Crotalus durissus collilineatus venom was isolated, structurally characterized and evaluated for its toxicity and ability to interfere with cell proliferation and migration in MDA-MB-231, a human breast cancer cell line.In recent decades, snake venom disintegrins have received special attention due to their potential use in anticancer therapy. Disintegrins are small and cysteine-rich proteins present in snake venoms and can interact with specific integrins to inhibit their activities in cell-cell and cell-ECM interactions. These molecules, known to inhibit platelet aggregation, are also capable of interacting with certain cancer-related integrins, and may interfere in important processes involved in carcinogenesis. Therefore, disintegrin from Based on previous studies, disintegrin was isolated by FPLC, through two chromatographic steps, both on reversed phase C-18 columns. The isolated disintegrin was structurally characterized by Tris-Tricine-SDS-PAGE, mass spectrometry and N-terminal sequencing. For the functional assays, MTT and wound-healing assays were performed in order to investigate cytotoxicity and effect on cell migration in vitro, respectively.p < 0.05), compared to negative control.Disintegrin presented a molecular mass of 7287.4 Da and its amino acid sequence shared similarity with the disintegrin domain of P-II metalloproteases. Using functional assays, the disintegrin showed low cytotoxicity  after 24 h of incubation and in the wound-healing assay, the disintegrin (3 μg/mL) was able to significantly inhibit cell migration (24%, C. d. collilineatus induces low cytotoxicity and inhibits migration of human breast cancer cells. Therefore, it may be a very useful molecular tool for understanding ECM-cell interaction cancer-related mechanisms involved in an important integrin family that highlights molecular aspects of tumorigenesis. Also, non-RGD disintegrin has potential to serve as an agent in anticancer therapy or adjuvant component combined with other anticancer drugs.Thus, our results demonstrate that non-RGD disintegrin from  Trimeresurus gramineus venom, called “trigramin”, presenting the arginine-glycine-aspartic acid (RGD) domain, was able to inhibit platelet aggregation, by preventing the connection between fibrinogen and platelets stimulated by ADP -2, 5 diphenyl tetrazolium bromide (MTT) assay according to Mosmann . Initial5 cells/well) were grown to 90% confluence on a 12-well plate. Then, with the aid of a sterile tip, a cicatrix was performed on the adherent monolayer of tumor cells. The culture medium was changed to remove the cell debris and a new culture medium added containing the lowest concentration (3.0 μg/mL) of disintegrin which demonstrated effects on the cell line in the MTT assay and PBS as CN. Images of the wounds were recorded with a camera coupled to the inverted microscope at times 0, 4, 8 and 24 h of incubation. The mean distance of the internal area was determined as the percentage migration using the software AxionVision according to the following equation:For the wound healing assay, 3.0 μg/mL of disintegrin was used, and the assay was performed according to Liang, Park and Guan . MDA-MB-n = 3) were statistically analyzed using the software GraphPad Prism 5 . After assessing normality of variable distributions using the Kolmogorov–Smirnov test, experimental data were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s test. Statistical significance was considered at p < 0.05.All the results were expressed as the mean ± standard deviation (SD). Data from three independent experiments . The electrophoretic profile revealed that Fraction 2 exhibits a protein band of apparent 7 kDa molecular mass and some contaminants Fig. . After tThe first 43 amino acid residues of the N-terminal region of the Fraction 6 were sequenced by Edman degradation and shared identity with others disintegrins Fig. . In addiC. d. collilineatus (C0L2T8) and C. d. durissus (Q2QA03), including the conservation of cysteine amino acid residues  of metalloproteases PII from ues Fig. . Comparit test - data not shown), we chose to use the concentration of 3 μg/mL in the wound healing assay.Cell viability of MDA-MB-231 was determined in the presence of disintegrin; it was observed that the concentrations of 3 and 6 μg/mL of disintegrin significantly decreased cell viability in approximately 15% and 17%, respectively Fig. . Considep < 0.05 compared to negative control), after 24 h of incubation  [C. simus (7.1 kDa) [C. d. collilineatus [Non-RGD disintegrin from 7.1 kDa) , disinte7.1 kDa)  and protlineatus , 40.Cdc non-RGD disintegrin isolated in this study is absent from the classical RGD motif or any other motif previously observed in this protein family or glutamate-cysteine-aspartate (ECD) motif which characterize disintegrin-like proteins that may inhibit tumor progression [C. d. collilineatus by transcriptome techniques [C. d. collilineatus (C0L2T8) and C. atrox (Q2QA03). On the other hand, these disintegrins, as well as that of our study, present the disulfide bond pattern of this protein family, another signature that characterizes disintegrins. This pattern of disulfide bonds has already been well reported and is closely related to the formation of the protein loop and activity. If disintegrins are reduced and alkylated their biological activity is decreased [As to the primary sequence obtained through Edman degradation and mass spectrometry procedures, gression . In the chniques , as wellecreased –53.IIbβ3 platelet integrins, preventing their binding to fibrinogen and consequently inhibiting platelet aggregation [IIbβ3) and breast cancer cells (αvβ3) are similar, disintegrins are considered candidates to be anticancer agents [Disintegrins are known to be potent inhibitors of platelet aggregation, and some drugs based on its structure were developed and approved by the FDA , such asr agents . Integrir agents . They arr agents , and arer agents .2 and β5 and moderate levels of αv, α3 and β1 integrins [5 and αvβ5 integrins when compared with non-breast cancer cell line (Hek-293) [C. d. colilineatus venom presents low cytotoxicity, although its toxicity increases with the concentration, having a dose-response effect. As in our results, the tzabcanin also showed dose-dependent toxicity against human malignant melanoma (A-357) and human colorectal adenocarcinoma (Colo-205) cell lines, but the viabilities of human breast adenocarcinoma (MCF-7) and human lung adenocarcinoma (A-549) cell lines were not affected [Since disintegrins are considered candidates to be agents of anticancer therapy, we performed cytotoxicity and cell migration assays on metastatic breast cancer cells, namely MDA-MB-231 cells. This cell line is able to express low levels of the subunits αntegrins . TaheriaHek-293) . Our resaffected , 60. Lebaffected , 61.Cdc non-RGD disintegrin was able to inhibit the migration of MDA-MB-231 cells after 24 h of incubation, demonstrating that it is a possible anticancer agent with potential to inhibit the formation of breast cancer tumors. Similar results were obtained from tzabcanin, which inhibited cell migration of A-375 and A-549 cell lines [Crotalus viridis viridis, and r-mojastin 1, from Crotalus scutulatus scutulatus, that inhibited the migration of human pancreatic adenocarcinoma cancer cells (BXPC-3) [The cell migration assay showed that ll lines , r-Virid(BXPC-3) .In addition, considering the potential of disintegrins as anticancer therapeutic agents, they can also be used in combination with known chemotherapeutics. For example, echistatin combined with cisplatin (CDDP) was capable of increasing the expression of nuclear factor kappa B (NFκB), caspase-9 and caspase-3, leading to increased apoptosis in MDA-MB-231 cells. These data suggest that the combination of this disintegrin with CDDP may serve as a new type of anticancer therapy .C. d. collilineatus venom. The studies performed on the human breast cancer MDA-MB-231 cell line demonstrate that this disintegrin is able to inhibit cell migration, without severely affecting cell viability. Therefore, it may be a very useful molecular tool for understanding ECM-cell interaction cancer-related mechanisms involved important family of integrin that would elucidate molecular aspects of tumorigenesis. Also, this non-RGD disintegrin has potential to serve as an agent in anticancer therapy or adjuvant component combined with other anti-cancer drugs.In this study, we described the isolation and characterization of the first disintegrin with an LVN motif from


