An Integrated Disease/Pharmacokinetic/Pharmacodynamic Model Suggests Improved Interleukin-21 Regimens Validated Prospectively for Mouse Solid Cancers
Cancer is a multi-faceted disease , involving complex interactions between neoplastic cells and the surrounding microenvironment 1 ., The prospect of immunotherapy , i . e . stimulating endogenous immune responses by various molecular and cellular factors , is emerging as a promising approach against this disease 1 , 2 , 3 ., One of the latest candidates for solid cancer immunotherapy is Interleukin ( IL ) -21 , a γc-signaling protein of the IL-2 cytokine family with versatile immune-modulating properties 4 , 5 , ., IL-21 has demonstrated substantial antitumor responses in several independent preclinical studies , in which mice inoculated with diverse transplantable syngeneic tumor lines were treated with the drug via cytokine-gene transfection , plasmid delivery , or injection of the recombinant protein 9 ., In Phase I and IIa clinical trials , IL-21 was well tolerated and triggered moderate antitumor activity in some renal cell carcinoma ( RCC ) and metastatic melanoma ( MM ) patients 10 , 11 , 12 , 13 , 14 ., More recently , clinical trials of IL-21 in combination with the tyrosine kinase inhibitor sorafinib for the treatment of RCC , and Rituximab for the treatment of non-Hodgkins lymphoma , have also been investigated with encouraging results 15 ., Yet , the intricate biology of IL-21 may set hurdles for its clinical development ., Produced mainly by activated CD4+ T cells , IL-21 induces anticancer immunity predominantly by stimulation of natural killer cells ( NKs ) and/or cytotoxic T lymphocytes ( CTLs ) 4 , 5 , 6 , 7 ., The cytokine regulates various cellular and humoral pathways of immunity , and exerts conflicting stimulatory and inhibitory effects on several cell types 9 , 16 , 17 ., Recent evidence for anti-angiogenic effects of IL-21 18 further complicates its dynamical influence on the tumor microenvironment ., Considering this biological complexity , traditional “trial-and-error” methodologies for clinical IL-21 therapy design are likely inefficient , and ought to be replaced by new guided approaches to maximize drug efficacy ., Rational and systematic planning of anticancer therapy may be directed by mathematical modeling and computer-aided analysis , which provides a better understanding of the involved dynamics ., Over the past 25 years , mathematical modeling strategies have been applied in oncology-focused studies investigating tumor progression , angiogenesis and interactions with the immune system 19 , 20 , 21 , 22 , 23 , 24 ., Models for cytotoxic , cytostatic and cytokine-based direct and supportive cancer drugs have been introduced , with some being subsequently validated in preclinical and clinical settings 23 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 ., These strategies have highlighted the importance of adequate selection of therapeutic regimens to achieve desired outcomes , by carrying out in-depth analysis of optimal times , dosages , and durations of treatment ., Pharmacokinetic ( PK ) and pharmacodynamic ( PD ) modeling of anticancer agents can be particularly useful for clinical design of treatment 37 , 38 ., We have previously developed a mathematical model for the local dynamic effects of IL-21 on solid cancers ., The model focused on interactions of IL-21 with NKs/CTLs , effector cytotoxicity against target cells , and immune memory , providing initial understanding of the optimal conditions for IL-21 gene therapy 39 , 40 ., Here , we have designed a new comprehensive PK/PD/disease model to predict clinically relevant scenarios of IL-21 treatment following intravenous ( IV ) subcutaneous ( SC ) or intraperitoneal ( IP ) administration in different cancer indications ., The model forecasts long-term effects of the drug by integrating newly described PK/PD processes together with a disease model , based on our initial in situ model 39 , 40 ., This new combined model was retrospectively and prospectively validated by in vivo experiments in IL-21-treated mice bearing melanoma ( B16 ) or renal cell carcinoma ( RenCa ) ., Model predictions provide substantial insights concerning adequate planning of systemic IL-21 therapy in solid cancers ., All experiments were conducted according to Novo Nordisk principles for animal studies , as approved by the Danish National Ethics Committee on Experimental Animals , and in accordance with National Institute of Health guidelines for the care and use of laboratory animals ., Data were collected from a published preclinical study in which mice bearing B16 and RenCa tumors were treated with IL-21 by various strategies 41 ., Briefly , tumors were induced at day 0 , and a daily ( B16 ) or 3×/week ( RenCa ) IL-21 regimen ( 50 µg/dose ) was applied SC or IP either at an “early” stage ( day 3 in B16; day 7 in RenCa ) , or at a “late” stage ( day 8 in B16; day 12 in RenCa ) of tumor development ., The tumor was measured several times until experiment termination ., Data were available from additional unpublished dose-titration experiments in RenCa: IL-21 was given SC , 1× or 3×/week , and groups of mice ( n\u200a=\u200a6 ) were assigned a dose between 1-50 µg ., The complete database was a priori divided into “training datasets” for model parameter estimation , and “validation datasets” for model verification ., In new prospective experiments designed to test model-suggested regimens , 7-8-week-old wild type C57BL/6 mice ( Taconic Europe A/S , Denmark ) were inoculated SC in the right flank with 1×105 B16F0 melanoma cells ( American Type Culture Collection ( ATCC ) , CRL-6322 ) on day, 0 . Recombinant murine IL-21 ( Novo Nordisk A/S , Denmark ) or PBS was injected SC from day 3 , when tumors were visible ., IL-21 was given at 12 µg/day , 50 µg/day , or 25 µg twice a day , each group including n\u200a=\u200a10 mice ., Tumor volumes were calculated by the formula based on the two perpendicular diameters d1 and d2 measured approximately 3×/week with digital callipers ., All experiments were carried out blindly , without the investigators knowledge of model predictions ., Animals were randomized and ear-tagged prior to treatment onset and euthanized when individual tumor volumes reached 1000 mm3 ., The new comprehensive systemic model for IL-21 immunotherapy contains PK/PD effects merged with disease interactions , as schemed in Fig ., 1 . The system is described hereafter , and the coupled ordinary differential equations ( ODEs ) are fully detailed in the Text S1 ( sections A-B ) ., The model ( Fig . 1 ) was implemented in C ( Microsoft Visual Studio . NET ) and MATLAB ( The MathWorks , Natick , MA ) programming platforms ., The system was solved by fourth-order Runge Kutta integration ., Model parameters were evaluated by a customized numerical method based on Hooke and Jeeves optimization 48 combining global and local search heuristics and least-squares curve-fitting ., Parameter sets achieving maximal model agreement with experimental training data were selected ( see Text S1 , Tables S1-S2 ) ., The model was simulated under numerous IL-21 regimens , differing in onset , duration , dose , inter-dosing interval , route , etc ., All simulations were repeated several times to ensure output consistency ., Retrospective verification of the model was accomplished by checking its prediction accuracy , via statistical comparison of its output with prior independent validation datasets ( see Experimental data and 41 ) : Model simulations were conducted under the specific tumor settings and treatment conditions of each prior experiment ., For prospective model validation , selected model-identified regimens were tested experimentally , and results were statistically compared to model predictions at the data sampling times ., The goodness-of-fit between the model output and experimental data was determined by calculating the coefficient of variation ( R2 ) ., To compare between experimental datasets , Students t-test ( two-tailed , assuming equal variance ) was applied ., A P< . 05 value was considered statistically significant ., First , we examined the sensitivity of the model to small variations in the value of the plasma-tissue correlation factor s , being that this pivotal parameter simplifies rather complex PK processes ., Simulations of the experimental early-onset IL-21 regimen ( 50 µg/day applied SC/IP ) in the B16-challenged setting were carried out under diverse s values , in the vicinity of those obtained through curve-fitting ( see Materials and methods and Fig . 2 ) ., After increasing or decreasing s values by two-fold , model predictions still accurately retrieved the murine data ( R2>0 . 90; Fig . 2 ) , and were comparable to the original fits ( Fig . 2 , “Model fit” ) ., Interestingly , model predictions remained precise even when modifying the values of the effector-tumor interaction coefficients k1 and k2 ( see Text S1 , section C , and also Fig . S3 ) ., These results indicate that model predictions are robust even when s , k1 and k2 values slightly diverge , meaning that different numeric combinations of these parameters , i . e . , multiple NK:CTL ratios , can accomplish the same therapeutic effect ., This implies a potentially wide window of IL-21 doses within which effects may be comparable ., Our primary goal was to validate the models predictive accuracy ., We therefore compared its output to the experimental B16 progression following a late ( day 8 ) onset regimen of IL-21 , given at 50 µg/day SC/IP for 3 weeks 41 ., All late treatment simulations were strongly in line with the independent validation data ( R2>0 . 90; Fig . 