Overexpression of PD-1 on T cells promotes tolerance in cardiac transplantation via ICOS-dependent mechanisms

The programmed death 1/programmed death ligand 1 (PD-1/PD-L1) pathway is a potent inhibitory pathway involved in immune regulation and is a potential therapeutic target in transplantation. In this study, we show that overexpression of PD-1 on T cells (PD-1 Tg) promotes allograft tolerance in a fully MHC-mismatched cardiac transplant model when combined with costimulation blockade with CTLA-4–Ig. PD-1 overexpression on T cells also protected against chronic rejection in a single MHC II–mismatched cardiac transplant model, whereas the overexpression still allowed the generation of an effective immune response against an influenza A virus. Notably, Tregs from PD-1 Tg mice were required for tolerance induction and presented greater ICOS expression than those from WT mice. The survival benefit of PD-1 Tg recipients required ICOS signaling and donor PD-L1 expression. These results indicate that modulation of PD-1 expression, in combination with a costimulation blockade, is a promising therapeutic target to promote transplant tolerance.


Introduction
Programmed death 1 (PD-1) receptor plays a major inhibitory role in T cell activation via interaction with its ligands PD-L1 and PD-L2, resulting in decreased T cell activation, proliferation, differentiation, and cytokine production (1). PD-1 is upregulated by T cells after antigen-mediated activation of the T cell receptor (TCR), and its expression is decreased once the antigen is cleared (2,3). Chronic stimulation of T cells by the antigen leads to high and continuous expression of PD-1, generating an exhausted T cell phenotype (4). PD-1 is expressed on thymocytes and all CD4 + and CD8 + T cells, including Tregs, exhausted T cells, and memory T cells (5). PD-1 can also be expressed by B cells, NK cells, NKTs, some myeloid cells, and cancer cells (5). In an attempt to induce immune regulation in transplantation, the PD-1/PD-L1 pathway has become an attractive target. PD-1 signaling is critical to protect against rejection in mouse heart (6)(7)(8)(9), liver (10), and skin transplant models (11), but the consequence of PD-1 overexpression on T cells is unknown.
In this study, we investigated whether overexpression of PD-1 on T cells could promote long-term graft survival. Using PD-1 Tg mice (12), we show that T cell-specific overexpression of PD-1 promoted tolerance in a fully MHC-mismatched murine cardiac transplant model in combination with a single administration of costimulation blockade. Mechanistically, PD-1 Tg conventional T cells (Tconv; CD4 + Foxp3 -) proliferated significantly less and produced fewer proinflammatory cytokines compared with WT Tconv. Also, graft-infiltrating PD-1 Tg Tregs expressed significantly higher levels of ICOS. In vivo blockade of ICOS prevented tolerance of PD-1 Tg recipients. Using PD-L1-KO donor allografts, we demonstrated that The programmed death 1/programmed death ligand 1 (PD-1/PD-L1) pathway is a potent inhibitory pathway involved in immune regulation and is a potential therapeutic target in transplantation. In this study, we show that overexpression of PD-1 on T cells (PD-1 Tg) promotes allograft tolerance in a fully MHC-mismatched cardiac transplant model when combined with costimulation blockade with CTLA-4-Ig. PD-1 overexpression on T cells also protected against chronic rejection in a single MHC II-mismatched cardiac transplant model, whereas the overexpression still allowed the generation of an effective immune response against an influenza A virus. Notably, Tregs from PD-1 Tg mice were required for tolerance induction and presented greater ICOS expression than those from WT mice. The survival benefit of PD-1 Tg recipients required ICOS signaling and donor PD-L1 expression. These results indicate that modulation of PD-1 expression, in combination with a costimulation blockade, is a promising therapeutic target to promote transplant tolerance.
To further investigate the effector T cell functions of PD-1 Tg recipients, ex vivo cytokine production upon mixed lymphocyte reaction was measured using the Luminex system. When stimulated with donor-derived irradiated splenocytes, PD-1 Tg splenocytes from heart-transplanted mice given CTLA-4-Ig produced significantly less IL-4, IL-6, and IL-17 compared with WT mice ( Figure 3A). Furthermore, there was a slight reduction in IFN-γ and IL-2 secretion by PD-1 Tg splenocytes, although the reduction did not reach significance. Thus, our data suggest that following heart transplantation, T effector cells from PD-1 Tg mice are less functional than T effector cells from WT hosts.