Silver nanoparticles (AgNPs) are widely used nanomaterials in both commercial and clinical biomedical applications, due to their antibacterial properties. AgNPs are also being explored for the treatment of cancer in particular in combination with ionizing radiation. In this work, we studied the effects of AgNPs and ionizing radiation on mitochondrial redox state and function in a panel of lung cell lines . The exposure to AgNPs caused cell cycle arrest and decreased cell proliferation in A549, BEAS-2B and Calu-1, but not in NCI-H358. The mitochondrial reactive oxygen species (ROS) and protein oxidation increased in a time- and dose-dependent manner in the more sensitive cell lines with the AgNP exposure, but not in NCI-H358. While ionizing radiation also induced changes in the mitochondrial redox profiles, in general, these were not synergistic with the effects of AgNPs with the exception of NCI-H358 and only at a higher dose of radiation.  Engineered nanoparticles, defined as materials having at least one dimension less than 100 nm in size, made of materials, including metals , are increasingly used for industrial application and as components of consumer products . BecauseAmong metallic nanoparticles, silver nanoparticles (AgNPs) are the most widely used for consumer, industrial, and biomedical applications because of their unique physical, chemical, optical, catalytic, antibacterial properties ,5,6. The2O2 to form highly reactive sulfenic acids [2-Coumarin (DCP-Net2C)) to specifically image sulfenylated proteins in mitochondria [Mitochondria are involved in the maintenance of cell viability and function and regulation of cell metabolism. Mitochondrial damage and dysfunction are implicated in aging ,29, and ic acids . As a fiic acids . Identifchondria , which eBecause the lung is a likely site for environmental and occupational exposure to AgNPs, in this work, we describe the effects of AgNPs and ionizing radiation on mitochondrial redox state and function in a diverse panel of lung cell lines. In order to understand and potentially mitigate long term adverse health effects that may occur as a result of exposure to AgNPs, especially following inhalation, it is important to examine the sublethal effects of AgNPs on mitochondria. As other ROS inducers, including ionizing radiation, can potentially increase the toxic effects of AgNPs ,39 we ha2, in Ham’s F-12K (Kaighn’s) medium  for A549, RPMI-1640 medium  for Calu-1 and NCI-H358 and DMEM medium  for BEAS-2B. Culture media for all cells was supplemented with 10% fetal bovine serum , 100 U/mL penicillin and 100 µg/mL streptomycin .The experiments were performed with a panel of four lung cell lines: Three cancer cell lines  and one transformed lung epithelial cell line (BEAS-2B). All cell lines were obtained from the American Type Culture Collection (ATCC). Cells were cultured at 37 °C under 5% COPrevious studies indicated that the size and coating of AgNPs could affect the toxicity profile of AgNPs in colorectal cancer and murine monocyte/macrophage cell lines . In cont137Cs (Cesium) irradiator  with indicated doses. The relative timing of AgNPs and IR treatment is described in each figure legend. The ionizing radiation (IR) was applied with a 444 TBq 12,000 Ci self-shielded 2O2) or 1 µM MitoSOX  and analyzed by flow cytometry. Changes in mitochondrial protein oxidation with AgNPs and IR exposure were similarly quantified by flow cytometry after treating the cells with 50 µM mitochondria-targeted probe DCP-Net2C [2. For analysis of mitochondrial ROS, the cells were treated with 10 µM MitoPY1  for 1 h then were washed thoroughly in PBS to remove AgNPs not bound or internalized by cells. Cells were fixed in 2.5% glutaraldehyde at 4 °C overnight. Next, fixed cells were scraped from the wells, pelleted, embedded in resin, cut into ultrathin sections (80 nm) and placed on copper coated formvar grids. All samples were imaged using an FEI Tecnai Spirit transmission electron microscope . Samples were imaged without additional staining to facilitate the detection of AgNPs.t-test was used to compare the mean fluorescence values between different conditions. After treatment, the cells were washed twice with Dulbecco’s phosphate-buffered saline (DPBS)  and detached from the plates with trypsin. For mitochondrial content and ROS analysis, the cells were resuspended live and kept on ice. For the analysis of mitochondrial protein oxidation and cell cycle, the cells were fixed for 5 min with cold (−20 °C) methanol or for 30 min with 70% ethanol, respectively. Fixed cells were washed with DPBS before resuspending for analysis. For the analysis of the cell cycle, cells were treated with 100 µg/mL RNAse  and stained with 50 µg/mL propidium iodide . The flow cytometry experiments were conducted with a BD FACS Canto II Cell Analyzer , and 10,000–50,000 cells were analyzed for each condition. Each experiment was repeated at least three times independently. Data were analyzed using the FCS Express 6 Flow software , and the Students’ t-test was used to compare the mean values between different conditions.The cell proliferation was analyzed with CyQUANT assay  according to the manufacturer’s protocol, and the fluorescence was read with a Spark microplate reader . The ATP content in cells was measured with CellTiter-Glo 3D assay  according to the manufacturer’s protocol, and the luminescence was read with the Spark microplate reader. All conditions were measured in three technical replicates, and each experiment was repeated independently three times. The Students’ 4 cells/well were plated on a Seahorse plate. The next day 10 µg/mL AgNPs were added to the assigned wells and incubated for 24 h. On the day of the experiment, the assay media, compounds , 1 μM for Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP)  and 1 μM Antimycin A /Rotenone  and cells were prepared according to Seahorse protocols. The experiment was run with Seahorse XF24 , following injection order: (1) AgNPs or vehicle media (1 h reading); (2) Oligomycin; (3) FCCP; and (4) Antimycin A/Rotenone. After the Seahorse analysis, the cells were lysed with modified RIPA buffer  protease and phosphatase inhibitor tablets) and protein concentration was measured with bicinchoninic acid assay  for data normalization. Each experiment was repeated at least two times independently. The data were analyzed with Wave software , and the Students’ t-test was used to compare the mean fluorescence values between different conditions.The effects of AgNPs on the mitochondrial function were measured using the Seahorse Mito Stress Test following the manufacturer’s protocol. Briefly, the day before the AgNP treatment, 1.5 × 10p = 0.106 for AgNP with 2 Gy irradiation and p = 0.014 for AgNP with 5 Gy irradiation as compared with AgNP only).Changes in cell proliferation were measured using CyQuant proliferation-assay. The response to the exposure to AgNPs was both time- a and dosTo determine if the varied sensitivity of the four cell lines to AgNPs was due to differential uptake of AgNPs into cells, we examined the uptake and subcellular localization of AgNPs using transmission electron microscopy (TEM). AgNPs were internalized within 1 h and were found in membrane bound vesicles, likely endosomes, in all cell lines after 1 h exposure to AgNPs . The celTP53 (A549 and BEAS-2B), whereas in the TP53 null cell lines  the cells accumulated mainly in the G2 phase. The population of cells in S-phase decreased after ionizing radiation in all cell lines. On the other hand, the AgNP exposure induced G2 arrest in A549 and Calu-1 cell lines, S-phase arrest in BEAS-2B and did not seem to have any effect on the cell cycle in the NCI-H358 cells. Only in the Calu-1 cells the exposure to combined AgNPs and ionizing radiation appeared to have a statistically significant effect on the increase in cell accumulation in G2 phase (p = 0.037 for 1 μg/mL AgNPs with 2 Gy irradiation and p = 0.028 for 10 μg/mL AgNPs with 2 Gy irradiation as compared with AgNPs only).The cell cycle was studied with flow cytometry using PI-staining. Exposure to AgNPs and ionizing radiation affected the cell cycle in all the cell lines studied , but the2O2 detection and MitoSOX as a more general sensor of superoxide and mitochondrial redox state. The cell lines in our study had different basal levels of mitochondrial superoxide whereas the mitochondrial H2O2 levels were more similar, except for