3A ) , thus verifying the model ., Notably , the model was able to recapitulate the biological behavior even under the aforementioned modifications in s , k1 and k2 parameter values ( data not shown ) ., Next we assessed the models generality by investigating whether it can predict IL-21 therapy outcomes in other solid cancer indications , such as RenCa ., Tumor growth and selected immune system parameters were set for RenCa , using training data in untreated mice and previously calibrated parameter values for moderately-immunogenic cancers ( see Materials and methods and 39 ) ., Other parameter values were set exactly as in the B16 case ., Simulations of the experimentally-applied IL-21 treatment of 50 µg at 3×/week , given for 3 weeks 41 , showed model predictions to be strongly akin to the observed dynamics under late ( day 12 ) therapy administered SC , as well as in early ( day 7 ) and late IP regimens ( R2>0 . 90; Fig . 3B ) ., Under the early SC regimen , predicted responses were slightly weaker than observed , yet still remained within the measurements standard deviation ( R2>0 . 73; Fig . 3B , upper panel ) ., To further validate the model for RenCa , we simulated it to predict the effects another experiment that applied lower IL-21 doses ( between 1–20 µg , SC 3×/week for 3 weeks ) ., Predictions were in agreement with the validation set readouts in most doses ( R2>0 . 94; Fig . 3C ) , collectively demonstrating a moderate dose-dependent decrease in IL-21-mediated tumor eradication ., The 10 µg ( 3×/week ) simulation experiment gave a good , but slightly lower , model-data correlation ( R2>0 . 83; Fig . 3C ) ., The model also successfully retrieved a retrospective experiment testing a 30 µg ( 1×/week ) IL-21 treatment schedule ( R2>0 . 90; Fig . 3C ) ., Having validated the model , we used it to gain insights into better IL-21 therapy in the B16 setting ., In particular , we searched for regimens that would be superior to the standard daily SC 50 µg treatment applied previously 41 ., First , we tested whether the treatment initiation time is a critical factor in determining IL-21 effects , by simulating different onsets of the standard daily regimen ., The model predicted that earlier therapy initiation results in stronger anticancer responses , as expected ( Fig . 4A ) ., The simulated tumor mass at the end of therapy ( day 20 ) was lowest under the earliest regimen , which began one day after B16 challenge: This final tumor load was roughly 15% lower than that obtained in the standard treatment initiated at day 3 ., In contrast to this early regimen , the tumor load resulting from a delayed regimen , initiated at day 10 , was doubled ( Fig . 4A ) ., Further delayed regimens ( with onsets as high as day 17 ) were even less favorable ( data not shown ) ., These results collectively emphasize the importance of early-onset therapies ., Notably , however , not even the earliest treatment onset was able to fully eradicate the tumor ., Simulations were performed also to see whether the anticancer response could be improved by fractionating the IL-21 regimen into a more intensive high-dosing protocol , as suggested for other drugs 34 , 49 ., To design alternative schedules , the daily IL-21 regimen ( 16 SC injections , 50 µg each , given from day 3; 41 ) was taken as a reference point: the same total dose ( 800 µg ) was distributed differently across the treatment window , using various doses and inter-dosing intervals , creating a collection of regimens to be tested ., Intriguingly , the model predicted that a more intensive schedule , applying two 25 µg doses per day at a 12-hour inter-dosing interval , would lead to a 45% lower tumor mass than that obtained under the standard daily 50 µg regimen ( Fig . 4B ) ., Fractionation into even smaller doses given every few hours produced slightly lower tumor sizes , yet these responses were not significantly better than the 25 µg regimen outcomes ( Fig . 4B ) ., In fact , not even the most fractionated schedule could arrive at full eradication of the tumor ., At the other end , less fractionated regimens comprising large IL-21 doses given every few days had significantly weaker efficacy ( Fig . 4B ) ., In order to verify our prediction that the fractionated 25 µg/12 hour regimen would be superior to the standard 50 µg/24 hour schedule , the two were experimentally applied in B16-challenged mice ., Even though both schedules effectively attenuated tumor progression as compared to control PBS-treated mice ( *p< . 001; Fig . 4C ) , the 25 µg/12 hour regimen was considerably more successful than the standard 50 µg daily regimen ( **p< . 05; Fig 4C ) , as mathematically predicted ., The observed tumor dynamics under the 25 µg regimen had an excellent fit with the prior model predictions ( R2>0 . 90; Fig . 