To further assess the cell-intrinsic inhibitory phenotype of PD-1-overexpressing T cells in vivo, we used a graft-versus-host disease (GVHD) model that allows us to track and measure polyclonal, alloreactive T cell proliferation and activation in vivo after adoptive transfer into an allogeneic recipient (17). PD-1 Tg or WT splenocytes were labeled with CFSE and adoptively transferred into sublethally irradiated BALB/c mice ( Figure 3B). Seventy-two hours after the transfer, the proliferation of transferred T cells was assessed by CFSE dilution ( Figure 3C). When compared with WT, PD-1 Tg Tregs ( Figure  3D), CD4 + cells ( Figure 3E), and CD8 + cells ( Figure 3F) proliferated significantly less. The analysis of T effector memory cell subsets demonstrated that proliferation of CD4 + effector memory cells was similar between the groups ( Figure 3E), but CD8 + effector memory cell proliferation was significantly decreased in the PD-1 Tg group ( Figure 3F). In PD-1 Tg animals, CD8 + T cells had a higher mean expression of surface PD-1 when compared with CD4 + T cells (MFI of surface PD-1 on CD8 + and CD4 + T cells, respectively: 13,972 ± 477 versus 8108 ± 364, P < 0.0001; Figure 1D). This finding suggests that PD-1 could have a stronger cell-intrinsic inhibitory phenotype in CD8 + T cells. Overall, the data suggest that PD-1 overexpression impairs T cell proliferation both in vitro and in vivo.
PD-1 upregulation is known to be a marker for exhausted T cells along with other coinhibitory molecules such as TIM-3, LAG-3, and CTLA-4 (18). Although graft-infiltrating Tconv and CD8 + T cells from  PD-1 Tg recipients expressed more CTLA-4 ( Figure 3G) and LAG-3 ( Figure 3H) when compared with naive hearts or WT recipients, the expression of other receptors, like TIM-3, was unchanged or decreased ( Figure 3I), and ICOS was increased ( Figure 3J) at 7 days after transplant. These data suggest that T cells from PD-1 Tg mice may have a partially exhausted phenotype.
To determine whether PD-1 Tg cells had diminished effector responses in settings other than solid organ transplantation, we used an influenza infection model, because the PD-1 pathway has important roles in viral infection. We infected naive WT or PD-1 Tg animals with a sublethal dose of influenza virus (strain A/Puerto Rico/8/1934 H1N1). PD-1 Tg animals were equally capable of controlling viral infection as WT controls, as determined by weight loss (Supplemental Figure 4A) and survival (Supplemental Figure  4B). Also, there was no difference in viral loads analyzed at day 7 after infection (Supplemental Figure  4C). Our data demonstrate that PD-1 Tg mice can mount an optimal immunologic response to acute viral infection despite the high PD-1 expression on T cells.
Tregs are fewer in PD-1 Tg recipients but express higher amounts of IL-10 and latency-associated peptide. Tregs are instrumental for the induction and maintenance of tolerance to auto-and alloantigens (19). To investigate whether Tregs were involved in the tolerance mechanism of PD-1 Tg mice, we analyzed Tregs in the spleen of WT and PD-1 Tg transplanted animals at day 7 after transplant. Treg percentages and counts (CD4 + Foxp3 + ) were significantly lower in PD-1 Tg recipients compared with WT ( Figure 4A). Naive PD-1 Tg animals also had a reduced percentage and absolute numbers of Tregs in the spleen and thymus compared with WT controls (Supplemental Figure 5).