BEAS-2B that had the highest basal level of both H2O2 and superoxide . Exposure to AgNPs increased both mitochondrial H2O2 .The mitochondrial ROS was measured in live cells using flow cytometry. MitoPY1-probe was used for Hperoxide . The celial H2O2 a and mitial H2O2 b in all 2C containing a protein sulfenic acid trapping moiety for detecting oxidized proteins and a coumarin moiety for the localization into the mitochondria and fluorescence detection [Mitochondrial protein oxidation was analyzed using the recently developed probe DCP-NEtetection . In lineMitochondrial protein oxidation also increased in the AgNP-sensitive cell lines as a function of increasing concentrations of AgNPs, and the increased oxidation was statistically significant in particular for concentrations greater than 10 μg/mL b. The 2 2C labeling, followed by S-phase and G1 having the lowest amount of labeling. Nevertheless, the changes induced by the AgNP exposure on mitochondrial protein oxidation could be seen in all cell cycle phases and are, thus, not linked to the mitochondrial content of cell cycle phases. The same results were obtained for the other three cell lines (data not shown).To verify that the changes in the level of protein oxidation labeling were not due to changes in mitochondrial content induced by treatment with AgNPs, we used MitoTracker Green to quantify mitochondrial content in the cell. We did not detect any significant change with AgNP treatment in the four cell lines studied here e. HoweveThe mitochondrial respiration was evaluated with the Seahorse MitoStress assay  after 1 Overall, while there appeared to be some increase in the basal respiration, proton leak and decrease in spare respiratory capacity and coupling efficiency with the AgNP treatment, these changes were not statistically significant d. Howevep = 0.019, BEAS-2B: p = 0.0.10, NCI-H358: p = 0.011).Thus, despite the changes in mitochondrial ROS and protein oxidation induced by AgNP treatment, mitochondrial respiration and ATP production/coupling efficiency were not impaired by treatment. In order to account for non-mitochondrial effects on ATP levels, we also measured the ATP content in the cells using Promega CellTiter Glo-assay . Treatme+ released from the nanoparticles, induce ROS and disrupt mitochondrial function [Several recent studies have shown that exposure to AgNPs is cytotoxic in different cell lines, but with variable response ,39,40,43function ,5,25,44.function ,45.The studies presented here were designed to mimic potential environmental exposure to AgNPs. In this case, both intracellular and extracellular exposure to AgNPs can have effects. Thus, all cell lines were exposed to equivalent doses of AgNPs, in equivalent volumes of culture media, and cells were plated in identically sized wells at similar cell density, ensuring comparable exposure to AgNPs. Furthermore, the data were collected at multiple time points and doses of AgNPs to be able to assess differences in the timing, magnitude, and type of responses detected among cell types.We initially evaluated the AgNP-induced cell toxicity using a battery of tests starting with the effects on cell proliferation. As others have reported in various cell lines ,5,15,43,TP53, and decrease of cells in the S-phase in all cell lines. Interestingly, the effects of ionizing radiation on cell cycle seemed to be different from the AgNP exposure, and there were no combined effects, even though in addition to DNA damage, ionizing radiation can also induce mitochondrial dysfunction and increase in cellular and mitochondrial ROS [AgNPs have been shown to induce DNA damage, cell death by apoptosis and necrosis, and also cell cycle arrest in different cell lines . The effrial ROS ,55.2O2 and MitoSOX as an indicator of superoxide and other ROS. Both of them showed an increase in fluorescence after 24 h exposure to AgNPs in the AgNP-responsive cell lines , but not in the AgNP-tolerant NCI-H358 cell line. In line with these results, there was also a time- and dose-dependent increase in the mitochondrial protein oxidation in the AgNP-sensitive cell lines, but not in the NCI-H358 cells, where a slight decrease was observed at higher concentrations of AgNPs. The TEM imaging showed internalized AgNPs in all cell lines after 1 h exposure, but at higher level in the AgNP-sensitive BEAS-2B and Calu-1 cells, correlating with the stronger increase in protein oxidation (occurring at earlier time points and at lower doses of AgNPs) compared with A549 and NCI-H358 cells. Interestingly, ionizing radiation increased mitochondrial superoxide and protein oxidation only in the A549 cells, which was not further increased with exposure to AgNPs. The different phases of the cell cycle have different redox states [Since the mitochondria appear to be an important target for the effects of AgNPs, we wanted to analyze the redox state of mitochondria by studying both the mitochondrial ROS and protein oxidation. We used two mitochondrial fluorescent probes to study ROS, MitoPY1 for Hx states ,56, and x states . In our We also analyzed the effects of AgNPs on mitochondrial respiration using Seahorse MitoStress assay. The AgNP-responsive cell lines had higher spare respiratory capacity both in the absence and presence of AgNPs, possible due to higher mitochondrial content, than the tolerant NCI-H358. Interestingly, Calu-1, the cell line most sensitive to AgNPs had also the lower capacity for ATP-production. The mitochondrial spare respiratory capacity seemed to decrease slightly with the AgNP treatment along with an increase in the proton leak pointing to mitochondrial dysfunction, whereas the basal respiration appeared to increase. In our study, the changes in the basal respiration, spare respiratory capacity, coupling efficiency or other key parameters of mitochondria function induced by AgNPs were not statistically significant in any of the four cell lines investigated. The effects of AgNPs on mitochondrial respiration have been little studied, but there has been at least one report also showing increased oxygen consumption after exposure to AgNPs in murine hippocampal neuronal HT22 cells [+ rather than direct interaction of AgNPs with mitochondria. There was only a small additional decrease in ATP with longer exposure to AgNPs, except for Calu-1 which showed almost complete loss of ATP and cell viability after 24 h. Interestingly, a similar decrease in ATP was observed in NCI-H358 cells and yet this did not impact its cellular proliferation. Other studies have also shown a decrease in ATP levels after exposure to AgNPs and loss of mitochondrial membrane potential . It While the clinical significance of our findings remains to be established, the data are consistent with the pathology of exposure to AgNPs. AgNPs are known to penetrate the lung structures deep into the alveoli through diffusion and to cross the blood-air barrier of the lungs leading to lung inflammation and fibrosis, as well as toxicity to other organs  . Interes+, intracellular relay of redox signaling from endosomes to mitochondria, and the intrinsic capacity of individual cell lines to interfere with this relay and/or respond to mitochondrial oxidative challenge. We have shown that exposure to AgNPs decreases cell proliferation and induces cell cycle arrest in A549, BEAS-2B and Calu-2, but not in NCI-H358 cells. The AgNPs increase mitochondrial ROS and protein oxidation in time- and dose-dependent manner in the sensitive cell lines, but without altering the mitochondrial respiration significantly. The change in mitochondrial redox state was accompanied by a decrease in cellular ATP levels, including in the NCI-H358 cells resistant to AgNPs treatment. The more sensitive cell lines had more mitochondrial content and spare respiratory capacity, potentially making them more susceptible to mitochondrial damage leading to increased ROS levels. Interestingly, ionizing radiation seemed to have separate effects from AgNP exposure, and there was no interaction with AgNPs in most cell lines with the exception of NCI-H358 which showed decreased proliferation with combined radiation and AgNPs treatment at higher doses of radiation.The results of the studies presented here reveal a broad variability in the sensitivity of lung cells to AgNPs determined by several contributing processes: cellular uptake of AgNPs and conversion to Ag


