presenting a covalent ligand to its nucleotide binding site. FASEB J22:861-869 • Krebs C, Adriouch S, Braasch F, Koestner W, Leiter EH, Seman M, Lund FE, Oppenheimer N, Haag F, Koch-Nolte F. 2005. CD38 controls ADP-ribosyltransferase-2-catalyzed ADP-ribosylation of T cell surface proteins. J Immunol174:3298-305 • Koch-Nolte F, Reyelt J, Schössow B, Schwarz N, Scheuplein F, Rothenburg S, Haag F, Alzogaray V, Cauerhff A, Goldbaum FA. 2007. Single domain antibodies fromm llama effectively and specifically block ecto-ADP-ribosyltransferase ART2.2 in vivo. FASEB J21: 3490-3498 • Scheuplein F, Schwarz N, Adriouch S, Krebs C, Bannas P, Rissiek B, Seman M, Haag F, Koch-Nolte F. 2009. NAD+ and ATP released from injured cells induce P2X7-dependent shedding of CD62L and externalization of phosphatidylserine by murine T cells. J Immunol. 182:2898-908 Acknowledgements supported by grants from the DFG to FH, FKN, SA, MS We thank Marion Nissen for technical assistance. We thank Tim Sparwasser for providing DEREG mice (see Lahl et al. 2007 J Exp Med 204:57-63 Selective depletion of Foxp3+ regulatory T cells induces a scurfy-like disease)