We next hypothesized that PD-1 overexpression could be affecting the suppressive function of the Tregs. Graft-infiltrating Tconv and Tregs from WT or PD-1 Tg recipients of fully MHC-mismatched cardiac transplantation receiving CTLA4-Ig were analyzed for the production of IL-10 and the expression of membrane-bound TGF-β1 through the analysis of the latency-associated peptide (LAP). Indeed, Tregs from PD-1 Tg recipients had higher expression of LAP ( Figure 4, B, D, and E) and increased production of IL-10 ( Figure 4, C-E) when compared with WT cells. Furthermore, there was a slight increase in LAP expression and IL-10 secretion by PD-1 Tg Tconv, although it did not reach significance ( Figure 4, B-E). Last, depletion of Tregs using anti-CD25 Ab before transplantation shortened graft survival of PD-1 Tg recipients treated with costimulation blockade (MST, 89 days versus > 100 days, P = 0.0174; Figure 4F), indicating the important role of Tregs for long-term graft acceptance in PD-1 Tg recipients. mTOR (20) and STAT5 signaling pathways are crucial for Treg function (21). Therefore, we next investigated whether they were responsible for the greater regulatory effects of PD-1 overexpression on T cells. We hypothesized that PD-1 overexpression could inhibit the mTOR pathway, thereby enhancing Treg function. We treated naive WT or PD-1 Tg T cells with PMA/ionomycin at different time points and analyzed the phosphorylation levels of S6K-1 (pS6K), a known mTOR downstream target (22). However, no differences were found in the levels of pS6K between PD-1 Tg and WT Treg cells, suggesting that the mTOR pathway is not affected by PD-1 overexpression (Supplemental Figure 6). We next investigated the phosphorylation level of STAT5, a transcription factor downstream of the IL-2 signaling pathway that binds to the Foxp3 promoter and supports Treg development (23). We treated WT or PD-1 Tg Tregs with 5 ng/mL recombinant murine IL-2 for 15 or 30 minutes and analyzed the expression of pSTAT5 by flow cytometry. In the presence of IL-2, PD-1-overexpressing Tregs had less phosphorylation of STAT5 than did WT Tregs ( Figure 4G).
In summary, Tregs are required for allograft tolerance in PD-1 Tg recipients and exhibit an increased expression of IL-10 and LAP, despite less Treg proliferation. This effect was not mediated by an increase in STAT5 activation or mTOR inhibition.
ICOS is upregulated in PD-1 Tg Tregs. Because PD-1 overexpression did not change the levels of mTOR and decreased pSTAT5, we further investigated PD-1 Tg Tregs, using an reverse transcription PCR array of sorted WT or PD-1 Tg Tregs (CD4 + Foxp3 + cells) from naive animals. The expression of Pdcd1, Icos, and Il13 were significantly upregulated, whereas Eomes, Csf2, and Il2 were downregulated in PD-1 Tg Tregs, compared with WT cells ( Figure 5A).
ICOS is required for the induction of prolonged immune modulation in PD-1 Tg recipients. Because ICOS was increased at both mRNA ( Figure 5A) and protein ( Figure 5C) levels, we assessed its potential role in vivo using a blocking Ab approach against ICOS at the time of transplant. We transplanted BALB/c hearts into PD-1 Tg recipients and treated tolerant recipients with the anti-ICOS Ab or isotype control on days 0, 2, 4, and 6 ( Figure 6A). This ICOS early blockade regimen has been demonstrated to have minimal effects on the improvement of graft survival (24). Prolongation of allograft survival in PD-1 Tg recipients was abrogated upon ICOS blockade, indicating a critical role for ICOS in tolerance induction ( Figure 6A).
We next investigated whether anti-ICOS therapy inhibited alloreactive T cell responses at day 21 after transplant by enzyme-linked immune absorbent spot (ELISPOT). Fewer IFN-γ-producing, splenic, anti-donor T cells from PD-1 Tg animals were observed when compared with those from WT recipients ( Figure  6B). In PD-1 Tg recipients, anti-ICOS treatment slightly increased the number of IFN-γ-producing alloreactive T cells, but the number was still significantly lower than in WT controls ( Figure 6B). ICOS blockade had no effect on CD4 + and CD8 + effector memory (CD44 hi CD62 lo ) T cells ( Figure 6C) or Treg frequency ( Figure  6D). Thus, our data suggest that anti-ICOS treatment has minimal effects on effector T cell responses.
We hypothesized that ICOS blockade could be affecting the suppressive function of the PD-1 Tg Tregs. Indeed, both intragraft and splenic Tregs from PD-1 Tg recipients treated with anti-ICOS had decreased production of IL-10 ( Figure 6, E and F) and LAP expression ( Figure 6, G and H). Anti-ICOS treatment had no effects on the IL-10 production and LAP expression by non-Tregs ( Figure 6, E-H). We verified by ELISPOT that the increased IL-10 production in PD-1 Tg recipients was allospecific, and it was inhibited by the ICOS blockade ( Figure 6I).