Esophageal cancer remains one of the hardest cancers to treat with rising incidence rates, low overall survival and high levels of treatment resistance. The lack of clinically available biomarkers hinder diagnosis and treatment stratification. While large scale sequencing approaches have uncovered a number of molecular makers, little has translated in the routine treatment of esophageal cancer patients.We evaluate the treatment response towards a panel of 215 FDA-approved and 163 epigenetic compounds of 4 established and 2 patient-derived esophageal cancer cell lines. Cell viability was evaluated after 72h of treatment using cell titer glow. The drug sensitivity testing results for gemcitabine and cisplatin were validated using clonogenic assays.The tested cell lines display different drug sensitivity profiles, although we found compounds that display efficacy in all of the tested established or patient-derived cell lines. Clonogenic assays confirmed the validity of the drug sensitivity testing results. Using the epigenetic library, we observed high sensitivity towards a number of epigenetic modifiers.Ex vivo drug sensitivity testing may present a viable option for the treatment stratification of esophageal cancer patients and holds the potential to greatly improve patient outcome while reducing treatment toxicity. Apart fA number of recent high-throughput investigations revealed histological, genetic and epigenetic changes typical for esophageal adenocarcinoma . While, Precision medicine approaches for the treatment stratification of patients with esophageal adenocarcinoma have recently come into the focus of the field in order to improve patient outcome. Apart from improvements in PET imaging techniques , 12, theWe have overcome these issues by adapting a drug sensitivity testing (DST) platform previously established for the stratification of patients with relapsed/refractory AML . Ex vivoOE33, OE19 and SKGT2 human esophageal adenocarcinoma cell lines were obtained from the European Collection of Cell Culture (ATCC). Flo1, EAC42 and EAC47 cells were established in the Capobianco Laboratory. EAC42 and EAC47 human esophageal adenocarcinoma cell lines are derived from patients undergoing surgery at the Miller School of Medicine, University of Miami . The CapThe normal esophageal epithelium cell line EACN42 was established in the Capobianco laboratory. The cell lines was derived from human normal epithelium and immortalized using HPV E6-E7.mod) function developed by Swords et al [mod (sDSSmod) for each drug in each patient screen is calculated according to the formula sDSSmod = DSSmod (patient cells)—DSSmod . Given in this way, the sDSSmod incorporated information on each drug’s potency, efficacy, effect range and therapeutic index, making it possible to prioritize compounds over multiple parameters using a single numerical metric. In addition, this methodology allows us to rank compounds by cancer-selective efficacy for each individual patient. For example, a large positive sDSSmod means that a compound is highly selective for esophageal cancer cells over normal cells in a given sample (favorable scenario), while a large negative score means that the effect was preferential for normal cells (unfavorable scenario). All calculations and scoring routines are implemented in MatLab and additional curve fitting and statistical analyses are performed in GraphPad prism.Drug sensitivity testing (DST) was performed as described previously . Brieflyds et al . As a fiClonogenic assays were performed as described previously . BrieflyWe tested a 163 compound epigenetic library that consists of inhibitors and activators of epigenetic modifying enzymes . All compounds were dissolved in 100% DMSO and tested in duplicate at a nominal test concentration of 6μM. Wells with assay buffer containing DMSO served as negative controls. 1000 exponentially growing cells were seeded per well in 384-well micro-titer plates and incubated in the presence of compounds in a humidified environment at 37°C and 5% CO2. After 72 hours of treatment, cell viability was assessed by measuring ATP levels via bioluminescence . Positive hits were defined as any compound that showed cell killing higher that 3 standard deviations of the negative control. Curve fitting and statistical analyses are performed in GraphPad prism.The normal esophageal epithelium cell line EACN42 was exposed to the 215 compound FDA-approved library cell viability was evaluated after 72 of treatment. The esophageal epithelial cells display toxicity to 71 compounds . SignifiBecause matched normal tissue is not available at sufficient quantities from patients with esophageal adenocarcinoma from either biopsies or surgical intervention, we compared the response of the esophageal epithelium to the treatment response of white blood cells derived from a healthy donor . While wDrug sensitivity of 4 established and 2 low passage patient-derived esophageal cancer cell lines towards a panel of 215 FDA-approved compounds was established using the DST platform.Similar to what we have previously observed in AML , the 4 tIn addition to the established esophageal cancer cell lines, we evaluated EAC42 and EAC47, two patient-derived esophageal cell lines at low passages. Although both lines differ in the magnitude of response, the drug sensitivity profiles are very similar Figs  and 3. Trd and 6th treatment suggestion in EAC42 and EAC47, respectively  was the 3ectively . Cisplatin EAC47 . Treatmein EAC47 .OE33, Flo-1 and EAC47 cells were exposed to a 163 compound library containing epigenetic modifiers at a concentration of 6μM. Positive hits were defined as any compound that showed cell killing higher that 3 standard deviations of the negative control.17 compounds displayed a significant reduction in cell viability in OE33, the majority of which were HDAC inhibitors . Flo-1 a10 compounds displayed a significant reduction of cell survival in all of the tested cell lines . With thEx vivo DST screening may present a new stratification approach for patients with esophageal adenocarcinoma that allows the identification of patient-specific treatments while reducing normal tissue toxicity [Treatment stratification of patients with esophageal adenocarcinoma is problematic due to the lack of molecular markers predicting outcome and treatment response, and the optimal patient management remains controversial. While the collection of large sequencing data sets revealed genetic changes, including alterations in DNA methylation patterns , 6–8, thtoxicity .The esophageal adenocarcinoma cell lines display a wide range of treatment responses and vastly different individual sensitivity profiles. In contrast to our previous observations in AML, we uncovered a small subset of compounds that displayed treatment efficacy in all of the tested samples. Specifically, Bortezomib displayed high activity in all lines. The DST screen revealed a number of treatment options not part of standard therapy in esophageal adenocarcinoma, while compounds commonly used in patients such as platinum drugs , taxanes , 5-FU, irinotecan, capecitabine and epirubicin display treatment efficacy in few or none of the lines. However, similar to previous observations, a treatment response can be observed in response to treatment with cisplatin when doses are escalated beyond the maximum dose of the DST screen. Due to high levels of normal tissue toxicity and numerous difficult to manage side effects, a similar dose escalation is not feasible in a clinical setting. The dose range used on the DST screen was chosen to represent a clinically relevant treatment dose. Therefore, a negative result on the DST screen does not imply a complete lack of treatment response. Nevertheless, as seen in the case of cisplatin treatment, the dose needed for a clinically relevant response lies outside of the dose range that is clinically feasible. The data derived from the FDA screen suggests that phenotypic-based treatment stratification may present a way to overcome the current issues with patient stratification and classification.The impact of epigenetic modifications has recently come into the focus of the esophageal adenocarcinoma community and studies have shown numerous changes in epigenetic marks in patient samples. A small number of studies have provided encouraging results for the use of HDAC inhibitors for the treatment of esophageal cancer –20. We oA pilot trial evaluating a set of patients with esophageal adenocarcinoma will be necessary to examine the clinical feasibility of this precision medicine approach. Clinically