4C ) , providing strong and quantitative prospective validation of the models precision ., We considered that the fractionated regimen may not be clinically practical , since it could involve increased costs of therapy , and , at least in IV delivery , would possibly require hospitalizing patients ., Therefore , the search for better treatment was limited to simple , widely-acceptable daily administration schedules ., Regimens of one IL-21 dose per day ( e . g . 16 SC injections given between days 3–20 following B16 inoculation ) were simulated under different dose intensities: A dose-dependent increase in the response , reflected by lowered tumor masses , was predicted for very low ( <5 µg ) or very high ( >50 µg ) levels ( Fig . 5A ) ., Yet interestingly , similar outcomes were predicted for the 5–50 µg dose range ( Fig . 5A ) ., This might be explained by the conflicting roles of IL-21 , enhancing CTL activation while drastically reducing NK numbers at the same time 39; It is likely that in this dosing range , IL-21-increased CTL responses fail to promote further tumor shrinkage due to the IL-21-inhibition of NK availability ., A prospective experiment in B16-induced mice examined whether a low dosing regimen in the plateau range ( i . e . 12 µg/day ) could indeed be as effective as the standard 50 µg/day treatment ., Beginning on day 3 following tumor challenge , the two doses were applied SC , and the tumor mass was measured until day 17 ., Both the 12 µg and 50 µg doses induced sufficient antitumor responses in the mice ( *p< . 05 and **p< . 001 compared with PBS-treated mice; Fig . 5B ) ., Although the 12 µg dose appeared slightly less potent , its effect was not significantly different from the 50 µg schedule ( ns , p> . 05; Fig . 5B ) , as anticipated by the model ., Indeed , the model prediction ( Fig . 5A ) fit the 12 µg/day outcome to a good degree ( R2\u200a=\u200a0 . 89 , Fig . 5B ) ., These findings further validate the model , and are the first indication of an IL-21 dosing range executing equally potent effects ., Immune-targeted therapy is increasingly apparent in the battle against cancer ., Several reagents are in development within this scope , some already approved for use in certain indications 1 , 9 , 50 ., In this study , we have devised and validated a clinically-relevant mathematical model integrating the PK/PD effects on immune and disease interactions of IL-21 , one of the recent immunotherapeutic drugs under focus in solid cancers 9 ., Following its verification , our model was used for suggesting beneficial IL-21 treatment policies ., Previous attempts to model cytokine-based immune modulation of solid malignancies have been mainly theoretical , helping to elucidate certain characteristics of the tumor-immune system cross-talk and providing important insights into treatment success ( see for example 23 , 31 , 33 ) ., Our former model focused on the heart of the IL-21 response , retrieving the effects of cytokine gene therapy to a good extent ., Yet , its predictions could not be extrapolated to the clinical realm ., The current work is thus among the first biomathematical studies accounting for practical treatment aspects of cytokine immunotherapy in general , and IL-21 treatment in particular ., Our current model deals with realistic PK and PD effects on disease progression , clinically-feasible scheduling , patient compliance , etc ., Moreover , in contrast to the customary stand-alone PK/PD modeling approach , we have integrated IL-21 PK/PD with specific effects on the involved biological processes , to give a mechanistic , yet minimal , model ., Particularly , our PD/disease model accounts for real entities of the IL-21 biological processes ( effector cells , etc . ) , which enabled us to use measurable data and make testable quantitative predictions ., At the same time , we kept our model concise thanks to condensation of other overly complex biological entities ( cytotoxic proteins , etc . ) which are less cardinal and often not measured experimentally ., Overall , our approach provides a robust model that can forecast the long-term anticancer effects of a specific immunotherapeutic cytokine , via a clinically-oriented prism ., Our integrated PK/PD model was constructed by an advanced “multiple-modeling” approach , which we found most suitable for the IL-21 scenario ., The selection of a favorable model out of many analyzed structures and complexities , and the use of non-linear kinetics , enabled us to explore significantly more functional possibilities , and allowed for flexibility in the design ., Moreover , rather than forming a model per scenario , we were able to create a generalized model by describing processes that are mutual to different therapeutic settings ( administration routes , etc . ) and tumor types ., This enhanced the robustness of the model , since it structure was subject to testing under diverse conditions ., Indeed , the model encompasses IL-21-induced outcomes in a wide range of treatment conditions , under different times and administration routes ., Despite its simplicity , the model accurately predicted IL-21-relayed effects in B16- and RenCa-challenged mice , both prospectively and retrospectively ., Moreover , the model demonstrated robust behavior , and predictions were largely insensitive to modulation of key parameters ., With this combined generality and accuracy , the model can potentially accommodate other clinical settings and solid cancers where similar immune processes apply and where IL-21 has been useful ( i . e . adenocarcinoma , glioma , neuroblastoma ) 7 , 8 ., A systematic design of clinically applicable IL-21 immunotherapy strategies has long been called for ., Considering the modest responses of MM and RCC patients to IL-21 therapy 10 , 11 , 12 , 13 , 14 , it is worthwhile to examine whether the drug can be more powerful under different treatment approaches ., Previous trial regimens of IL-21 were determined based on the US Food and Drug Administration guidelines for high-dose IL-2 therapy in MM patients 13 , as the two cytokines share homology and certain effector-inducing functions ., Yet , recent findings demonstrate that IL-2 and IL-21 do not entirely align in their actions 17 , 51 , 52 , inferring that the optimal administration strategies ( administration routes , dose intensities , inter-dosing intervals , etc . ) likely vary between the two agents ., Local IL-21 delivery or expression have been proposed , by us and others , to be potentially effective and safe approaches 17 , 39 , 53 , yet such therapeutic methods are not yet available for clinical use ., Our systemic model analysis therefore represents a new effort to identify improved , clinically-appropriate IL-21 therapies , using the preclinical tumor models B16 and RenCa as case studies ., Simulations of differently dosed IL-21 schedules gave rise to central new insights ., According to the model , comparable antitumor responses are induced by daily IL-21 doses within the 5–50 µg range ., This was prospectively confirmed in B16-challenged mice , in which a substantially lower IL-21 dose ( roughly 12 µg/day ) was as effective as the standard 50 µg/day treatment ., An insensitive range of IL-21 doses with similar efficacy is not unreasonable , considering that the drug respectively inhibits or induces NKs and CTLs , two cells which complement one another in the process of cancer targeting ., This model-aided identification of smaller doses with similar therapeutic efficacy could have immense clinical value , possibly reducing putative IL-21-associated toxicities ., Adverse events have indeed been reported in IL-21-treated patients 10 , 11 , 12 , 13 , 14 ., IL-2 and interferon-α , other cytokine drugs , are associated with severe hematological and neuropsychiatric side effects complicating their use 2 ., Recent PK/PD models of toxic IL-21 effects on body temperature and red blood cell regulation 46 , 47 present a possible framework in which our improved regimens can be confirmed for clinical safety ., Another interesting concept surfacing from our simulations addresses IL-21 fractionation ., The model predicted improved antitumor responses by simple partitioning of the experimental regimen ( a single 50 µg dose/day ) into an equally intense regimen of 25 µg doses given twice daily ., This was prospectively validated by experiments in which the fractionation-treated mice ended therapy with ca ., half of the tumor load observed after the standard treatment ., Model-predicted halving of a daily dose was sufficient to significantly enhance IL-21 efficacy , and further division of the doses was not imperative ., Indeed , fractionation of cancer therapeutics was recommended in the past by mathematical modeling 29 , and its beneficial effects have been validated preclinically for a chemotherapy supportive drug 34 ., This strategy has mostly been applied in the context of radiation therapy and chemotherapy 54 , yet our results , which clearly indicate the benefit of fractionated IL-21 dosing , propose its relevance also to immune-modulating drugs ., Notwithstanding , fractionation may be impractical , reducing patient compliance and requiring hospitalization in certain cases ., Moreover , embarking on new clinical studies to test fractionation therapy is a large and expensive task , and further adjustment of the mathematical model to humans is needed before engaging in such endeavors ., Our findings also raise the question whether IL-21 ought to be administered by available “slow and continuous release” drug delivery methods , which can be viewed as regimens of maximal partitioning ., Past cytokine-gene therapy experiments in mice showed complete eradication of IL-21-secreting tumors in which the drug was released in low