Last, we observed that flow-sorted CD4 + Foxp3-GFP + Tregs from PD-1 Tg heart recipients had a superior ability to inhibit T cell proliferation in an in vitro suppression assay than did WT Tregs ( Figure 6J). This effect was abrogated by the anti-ICOS treatment ( Figure 6J). Altogether, our data demonstrated that the anti-ICOS treatment has a dominant inhibitory effect on Treg suppressive function.

Discussion
In this work, we show in a mouse cardiac transplant model that the overexpression of PD-1 on T cells significantly prolongs allograft survival and protects against chronic rejection in the setting of early B7:CD28 blockade with CTLA-4-Ig. The importance of the PD-1/PD-L1 pathway in mediating alloimmune regulation has been investigated in several prior studies, most of which used either a blocking Ab or genetic deletion. For example, CTLA-4-Ig-induced tolerance was abrogated in PD-L1 KO recipients of BALB/c hearts or WT recipients treated with anti-PD-L1 blocking Abs (7). On the other hand, the use of PD-1 agonistic agents such as PD-L1-Ig in addition to cyclosporine or rapamycin resulted in prolonged allograft survival (6).
The requirement of the early blockade of the B7:CD28 signal to achieve the significant survival difference is most likely related to the competitive opposite signaling effects of PD-1 and CD28 on T cells (3). PD-1 transduces an inhibitory signal in part via the recruitment of phosphate SHP-2, which downregulates CD28-mediated PI3K activity (25). A strong CD28 signal can easily overcome PD-1 inhibition and simultaneous blockade of B7:CD28 with an enhanced PD-1 signal demonstrated synergism in regulating the alloimmune response. Another mechanism of PD-1 inhibition of T cells is by affecting the stability of the antigen-presenting cell-T cell (APC-T cell) synapse. Through intravital microscopy, PD-1 signaling was shown to block the TCR "stop signal" required for an effective and prolonged APC-T cell interaction and consequent T cell activation (26)(27)(28). Therefore, overexpression of PD-1 may promote transient and incomplete APC-T cell interactions, leading to a significant reduction in T cell activation and proliferation. Nonetheless, these PD-1 Tg T cells are still capable of mounting an effective immune response in the absence of costimulation blockade, as evident in both an acute murine cardiac transplantation model and a model of influenza A infection.
A unique feature of PD-1/PD-L1 coinhibitory signal is the expression of PD-L1 by nonhematopoietic cells, such as vascular endothelium (9). Although CTLA-4 signaling plays a predominant role in early T cell activation in secondary lymphoid organs, PD-1 may have a dominant regulatory function in peripheral tissues, based on the local PD-L1 expression (27,29). Supporting the role of PD-L1 on the allograft, the survival advantage was abrogated if PD-1 Tg mice were transplanted with BALB/c hearts that lacked PD-L1. This finding reinforces prior observations suggesting a protective effect of overexpression of PD-L1 on donor grafts (30) and the deleterious effect of the absence of PD-L1 in either hematopoietic or nonhematopoietic cells in the graft in a chimeric PD-L1 mouse model (9). However, other reports show that allografts lacking PD-L1 are still accepted in mice treated with repetitive doses of CTLA-4-Ig but exhibited severe chronic rejection and vasculopathy (7). Our histopathology analysis of the BALB/c and bm12 grafts at 100 days and 8 weeks after the transplant, respectively, support the critical role of PD-1/PD-L1 signaling in preventing chronic rejection. In sum, PD-1/PD-L1 signaling in the graft seems to play a major protective role in the allograft. We provide evidence that despite the decrease in Treg numbers, Tregs were required for the long-term graft acceptance in PD-1 Tg recipients. Known to regulate both central and peripheral tolerance, PD-1 is highly expressed on regulatory T cells and is involved in inducible Treg generation (16).We have demonstrated an increase in IL-10 and TGF-β signaling in PD-1 Tg Tregs in our transplant model, both of which are important pathways for Treg suppression. We also have shown that, compared with WT T cells, PD-1 Tg effector T cells proliferate significantly less in vitro and in vivo when exposed to alloantigens, and their cytokine secretion is blunted when stimulated ex vivo. This is in agreement with reports showing that PD-1/PD-L1 interaction results in inhibition of TCR-mediated lymphocyte proliferation (31,32). Our findings also consolidate data showing that PD-1 activation in a fully MHC-mismatched cardiac transplant in mice results in the reduction of intragraft cytokine secretion such as IFN-γ (6).