the treatment of esophageal adenocarcinoma consist of a multimodal approach. Although our platform evaluates the response towards single treatments only, our experience using the DST platform for the treatment stratification of patients with relapsed/refractory AML  suggestsex vivo screen of this magnitude. This can be overcome by the use of surgical specimen which yield sufficient cellular material to support the DST screen. Although a matched normal tissue sample represents the ideal toxicity control for the DST screen, normal esophageal epithelium is not obtainable from either biopsies or surgical samples. White blood cells, while a matched control, display a vastly different sensitivity profile than the esophageal epithelium and cannot be used to evaluate epithelial toxicity in the esophagus. This can be overcome by using a catchment area matched panel of normal esophageal epithelium samples as a normal reference for future patient screens.Although all patients receive at least one biopsy, the material obtained during the procedure is essential for diagnostic purposes and the material superfluous to pathological staging is rarely of sufficient quantity and quality to support and This precision medicine approach has the potential to improve treatment stratification and outcome for patients with esophageal adenocarcinoma. Additionally, DST screens on patient populations may allow us further insight into the biology of the disease through the accumulation of sensitivity information on different classes of therapeutic compounds.S1 FigBar graphs of clinically actionable drug responses for (A) Flo1 and (B) SK-GT2.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


The first ideas leading to The Two‐Signal Model of lymphocyte activation were published 50 years ago, but the model was not realized in one sitting. I describe the three phases that led to its contemporary formulations. A motivation underlying all these models was to generate a minimal description of what is required for antigen to inactivate and activate mature lymphocytes that, at the same time, accounts for how peripheral tolerance is achieved. I suggest the two signal model has not only provided a substantiated framework for understanding how antigen interacts differently with B cells and CD8 T cells, to result in their inactivation and activation, but its postulates are pertinent to contemporary issues concerning the inactivation and activation of CD4 T cells. The most obvious and prosaic is to give credit where credit is due. The second is more important and interesting. Knowledge of how important advances are made is germane to understanding how progress occurs. Such understanding can be empowering.I clarify at the outset what different people mean by “two signal models” in the context of lymphocyte activation. There are two different and sometimes confusing uses. Dresser and Mitchison clearly stated in 1968 the idea that competent lymphocytes can interact with antigen in two ways, one way leading to their “paralysis”, or inactivation, and the other leading to their “induction” or activation.2This account was stimulated in part by Cohn's accounts of how the two‐signal model of lymphocyte activation came about. I think these accounts are inaccurate in some respects and so obscure the considerations underlying the initial proposal. They therefore undermine an understanding of the imaginative, analytical and observational considerations involved. A correct appreciation of these considerations might stimulate others to be brave, a quality that fosters creativity. My intellectual braveness, such as it is, has been fostered by appreciating that of others; an appreciation gained by closely following their intellectual journeys. This fostering led me to believe in the subtlety of argument. I felt I was part of a community that recognized sophisticated thought. I hope a correct account of the origin of the two‐signal model can help others to be brave and thus contribute to our culture. The most effective type of braveness is probably unselfconscious; it arises from intense engagement with a problem and a faith, nurtured by experience, that subtle considerations can yield enormous dividends.Cohn wrote an invited article of reminiscences for the 1994 Annual Review of Immunology, entitled the Wisdom of Hindsight.My primary aim, for most of my career, has been to do the best science I can, anticipating this would speak for itself. I found support for this course with my happiness at how Cohn and I had expressed our vision in the 1970 Science article,I started a year of administrative leave in June 2013. I reconsidered my priorities, particularly in view of my age of 70, and my frustration, over decades, that ideas, that I considered may be valuable to the field, were barely considered by immunologists. Some were pertinent to the two‐signal model, others to immune class regulation. I wrote two “Discussion Forums”,The first, on the inactivation/activation of CD4 T cells, drew a long response from Cohn that was published.Peter Bretscher joined my laboratory in 1967; he was, by way of background, an X‐ray crystallographer who had a passionate curiosity about immunology as well as a clear, crisp way of analysing complex problems. This made the difficult question of the self‐nonself discrimination an ideal one to answer; we were aware that it had to be analysed correctly if a next step was to be made in immunology.“We had two prior formulations, those of LederbergCohn, in his 1994 “The Wisdom of Hindsight” article,I now describe my recollections concerning the origins of the two‐signal model of lymphocyte activation. I apologize for going into considerable detail; such an exposition seems necessary to both understand the basis, and assess the legitimacy, of what I say. In addition, this model was not developed at a single sitting. Indeed, the formulations given by Cohn and myself started going in different directions, perhaps not unnaturally, when I left Mel's laboratory in 1972, as I shall indicate.2.1I became interested in immunology as a graduate student, while doing research in protein X‐ray crystallography, at the UK Cambridge Laboratory of Molecular Biology. I started graduate studies in 1964. I was much more interested, from the beginning, in what was going on in the Division of Molecular Genetics than in that of Structural Studies, to which I belonged. Brenner and Crick jointly headed the former division. About five graduate students and postdoctoral fellows, primarily from this division, met regularly on Saturday mornings for a few hours to chat, with Sydney Brenner and over Nescafe, about diverse scientific topics. I joined my brother Mark, on becoming a graduate student, in attending these get‐togethers. Mark soon left for postdoctoral studies. I continued attending these get‐togethers.One Saturday morning, in 1966, Sydney talked about a paper by himself and Cesar Milstein, on a model for “The origin of antibody variation”, just accepted by Nature.In time, I made use of the invitation, placed on a noticeboard outside Francis Crick's office, to discuss science. I knocked on Crick's door on at least ten occasions. One of my earliest immunological ideas is now obvious. However, at the time, Francis appeared to think it new and was kindly enthusiastic. Some reasonable people were then unsure about the plausibility of the Clonal Selection Theory. My ideas buttressed the plausibility of some of the critical features of this theory.uniquely only activate the transcription of genes coding for the antibody receptor that had interacted with antigen. I was thus led to the proposition one cell “must” be committed to the synthesis of antibody of just one specificity. These considerations were obviously another way of making plausible some essential aspects of the Clonal Selection Theory. They formed part of the two papers Cohn and I subsequently published in Nature in 1968I supposed that, when antigen interacted with antibody receptors on an antibody precursor cell, there must be “an interaction sensing site” that “recognizes” this interaction via changes in the structure/accessibility of the antibody receptor. I considered the situation that would occur as the genes coding for immunoglobulin chains are generated, in evolutionary time or somatically. I considered that this “interaction sensing unit” must be complementary to an invariant part of the antibody molecule; if it was complementary to a variable part, there would