Recognition of synthetic polyanionic ligands underlies “spontaneous” reactivity of Vγ1 γδTCRs

Abstract Although γδTCRs were discovered more than 30 yr ago, principles of antigen recognition by these receptors remain unclear and the nature of these antigens is largely elusive. Numerous studies reported that T cell hybridomas expressing several Vγ1‐containing TCRs, including the Vγ1Vδ6 TCR of γδNKT cells, spontaneously secrete cytokines. This property was interpreted as recognition of a self‐ligand expressed on the hybridoma cells themselves. Here, we revisited this finding using a recently developed reporter system and live single cell imaging. We confirmed strong spontaneous signaling by Vγ1Vδ6 and related TCRs, but not by TCRs from several other γδ or innate‐like αβ T cells, and demonstrated that both γ and δ chains contributed to this reactivity. Unexpectedly, live single cell imaging showed that activation of this signaling did not require any interaction between cells. Further investigation revealed that the signaling is instead activated by interaction with negatively charged surfaces abundantly present under regular cell culture conditions and was abrogated when noncharged cell culture vessels were used. This mode of TCR signaling activation was not restricted to the reporter cell lines, as interaction with negatively charged surfaces also triggered TCR signaling in ex vivo Vγ1 γδ T cells. Taken together, these results explain long‐standing observations on the spontaneous reactivity of Vγ1Vδ6 TCR and demonstrate an unexpected antigen presentation‐independent mode of TCR activation by a spectrum of chemically unrelated polyanionic ligands.

transcription factor promyelocytic leukemia zinc finger protein (PLZF), 16,17 and innate-like functional properties 13 similar to that of NKT T cell subsets, suggesting that these cells, like their counterparts, undergo agonist selection on endogenous ligands during thymic differentiation. [18][19][20] Although NKT cells are often defined by expression of a TCR composed of V 1 (V nomenclature here and below after 21 ) and V 6.3 chains in C57BL/6 mice (or allelic variants of the latter-V 6.4 in DBA or V 6.2 in Balb/c 22 ), few PLZF-expressing T cells are also detected among V 6.3cells, many of which express V 1 with V 6 12 and V 4 chains. 23 Numerous studies on the characterization of T cell hybridomas described spontaneous cytokine secretion [24][25][26][27][28][29][30][31][32][33] or nuclear factor of activated T cells (NFAT) reporter expression 34 by some clones. Characterization of the TCR repertoire of such hybridomas revealed that nearly all spontaneously reactive clones expressed V 1, and the majority were V 6.3 positive, whereas a minority expressed other chains, often homologous to V 6.3-such as V 6 12 and V 4. 28,30,35 Expression of such TCRs conferred spontaneous cytokine secretion ability to TCRhybridomas, formally demonstrating that this property was driven by TCR. 29,36 These results led to the idea that these TCRs recognize a self-ligand expressed by hybridoma cells themselves. [24][25][26][27][28][29][30][31][32][33][34][37][38][39][40] Early studies also demonstrated that cytokine secretion by such hybridomas was enhanced by mycobacterial Hsp60. 24 It was later demonstrated that in addition to Hsp60 itself and peptides derived from this protein, 36 cytokine secretion by these hybridomas can be enhanced by synthetic peptides such as poly(Glu, Tyr), 41 several lipids including cardiolipin 33 and even cultivation under reduced serum concentrations. 33 Although "TCR transfer" experiments confirmed that cytokine secretion in response to these stimuli was specific to this group of TCRs, it remained unclear how such a variety of treatments can trigger TCR signaling and how this signaling is related to spontaneous activation of cells with the very same TCRs. As a possible explanation of these phenomena, it was suggested that these treatments may in fact result in further up-regulation of a self-ligand, possibly as a result of cellular stress, rather than serve as ligands themselves. 40 It was also noted that a lot of these candidate ligands are negatively charged, and it was suggested that polyanionic properties may mediate this broad reactivity. 42,43 Here, we report that spontaneous reactivity of V 1 TCR-expressing cells is unrelated to recognition of a self-ligand, is independent of contacts between cells and is explained by activation of these TCRs by negatively charged cell culture surfaces such as tissue culture-treated polystyrene and glass. Taken together with earlier studies, these results demonstrate that several V 1 TCRs, including that of NKT cells, exhibit broad polyreactivity against anionic ligands.