Although STAT5 is known to support Treg function (21,23), we observed that PD-1 overexpression decreased the activation of STAT5 upon IL-2 stimulation. Our data suggest that alternative intracellular molecular mechanisms exist by which Tregs can exert their inhibitory effects. Indeed, our data show that  Tregs overexpressing PD-1 have a higher expression of coinhibitory molecules upon transplantation. We found that PD-1 Tg Tregs exhibited upregulation of ICOS both at mRNA and protein levels. Also, all graft-infiltrating Tregs from PD-1 Tg recipients were ICOS + . ICOS has a key role in controlling effector functions and survival of Tregs in models of oral (33) and respiratory tolerance (34), as well as type 1 diabetes (35). However, it was reported to be dispensable for the in vitro generation of Tregs from CD4 + CD25cells (36). ICOS was also reported to be a marker for highly suppressive, differentiated, antigen-specific Tregs that can inhibit CD8 + T cell responses (37). Moreover, ICOS hi Tregs are accumulated in the microenvironment of different tumors (38)(39)(40) and present a higher suppressive ability than ICOS lo Tregs (40). In humans, Ito et al. (41) identified a population of thymic-derived ICOS + Tregs that overexpressed CTLA-4 and produced high amounts of IL-10. The authors also found that CD28 engagement strongly inhibited the in vitro proliferation of ICOS + Tregs. This suggests that in our model, early costimulation blockade in PD-1 Tg recipients would favor the proliferation and suppressive effects of ICOS + -expressing Tregs.
The costimulatory molecule ICOS has also been reported to play a critical role in effector T cell activation and differentiation. Therapies targeting the blockade of the ICOS/ICOSL pathway have been used to treat rejection in mice (24,42). However, these therapies have failed to improve cardiac graft survival in monkeys (43). We hypothesized that unexpected effects on ICOS blockade on highly suppressive ICOS + Tregs could contribute to these disparate results. Indeed, anti-ICOS treatment negatively impacted the suppressive function of Tregs in PD1-Tg recipients, as evident by reduced production of IL-10 and TGF-β as well as by reduced suppressive capacity in vitro.
Anti-CTLA-4 (44), anti-PD-1 (45), and anti-PD-L1 (46) Abs and their combinations have reached clinical use and have proven effective for cancer immunotherapy. PD-1-pathway inhibitors are now FDA-approved for therapeutic use in more than 20 types of cancer. However, in regard to immunosuppression, CTLA-4 fusion proteins are clinically approved for use in rheumatoid arthritis (47) and kidney transplantation (48), and a potentially novel PD-1 agonist is being tested in humans in a phase 1 trial (Clin-icalTrials.gov identifier NCT03337022).
Given the complexity and the myriad escape mechanisms that the immune system possesses, immunosuppression for solid organ transplantation continues to require a multipronged approach (49), and efforts to identify novel potential targets are essential. The preclinical data we present in this report suggest that the PD-L1/PD-1 pathway is a promising target in clinical transplantation and warrants further investigation, especially in synergy with costimulation blockade.
Mouse abdominal heart transplantation. Heterotopic intraabdominal vascularized cardiac transplant was performed using microsurgical techniques, as described by Corry et al. (52). When indicated, recipient mice were injected i.p. with a single dose of human CTLA-4-Ig (5 mg/kg on day 2 after transplantation; Abatacept; Bristol Myers Squibb). Graft survival was assessed by palpation of the heartbeat. Rejection was determined by complete cessation of palpable heartbeat and was confirmed by direct visualization after laparotomy. Graft survival is shown as the MST in days. Anti-CD25 (PC-61.5.3, 100 μg; catalog BE0012; Bio X Cell) was administered i.p. on days -7 and -1 before transplantation. Anti-ICOS (clone 7E.17G9; catalog BE0059; Bio X Cell) or rat IgG2b isotype control (clone LTF-2; catalog BE0090; Bio X Cell) was administered i.p. on day 0 (375 μg), followed by 250 μg on days 2, 4, and 6 after transplantation.