have to be two “complementary” mutations, one in the antibody genes, the other in the gene encoding the interacting sensing unit, an unlikely event. Assume, as seemed plausible, that the “interaction sensing unit” was complementary to an invariant part of the antibody. In this case, if the cell had two chemically different antibody receptors, the cell could not distinguish which of the antibodies interacted with antigen, and so could not, for example, Crick encouraged me, once he understood what I was driving at. Francis could initially not understand what I was saying, given my nervousness. He recommended I put my ideas on one side of a page. I gave him my effort. The argument was dense, and as rigorous as I could make it. He in time understood my considerations. I remember the details of this interaction, as it was so important to me. Francis chastised me, in his jovial way, a style he employed to lessen the sharpness of his advice. I did not need to take his recommendation of one page so literally, and so make the argument so dense. I did not have to prove my proposal, just make it plausible. My interactions with Francis determined to a considerable extent my scientific fate.On getting interested in immunology, I asked Cesar Milstein for advice on what to read. He suggested Burnet and lent me his copy of The Clonal Selection Theory of Acquired Immunity.3This accident occurred in mid‐1966. I was using a chemical that reacted strongly with proteins and readily permeated skin. I wore gloves when using it. The tip of my right index finger started throbbing one night as I fell asleep, on a day I had used this chemical. The next morning the tip was white and quite without feeling. In the long run, the tip was surgically removed and skin from my arm was grafted onto the raw end. However, before this, during the days after the mishap, a sharp line developed between what I imagined was healthy me and the proteins of my finger tip, altered by reacting with this chemical and so becoming “foreign”. This personal experience stimulated my interest in how the immune system managed to respond to foreign but not to self‐antigens. My limited reading, or a conversation with Cesar Milstein, I now do not know which, led me to believe that antigen could interact with antibody precursor cells in two ways, one leading to the antibody precursor cell's inactivation, the other to its activation, that is, multiplication and the differentiation of its progeny into antibody‐secreting cells.I had already decided to try to become an immunologist if I managed to get my PhD. In talking about this with Francis, he suggested I might do postdoctoral studies with Avrio n Mitchison, at that time at Mill Hill, London. I therefore visited Av, as he was known to all, probably sometime in 1966. Av explained his laboratory was very full but that, if I really wanted to come, he could probably squeeze me in.4I had some ideas, probably in 1967, and certainly after my meeting with Av, about how antigen interacts differently with antibody precursor cells to result in their activation and inactivation. I realized there were three appealing characteristics of the proposal that single precursor cells are inactivated by antigen, but that their activation requires the antigen‐mediated interaction between specific lymphocytes. Firstly, it was the only proposal I knew of that attempted to account for how antigen interacted differently with the same antibody precursor cell to result in either its inactivation or its activation. Secondly, it “explained” why antigens had to be macromolecular to be immunogenic. A small, univalent molecule, could not mediate the interaction between lymphocytes. Thirdly, it accounted for how self‐nonself discrimination could be achieved, as outlined elsewhere.immunogenic carrier, namely bovine serum albumin (BSA).I searched the literature, once I had formulated the one lymphocyte/multiple lymphocyte model, for observations bearing on it. I had no idea what journals to look at, so I randomly leafed through various periodicals on the library shelves. I found a paper in the Journal of Experimental Medicine that seemed to strongly support the proposal I envisaged. This was a paper from Benacerraf's group on “non‐responder” guinea pigs that did not produce antibody upon immunization with the antigen, poly‐L‐lysine (PLL). These animals produced anti‐PLL antibody when immunized with PLL coupled to an 5Two articles were very important to the further development of my ideas as a graduate student. Firstly, the articleI also came across an article by Gell and Kelus, entitled “Anti‐Antibodies”, in Advances in Immunology.I kept Francis informed on how my immunological ideas progressed. He changed his advice on where to do my postdoc. Francis was now annually visiting the Salk Institute, in southern California, as a Non‐Resident Fellow. Given his appreciation of my interest in theory, he suggested I might do a postdoc with Mel Cohn at the Salk Institute. Naturally, I accepted Francis' advice.Mel Cohn also accepted me on Francis' recommendation. Cohn had a major, NIH‐sponsored, programme for producing myeloma tumours in BALB/c mice. He already had a substantial collection. I was encouraged to apply for a Damon Runyon Postdoctoral Fellowship while a graduate student in Cambridge. I had to outline a plausible research proposal. I proposed testing the hypothesis that myeloma proteins have an abnormal structure, reflecting a structure representative of antibody complexed with antigen. I hypothesized that if such abnormal “myeloma antibodies” were generated, in an antibody precursor cell, the cells might behave as continually activated, and so constitute a cancer of this cell. The prediction was that myeloma proteins, not complexed with antigen, would react with antibodies that normally only recognize antibody sites present on antibodies present in antigen/antibody complexes. Although this idea was not experimentally explored, as I came to feel it implausible once I had been in Mel's laboratory for a few months, it was part of my successful application for a postdoctoral fellowship.Before leaving Cambridge for the Salk Institute, I submitted a letter6My impression, on arriving at the Salk Institute, was that Cohn was primarily occupied with the problem of the origin of the diversity of antibody genes. He was less aware of some of the literature on the requirements to generate antibody responses and generate unresponsive states than I was. I in turn had not come across, as a graduate student, Lederberg's classic Science paper of 1959.I was very motivated, on arriving in Cohn's laboratory, to review the literature; to see how my ideas stood up to current findings and other people's conjectures, and to explore how they might be further developed before “going public”. This was not to be.Mel told me, shortly after my arrival, that he had been invited to a small meeting of about 40 individuals, on Immunological Tolerance, to be shortly held at Brook Lodge, Michigan. Moreover, Mel was asked to summarize the meeting. The invited participants included many leading investigators, including Av Mitchison, Klaus Rajewsky, Bill Weigle and Baruj Benacerraf. The meeting was held from September 18 to September 20, 1968, surely <2 months after I arrived in Mel's laboratory. It was natural for Cohn, in his summary of the meeting, to try to account for major findings in terms of the ideas we were developing.argued that the evidence showed there were also carrier effects in generating unresponsiveness. Cohn, in his recounting the origin of our ideas,Cohn's description of some reported observations was challenged by other attendees at the meeting, as evident from the original transcript, a copy of which Mel gave me. In particular, Cohn was unclear about the nature of Weigle's observations that showed that immunization with an antigen, that crossreacts with the tolerogen, could break the unresponsive state.Both Cohn and I were uneasy about how Cohn had presented our ideas at Brook Lodge. Moreover, the proceedings of the meeting were to be published as a book.non‐immunogenic molecules were also non‐tolerogenic. We argued that such observations were likely misinterpreted, as quantitative considerationstolerogenic but not immunogenic, in accordance with the one lymphocyte/multiple lymphocyte model.I received a personal letter from Av Mitchison, shortly after our Nature paper was published. He said “I think you deeply cloud the issue by using