continuous levels directly in the target tissue 7 , 9 , supporting the possible advantage of fractionated regimens ., Future implementation of such routes of drug delivery within our model can allow to specifically analyze the benefit of such strategies for IL-21 therapy ., Our present results set the stage for constructing a humanized IL-21 model , to serve as a tool for streamlining development of the drug , and in the future , hopefully , also for personalizing cytokine immunotherapy ., The model , up-scaled to the clinical arena , can entertain diverse cancer indications , patient-specific characteristics , and different modes of therapy ., Newly-discovered IL-21 properties of relevance to the anticancer response , such as modulation of T regulatory cell functions 17 and anti-angiogenic properties 18 , may be introduced in the evolving IL-21 model ., Finally , considering the growing interest in combination therapies for solid cancers , and the promising preclinical and clinical responses observed when applying IL-21 with monoclonal antibodies or signaling inhibitors 9 , 50 , a future model will also study IL-21 therapy in combination with additional therapeutic reagents .
Interleukin ( IL ) -21 is an attractive antitumor agent with potent immunomodulatory functions ., Yet thus far , the cytokine has yielded only partial responses in solid cancer patients , and conditions for beneficial IL-21 immunotherapy remain elusive ., The current work aims to identify clinically-relevant IL-21 regimens with enhanced efficacy , based on mathematical modeling of long-term antitumor responses ., For this purpose , pharmacokinetic ( PK ) and pharmacodynamic ( PD ) data were acquired from a preclinical study applying systemic IL-21 therapy in murine solid cancers ., We developed an integrated disease/PK/PD model for the IL-21 anticancer response , and calibrated it using selected “training” data ., The accuracy of the model was verified retrospectively under diverse IL-21 treatment settings , by comparing its predictions to independent “validation” data in melanoma and renal cell carcinoma-challenged mice ( R2>0 . 90 ) ., Simulations of the verified model surfaced important therapeutic insights: ( 1 ) Fractionating the standard daily regimen ( 50 µg/dose ) into a twice daily schedule ( 25 µg/dose ) is advantageous , yielding a significantly lower tumor mass ( 45% decrease ) ; ( 2 ) A low-dose ( 12 µg/day ) regimen exerts a response similar to that obtained under the 50 µg/day treatment , suggestive of an equally efficacious dose with potentially reduced toxicity ., Subsequent experiments in melanoma-bearing mice corroborated both of these predictions with high precision ( R2>0 . 89 ) , thus validating the model also prospectively in vivo ., Thus , the confirmed PK/PD model rationalizes IL-21 therapy , and pinpoints improved clinically-feasible treatment schedules ., Our analysis demonstrates the value of employing mathematical modeling and in silico-guided design of solid tumor immunotherapy in the clinic .
Among the many potential drugs explored within the scope of cancer immunotherapy are selected cytokines which possess promising immune-boosting properties ., Yet , the natural involvement of these proteins in multiple , often contradicting biological processes can complicate their use in the clinic ., The cytokine interleukin ( IL ) -21 is no exception: while its strength as an anticancer agent has been established in several animal studies , response rates in melanoma and renal cell carcinoma patients remain low ., To help guide the design of effective IL-21 therapy , we have developed a mathematical model that bridges between the complex biology of IL-21 and its optimal clinical use ., Our model integrates data from preclinical studies under diverse IL-21 treatment settings , and was validated by extensive experiments in tumor-bearing mice ., Model simulations predicted that beneficial , clinically practical IL-21 therapy should be composed of low-dose schedules , and/or schedules in which several partial doses are administered rather than a single complete dose ., These findings were subsequently confirmed in mice with melanoma ., Thus , future testing of these strategies in solid cancer patients can be a promising starting point for improving IL-21 therapy ., Our model can thus provide a computational platform for rationalizing IL-21 regimens and streamlining its clinical development .
medicine, immune physiology, immune cells, cytokines, clinical research design, immune activation, animal models of disease, immunology, anatomy and physiology, preclinical models, adaptive immunity, genetics and genomics, immunoregulation, immunomodulation, immunotherapy, t cells, biology, immune response, immune system, systems biology, clinical immunology, nk cells, immunity, physiology, computational biology, modeling