JCI Insight 2021;6(24):e142909 https://doi.org/10.1172/jci.insight.142909 Mouse cervical heart transplantation. Cervical heart transplantation was done by end-to-side anastomosis of donor ascending aorta to recipient carotid artery and end-to-side anastomosis of donor pulmonary artery to recipient external jugular vein. Rejection was determined by complete cessation of palpable heartbeat and was confirmed by direct visualization after laparotomy.
Histopathology. Cardiac graft samples from transplanted mice were harvested from both PD-1 Tg and WT groups at 14, 60, or 100 days after transplantation. Grafts were then fixed in 10% formalin, embedded in paraffin, transversely sectioned, and stained with H&E or elastin stain. Using the revised ISHLT classification (53), a transplant pathologist blinded to treatment graded the degree of rejection (from 0 to 3).
In vivo proliferation analysis using a GVHD model. WT or PD-1 Tg splenocytes were labeled with CFSE and adoptively transferred (~6 × 10 7 /mouse) into sublethally irradiated (1000 rad) BALB/c mice. Lymphocytes were harvested 3 days afterward, and proliferation was assessed by flow cytometry, using a FACS Canto II with FACS Diva software (both from BD Biosciences).
Influenza infection. WT or PD-1 Tg mice were infected intranasally with 0.3 LD 50 influenza A strain PR8 (strain A/Puerto Rico/8/1934 H1N1; Charles River Laboratories). Weight loss was measured daily, and mice were euthanized when weight loss reached 20% of starting weight. Viral loads were determined from RNA isolated from mice lung homogenates using the Direct-zol RNA Miniprep kit (Zymo Research). RNA (40 ng) was reverse transcribed into cDNA using the iScript cDNA Synthesis Kit (Bio-Rad). In a final volume of 20 μL, cDNA was amplified for the acidic polymerase gene of influenza A/PR8 using the primers and probe: forward primer, 5′-CGGTCCAAATTCCTGCTGA-3′; reverse primer, 5′-CATTGG-GTTCCTTCCATCCA-3′; probe, 5′-6-FAM-CCAAGTCATGAAGGAGAGGGAATACCGCT-3′. Quantitative PCR was performed with a QuantStudio 3 PCR system (Applied Biosystems). Relative mRNA levels were calculated using the comparative Ct method, using the housekeeping gene Hprt as an internal control. All the harvested lungs were weighted, and data were expressed per mg of tissue.
Isolation of organs lymphocytes. To isolate cells from the native hearts, cardiac allografts, spleens, and draining lymph nodes, organs were excised, minced, and digested with 500 U/mL collagenase (Roche) for 30 minutes at 37°C, followed by incubation with 0.1 M EDTA in PBS, pH 7.2, buffer for 5 minutes before final suspension in 5 mM EDTA, 1% FBS in PBS, pH 7.2. Isolated cells were then mechanically dissociated through a Falcon 70 μm cell strainer (Corning), and RBCs were lysed using hypotonic ACK buffer (Lonza).
Cytokine measurement by Luminex assay. Splenocytes harvested at 2 weeks after transplantation from recipient mice were restimulated ex vivo by irradiated donor-type splenocytes (0.5 × 10 6 responder cells with 0.5 × 10 6 irradiated donor splenocytes) for 48 hours. Cell-free supernatants were analyzed by a multiplexed cytokine bead-based immunoassay using a preconfigured 21-plex mouse cytokine detection kit (Millipore). All samples were tested in triplicate wells.
ELISPOT assays. Mouse IFN-γ and IL-10 ELISPOT assays were performed using kits from BD Biosciences according to the manufacturer's protocol. Briefly, 0.45 μm hydrophobic, high-protein-binding Immobilon-P membrane plates (Millipore) were coated with either IFN-γ or IL-10 capture Ab at 4°C overnight, followed by blocking with 10% FBS-supplemented RPMI1640 for 1 hour at room temperature. Lymphocytes were isolated from transplanted mouse spleens at day 21 after transplant by magnetic separation using the EasySep Mouse T Cell Isolation Kit (StemCell Technologies). We incubated 3 × 10 5 T cells with 5 × 10 5 irradiated donor (BAL-B/c), host-derived (B6), or third-party (C3H) splenocytes at 37°C and 5% CO 2 for 24 hours for IFN-γ or 48 hours for IL-10 detection. Spots were detected and counted using an ImmunoSpot analyzer (Cellular Technology).