the carrier effect to account for the immunity/tolerance decision. I do not at all see why one cannot have all sorts of lovely carrier effects and also something else, for example, non‐specific stimulation from macrophages” (to induce an antibody precursor cell). Klaus Rajewsky had come to the Salk Institute straight from the Brook Lodge Meeting to talk science, a decision taken on the spur of the moment, as I understood at the time. I recall Klaus' uneasiness about the one lymphocyte/multiple lymphocyte model. As clear from our Nature and Science papers, we disagreed with a conclusion Klaus had come to in his studies in rabbits with porcine LDH. His observations were a particularly clear example of those employed by immunologists to support the proposition that This description gives my best recollection of the events leading up to the publication of our Nature paper, representing the first phase in the development of the Two Signal Model.Our paper was submitted to Nature on October 17, 1968, less than a month after the Brook Lodge Meeting. It was published in the November 2, 1968, issue. I had been in Cohn's laboratory for <4 months, probably closer to two. Our Nature paper pretty well reflected the ideas I brought with me, except that there were references to the ideas of Mitchison, Rajewsky and Jerne on cell cooperation in the generation of antibody responses, and to Rajewsky's observations on tolerogenicity, referred to above. We (meaning I) thanked Crick for discussions related to this paper. Cohn was 46 and I was 25.Cohn said, in his 1994 Annual Reviews article,I have never met Donald Forsdyke, but he emailed me in 2002 over some matter. When he emailed me, I felt I had to let him know of my ignorance of his 1968 Lancet paper when we formulated our ideas.6.1I think it helpful if I explain at this point the three distinct phases in the history of the Two Signal Model of Lymphocyte Activation, as I perceive it. The first phase was the one outlined above, that culminated in our November 1968 Nature paper.6.2The 1968 Nature paper was written fast and under considerable pressure. We were very soon uneasy with part of its content. I consider its strengths to be its articulation that antibody precursor cells would be inactivated upon interacting with antigen alone, and that their activation required a second site be recognized by antigen‐specific “carrier antibody”, derived from or present on another antigen‐specific cell, that is, a lymphocyte. However, its weakness was the envisaged mechanism by which the activation of the antibody precursor cell was “made to depend” on the recognition of a second site on the antigen by the carrier antibody. We suggested that the divalent carrier antibody held the antigen in a way that, when the antibody receptor of the antibody precursor cell bound to two bound antigen molecules, the receptor took on a “distorted” conformation, recognized by an “interaction recognition unit” of the cell. This proposal reflected ideas in my research proposal for a Postdoctoral Fellowship, written when I was in Cambridge. The model predicted that self‐antigens, with repeating epitopes that could distort the antibody receptors in the appropriate way on interacting with them, would induce autoantibodies, an awkward and implausible conclusion. The recognition of this weakness led to the second phase.The “Miller papers”,These circumstances set the scene for the second phase. I was now a Postdoctoral Fellow in Mel's laboratory. Mel and I talked science an average of at least an hour a day. I have never had this type of interaction with anyone before or since. We talked much more broadly than on lymphocyte activation. We were invited in 1970 to give an exposition of our views in Science, an article that went through more than 20 drafts. The articleFirstly, we adhered to the proposition that lymphocyte cooperation was essential for lymphocyte activation and that antigen would, if at a sufficient concentration, inactivate single lymphocytes. We had no idea as to what the signals involved might be in chemical terms, so we formulated an abstract symbolism to express our thoughts. We proposed that when antigen interacts with a lymphocyte's antigen‐specific receptor a signal, signal 1, is generated that, when generated alone for a sustained time, leads to the inactivation of the lymphocyte. The activation of a responder lymphocyte, such as a B cell, also requires its antigen‐specific receptor to bind to antigen, also resulting in the generation of signal 1; its activation required the activity of helper lymphocytes that recognize the same antigen. There was no indication at this time of the MHC‐restricted recognition of antigen by T cells. The T cell receptor was envisaged to be an antibody‐like molecule. In order to make the recognition of antigen by the helper lymphocyte mandatory for the activation of the antibody precursor lymphocyte, we proposed that this recognition by the helper lymphocyte resulted in the initiation of the generation and the delivery of a second signal, signal 2, to the responder lymphocyte. We postulated that this second signal was mandatory for the activation of the responder lymphocyte, a B cell in the case of an antibody response, so that the activation of all lymphocytes was envisaged to be T helper cell‐dependent. Jacques Monod fostered this formulation of our model.Jacques was a Non‐Resident Fellow of the Salk Institute and visited it for a few weeks in the summer of 1969. Jacques was very interested in immunology. He and Mel knew one another well. Mel had done a postdoc with Jacques, working on what became known as the lac operon at an early and critical stage of their research. I very much enjoyed Jacques' scientific company, and I think he enjoyed mine. Mel and I had ideas as to what events might constitute signal 2, as outlined in the next paragraph. Jacques suggested to us the importance, in getting our message over, that we develop a way of expressing the essence of our views, independently of the particular details of how these might be realized. We recognized the wisdom of this suggestion. It was the result of Jacque's prompting that we started to say that the interaction of antigen with the antigen‐specific receptor of the antibody precursor cell resulted in the generation of signal 1, and the recognition of a second site on the antigen, by the T helper cell, or an antigen‐specific molecule it produced, resulted in the generation of signal 2.Our second consideration was directed at how signal 2 might be realized in cellular/molecular terms. We proposed that signal 2 was mediated by short‐range molecules, produced by the helper lymphocyte, similarly as a neuron stimulates or inhibits a cell with which it forms a synapse by the action of neural transmitters it produces, an analogy we employed at the time; alternatively, or in addition, we supposed signal 2 might involve a membrane/membrane interaction between the two interacting cells. These proposals were substantiated once interleukins were discovered and costimulatory systems established as a means by which cells of the immune system communicate. For example, it is now recognized that the activation of B cells requires both the delivery of cytokines to B cellsI should add a comment on the designation of the T cell required to activate a B cell as a helper T cell. MitchisonOur third consideration addressed how the activity of helper T lymphocytes might be controlled. Mitchisonoptimal antigen‐dependent activation of a responding precursor T helper cell required the activity of effector T helpers. This proposal led to the question of how are the first effector T helper cells generated? I call this problem the “priming problem”.The fourth issue was a problem, the existence of which we drew attention to. B cells are precursors of antibody‐secreting cells; their activation results in their proliferation and the differentiation of their progeny into antibody‐secreting cells. This life style of the B cell was envisaged in the formulation of the Clonal Selection Theory, as it explained not only a striking observation, namely the extreme sensitivity of in vivo immune responses to radiation of the animal around the time of immunization, but how very scarce B cells, specific for a particular antigen, could be triggered by antigen to produce substantial antibody after a lag period of a few days. Similar considerations seemed even more critical in the case of the activation of precursor