TCR constructs
TCR -P2A-TCR cassettes for V 5V 1, V 6V 1, and V 1V 6.4 (DTN40) were generated by overlapping PCRs as described 44 and cloned into pMIG vector containing IRES-GFP using BglII and XhoI sites. DTN40 TCR chains were PCR-amplified from DNA of DTN40 TCR-transgenic mouse. 16 V 5V 6.4, V 6V 6.4, and V 1V 1 TCR were cloned from the constructs described above using BspEI and BglII or BspEI and XhoI sites.
V 4 and V 6 12 TCR chains were cloned from V 1-expressing spontaneously reactive hybridomas generated as previously described 34

Retroviral infections
Retrovirus-containing supernatants were generated by transient cotransfection of PlatE packaging cells with retroviral constructs described above and pCL-Eco packaging vector using calciumphosphate transfection as described. 46 Two or three rounds of spin-infection (500 ×g, 45 min) were performed.

Cell culture
All cells were kept in humidified atmosphere at 37 • C and 5%  All stimulation experiments were performed overnight unless stated otherwise.

Mice
All mice used in this study had C57BL/6 genetic background. Mice were bred and maintained at Comparative Medicine Biomedicum facility of Karolinska Institutet (Stockholm, Sweden). All animal experiments were carried out according to valid project licenses, which were approved and regularly controlled by the Swedish Veterinary Authorities.

Flow cytometry
Samples were acquired on BD LSR Fortessa flow cytometer and ana-

Live cell imaging
Single cell imaging was performed using a microwell chip (with a glassbottom surface and silicon walls of the wells) 50 placed in a confocal microscope (Zeiss LSM880), Oberkochen, Germany equipped with a motorized stage and an incubation chamber allowing physiologic conditions (37 • C, 5% CO 2 ). Time-lapse images were acquired at multiple positions every 90 min. For signal quantification, images from single cells trapped in individual wells were exported as TIFF files every 5 frames (7.5 h) and analyzed in ImageJ/Fiji (version 1.52 h). Cell area was marked manually using the freehand selection tool and the mean fluorescence intensity from sFT-blue and sFT-red within the region was quantified.

Modeling and alignment
Homology modelling using SWISS-MODEL 51

Statistics
Statistical analyses were performed in GraphPad Prism 8 (GraphPad Software, Inc.) using unpaired two-tailed Mann-Whitney test for comparison of sFT-blue levels in reporter cells transduced with different TCRs.

Unique spontaneous reactivity of the NKT TCR
To study signaling properties of TCRs, we utilized BW5147 murine thymic lymphoma cells engineered to express subunits of the CD3 complex and a sFT reporter under the control of an NFAT-dependent promoter. In this system, TCR signaling induces expression of the sFT reporter and this protein shifts its fluorescence from blue to red over time, thereby allowing to distinguish recent and past NFAT activation 47,53 (Fig. 1A). We used these cells to express a panel  in any measurable induction of the reporter in complete or serumfree media (Fig. 1), whereas sFT expression was readily activated by addition of GalCer to iNKT TCR-expressing cells in a cell densitydependent manner (Fig. 1J)   It was previously reported that V 1 + hybridomas with several TCR chains distinct from but related to V 6.3/V 6.4 also exhibit spontaneous cytokine secretion. 25,28 In line with these findings, we also observed spontaneous cytokine production by hybridomas expressing V 1V 4 and V 1V 6 12 TCRs (data not shown). Expression of the DTN40 TCR-derived V 1 chain in combination with chains cloned from these hybridomas conferred spontaneous reactivity in our reporter system (Fig. 2D). Interestingly, whereas the two V 1V 6 12 TCRs only differed in their CDR3 regions (Table 1), their expression resulted in different levels of reporter up-regulation even when gating on identical levels of TCR expression was applied (Fig. 2E)