helper T cells, as effector helper T cells have to be generated before B cells can be activated. Recognizing this situation, Cohn and I postulated that precursor T helper cells had to be activated by antigen to multiply and their progeny differentiate to give rise to effector helper T cells. We also proposed that lymphocyte cooperation, presumably between cooperating T cells, was required to activate T cells, thus accounting for how  tolerance is achieved at the T cell level. In more detail, we proposed that the This type of problem is of course not unique to immunology. Life begets life, so how did life originate? Ribosomes contain proteins, and ribosomes are required for protein synthesis, so how were the first proteins produced? We shall see this “priming problem” was important in the third, last phase, of the evolution of two signal models for CD4 T cell activation. Cohn and I suggested different solutions to the priming problem.A final point for clarity is needed. It became apparent from various observations, made after 1970, that there are at least two different processes contributing to self‐tolerance. There is central toleranceI would like to add that my time in Mel's laboratory as a Postdoctoral Fellow, during this second phase, was wonderful. There was a lot of lively, scientific discussion. I could think about anything I wanted to without pressure of who would provide bread the next day. As a graduate student in England I had to count my pennies. The support from my Postdoctoral Fellowships made me feel wealthy. I had the freedom to think about anything I thought interesting and important. I still think my theory of generalized autoimmunity6.3I left Cohn's laboratory in 1972. Cohn and I no longer communicated regularly. It was natural that we worked on different problems and developed different views. I naturally sought and looked out for Cohn's papers. However, I found them often difficult to understand and their contents, when I thought I understood them, often seemed implausible to me. This, on my side, naturally led me to be uncommunicative with Cohn. I believe it would have been natural, if I had liked the ideas that Cohn expressed, that we would have kept in closer contact. Until recently, and after leaving Cohn's laboratory, I only wrote a paper on the two signal model when I had something really new to say, either of a theoreticalCohn responded6.4Studies by Norman KlinmanStudies by LaffertyCharlie Janeway recognized that the presence of helper T cells was required to activate B and CD8 T lymphocytes and that antigen could inactivate these lymphocytes in the absence of helper T cells. He therefore, in 1989, naturally focussed his attention on the circumstances that lead antigen to either inactivate or activate CD4 T helper cells. As is well known, he postulated that the expression of the critical CoS molecule on the APC, needed to generate the second or CoS signal and required to activate CD4 T cells, was only upregulated if a pattern recognition receptor (PRR) of the APC recognizes a pathogen‐associated molecular pattern (PAMP). In the absence of an appropriate PAMP, and consequently inadequate expression of the critical CoS molecules, antigen is envisaged to inactivate the CD4 T cell.Three major and related advances post‐1970 had an impact on our ideas, without undermining them. Firstly, the roles of processing and presentation of antigen by antigen presenting cells (APC) were clarified. Secondly, the fact that nominal antigen is processed and its peptides presented on MHC molecules fitted in with the realization that the recognition of antigen by the T cell receptor is MHC‐restricted.Lanzavecchia's observations, together with these advances, led him to propose a new model of the B cell/helper T cell interaction. He employed a hapten (h) carrier system and studied the requirements to induce anti‐hapten antibody. He postulated that an anti‐h B cell endocytoses an h‐Q conjugate, where Q represents the carrier protein; the conjugate is then processed inside the B cell, and the peptide q/MHC complexes are subsequently presented by the B cell. The T helper cell specific for the nominal antigen Q can then recognize these peptide q/MHC complexes and deliver signal 2. The critical point is that the anti‐h B cell would not present peptide q/MHC complexes in the presence of Q and hR, but rather it would present peptide r/MHC complexes. The hapten must be coupled to Q if the anti‐h B cell is to endocytose Q via its antigen‐specific receptors.6.5Somewhat similarly, our extensive studies on the requirements to activate CD4 T cells show that the activation of Q‐specific responder T helper cells can be facilitated by R‐specific T helper cells in the presence of the conjugate Q‐R, but not when Q and R are present as separate molecules.Cohn disagreed with this solution for how linked recognition between cooperating CD4 T cells might be realized, and has discussed other possibilities,6.6We had proposed that effector helper T cells are required for antigen to optimally activate responder T helper cells, thus generating more effector helper T cells. How then are the first effector helper T cells generated? We refer to this as the priming problem.I proposed in a 1972 invited article,in the absence of antigen, slowly differentiate into effector helper T cells. Cohn envisioned that this pathway would not happen for CD4 T cells specific for self‐antigens, as self‐antigens are always present and their corresponding precursor helper T cells would be quickly inactivated by the mechanisms incorporated in our two‐signal model.Cohn stated7I would like to thank the four reviewers for their careful and considered comments. There is no doubt that these comments allowed me to improve the manuscript. Two comments made by different reviewers call for a response.One comment by one reviewer was: “What about B cell deficiency, where the (human) patient altogether lacks B cells? If B cells are important for providing the second antigen specific signal, how can these patients have a relatively adequate defense against viruses? Some practical examples based on current immunological thinking …would have made this account much more relevant and interesting.” My initial response to this comment, before discussing the science, is to say that the primary purpose of the article was to give my perspective on the history of a model that has remained pertinent for almost 50 years.Now for the science, I personally never expect all observations to fit into one picture, as no picture is complete, and because some of the frameworks we employ in “obtaining” our observations are likely wrong. The evidence for a mandatory role of B cells in generating murine immune responses appears to be highly contradictory. What to do about this situation was indeed the subject of recent discussions between Cohn and myself.Another reviewer felt the omission of discussing the role of co‐inhibitory signals, in the negative control of lymphocyte activation, was unbalanced. My perspective is that whether the one lymphocyte/multiple lymphocyte model holds is a fundamental question. This minimal model can accommodate a requirement or a lack of requirement for a co‐inhibitory signal in lymphocyte inactivation. What is particularly appealing to me is the clear role of co‐inhibitory signals in feedback regulation. I believe Sinclair originally proposed that antigen/IgG complexes could mediate an Fc‐mediated, negative signal to B cells, requiring the antigen to also bind to the B cell's antigen‐specific receptors.One reviewer pointed out that Cohn had a paper coming out in this journal, entitled ‘The real “danger” lies in failure to confront fundamentals',8Mel Cohn died in October 2018. A first version of this article was submitted before Cohn's death. His death occurred during the reviewing process. I asked the Associate Editor, who was dealing with this manuscript, if he could delay its consideration for a few months.I wish to give a brief explanation of the way I have responded to what I perceive as inaccuracies in the history Cohn gave of his and my involvement in the formulation of The Two Signal Model of Lymphocyte Activation. I have described above why Cohn's 1994 Wisdom of Hindsight articleFor years, I did not criticize in print Cohn's revisions or his further development of our ideas. I thought it more worthwhile that my students and I concentrate on experimentally testing predictions of the model we favoured,My 2014 article,