Spontaneous reactivity of the NKT TCR does not require cell-cell contact
It was long thought that spontaneous cytokine production by the NKT TCR-expressing hybridomas reflects recognition of a self-ligand expressed by the hybridoma cells themselves. [24][25][26][27][28][29][30][31][32][33][34][37][38][39][40] We reasoned that if this was the case and this process required interaction with a ligand on neighboring cells, then reporter induction will be decreased when cells are cultured at a lower density due to a decrease in cell contacts. Unexpectedly, and in stark contrast with iNKT TCRexpressing cells pulsed with GalCer (Fig. 1J), the reporter in V 1V 6.4 cells was expressed at comparable levels in cells cultivated at very low density in flat-bottom wells and at high density in U-bottom wells both in complete medium and upon serum starvation (Fig. 3A). These results suggested that NKT TCR activation in this system may not involve recognition of a ligand expressed on neighboring cells.
To formally test if induction of the reporter can occur without contacts between cells, we next performed live cell imaging using a silicon-glass microchip that allows to spatially confine single cells in microwells. 54 Reporter expression was not detected in this system in unstimulated control V 5V 1 cells (Fig. 3B). V 5V 1 cells stimulated overnight with plate-bound anti-CD3 and deprived of this stimulus upon plating on the chip exhibited high starting levels of the sFT-blue reporter that declined over time, concomitant with an increase in sFT-red signal (Fig. 3C). Expression of the reporter was also readily detected in unstimulated V 1V 6.4 cells even when analysis was restricted to single cells trapped in a well that did not undergo cell division during the observation period (Fig. 3D).
The reporter levels were not enhanced when multiple cells were present in the same well (data not shown) and the level of reporter expression fluctuated over time (Fig. 3D). Reporter up-regulation by V 1V 6.4 cells was often followed by loss of round shape and cell stretching (Fig. 3D). Similar morphologic changes were observed in stimulated ( Fig. 3C) but not unstimulated (Fig. 3B) V 5V 1 cells, indicating that this change in morphology reflects an activated status of the cell. Taken together, these results demonstrate that activation of V 1V 6.4 TCR signaling in this system does not require any form of intercellular communication.

Spontaneous reactivity of V 1 TCRs is induced by interaction with negatively charged cell culture surfaces
Two models could explain cell interaction-independent induction of the reporter described above. First, it was conceivable that V 1V 6 TCR is capable of cell-autonomous signaling similar to that of pre-TCR or pre-BCR. Second, it was possible that the TCR is triggered surfaces, the reporter expression was close to background level ( Fig. 4D), but it was readily up-regulated upon replating to negatively charged plastic (Fig. 4D). We therefore conclude that "spontaneous"

DISCUSSION
T cell hybridomas expressing V 1V 6 and few related TCRs were reported to exhibit two modes of reactivity. First, cells with these TCRs were known to spontaneously secrete cytokines in culturea phenomenon that was always interpreted as recognition of a self-ligand. [24][25][26][27][28][29][30][31][32][33][34][37][38][39][40] Second, this secretion was enhanced by a wide variety of chemically unrelated compounds, including bacterial Hsp60, 24,25 negatively charged amino acid-containing peptides derived from Hsp60, 36 synthetic poly(Glu, Tyr) peptide, 41 and several negatively charged lipids such as cardiolipin 33 that were all suggested to function as ligands for these TCRs. Our findings reported here demonstrate that spontaneous signaling by the V 1V 6 TCR is unrelated to self-antigen recognition and is instead induced by commonly used negatively charged cell culture surfaces. Taken together with earlier reports, these results suggest that both "spontaneous" and "induced" modes of V 1V 6 activation are likely to be underlain by the same mechanism-recognition of a broad spectrum of anionic ligands.
Although the exact mechanisms that activate this TCR upon exposure to such a wide range of organic and even inorganic compounds remain to be determined, the presence of negative charges is a common feature, and, as previously noted, 42 many of these compounds represent polyanions. It is therefore plausible that anionic properties are required for interaction of a compound with the V 1V 6 TCR.
Although the direct physical interaction of this TCR with the polyanionic ligands remains to be shown and the responsible paratope has to be mapped, our results reported here demonstrate that specific and chains both contribute to this reactivity. It was previously suggested that structural properties of V 1 chain may mediate recognition of polyanionic ligands. 42 Indeed, molecular modeling suggests that several positively charged amino acids in V 1 framework 3 region (R74, K79, K81, K82) are exposed at the "apical" surface of the TCR (Supporting Information Fig. S1E and F). These amino acids are absent from V 5, V 6, and V 7 sequences but are present in V 2 and/or V 4 chains (Supporting Information Fig. S1G). However, lysins 59 and 60 in framework 2 region, that are, according to the model, also exposed on the "lateral" TCR surface, are unique for the V 1 chain (Supporting Information Fig. S1G).
Although it remains to be tested if any of these positively charged amino acids in the framework regions are involved in recognition of the polyanionic ligands, it is interesting to note that germline-encoded V sequences were recently shown to be involved in recognition of butyrophilin family members by mouse V 7 and human V 4 TCRs. 8,9 Results reported here also demonstrate that the amino acid sequences of CDR3 regions impact the strength of spontaneous reactivity. Although the exact features of CDR3 regions responsible for that phenomenon remain to be characterized, it is interesting to note that 1A1 V 1V 6.3 TCR that has an arginine in its CDR3 loop exhibited higher spontaneous reactivity than 1B1 TCR that does not have positively charged amino acids in this region. Although for these TCRs minor differences in CDR3 may also contribute to differences in spontaneous signaling, the two V 1V 6 12 TCRs used here only differed by their CDR3 regions. Again, the 68 TCR that has three arginines in CDR3 exhibited spontaneous reactivity stronger than that of A2 TCR with no positively charged amino acids in its CDR3 loop. Thus, CDR3 sequence modulates the intensity of the signaling, possibly through positively charged amino acids in that region.
Overall, the results reported here with a limited number of naturally occurring and chimeric TCRs suggest that responsiveness to polyanionic ligands is "a quantitative trait" that can be influenced by both and chain sequences, with V 1 possibly making a more important contribution to this interaction.
Although the polyanionic nature seems to be a common property of V 1V 6 TCR activators, not all negatively charged substances can induce this signaling and a number of polypeptides containing negatively charged amino acids failed to activate this receptor. 41  Although recognition of polyanionic ligands is restricted to a narrow subset of TCRs, these few TCRs seem to be capable of recognizing a remarkably broad spectrum of ligands ranging from glass surfaces to lipids and synthetic peptides. This breadth of reactivity of these TCRs is reminiscent of polyreactivity suggested for antigen receptors of another innate-like lymphocyte subset-B-1 cells. 58 Polyreactive antibodies produced by these cells are thought to provide a first line of defense against pathogens by binding to their membranes and cell walls. 58 Although the physiologic relevance of V 1V 6 TCR polyreactivity remains to be determined, it is interesting to note that whereas occurrence of polyanionic surfaces in mammals is limited, 59 cell walls of Gram-positive and Gram-negative bacteria are negatively charged. 60 Whether V 1 TCRs can recognize negatively charged cell walls and whether such recognition could be physiologically relevant remains to be investigated. Nevertheless, it is interesting to note that V 1V 6.3 TCR-expressing cells are crucial in the immune response to several pathogens, 61,62 including response to Listeria. 63,64 Ample evidence, including expression of the TCR-inducible transcription factor PLZF, activated cell-surface phenotype and ability to rapidly produce cytokines upon stimulation, 13,16,17 suggests that NKT cells, similarly to many other T cell subsets as well as