Given the rising popularity of post-retirement work and its potential benefits for older adults’ mental health, this study examined older adults’ adaptation ability as a conditional factor for the impact of post-retirement work on depression. Quantitative data from 1433 working older adults and 1433 non-working older adults were analyzed using the PROCESS macro in SPSS to test a moderated regression model with adaptation ability as the moderator. Results showed that older adults with lower adaptation ability demonstrated significantly lower depression if they worked (vs. did not work). Older adults with higher adaptation ability exhibited significantly higher depression if they worked (vs. did not work). These findings were subsequently verified with a robustness check. Overall, post-retirement work did not prevent depression for the entire sample; working only alleviated depression among older adults with limited adaptation ability. Older adults with stronger adaptation ability could better maintain their mental health by staying retired. This study fills a knowledge gap regarding the relationship between post-retirement work and mental health. Implications for aging societies are also discussed. The aging of the population accompanies the decline of the working-age population  which prIn addition to fulfilling older adults’ financial need, post-retirement work is found to benefit the health of older adults, including preventing depression ,17. DeprThe dramatic process of social change and technology development in the 21st century presents challenges and stresses to older adults. In addition to these macro-level social and technological challenges, micro-level challenges are present for older adults as well. For instance, for older workers who engage in non-career jobs, they may have to learn new skills and build new social networks. Even for those who stay in their career jobs, they may face challenges such as ageism, concerns of stealing jobs or blocking promotion for younger generations, gossip of why they do not enjoy later life with more leisure, etc. . For oldOlder adults pursue post-retirement work for various reasons. In addition to meeting financial need, older adults expect social, personal, and generative rewards from work , such asOlder adults’ pursuit of post-retirement work is associated with four categories of antecedents: individual attributes, family-related variables, job-related variables , and macPost-retirement work has been found to bring favorable outcomes for older adults. For example, financial stability for later life is apparent . As postWith the foundational support of continuity theory, role theory, and the majority studies that have demonstrated positive impact of post-retirement work on mental health, we hypothesize that:H1: Post-retirement work is negatively associated with depression.Although the literature lacks conditional factors for the underlying relationship between post-retirement work and older adults’ mental health, the person–environmental fit theory  and the According to the person-environmental fit theory, when one fails to fit with environmental demands, one will experience a higher level of stress. The cumulative stress caused by the misfit may impair one’s mental health . If oldeThe trait activation theory describes trait–situation relevance to explain outcomes . The theAccording to the ecological theory of aging, successful aging occurs when one finds the best balance between competence and environmental press. Competence is defined as the theoretical upper limit of capacity of the individual to function in the area of biological health, sensation perception, motoric behavior, and cognition . EnvironIn accordance with the three theories, it is presumable that post-retirement work would benefit the mental health of older adults when they have higher level of adaptation ability but would boost mental stress of older adults who have lower level of adaptation ability. We therefore develop the hypotheses as follows:H2: Post-retirement work increases depression in older adults who have a lower level of adaptation ability.H3: Post-retirement work decreases depression in older adults who have a higher level of social adaptation.The hypotheses were tested with the secondary data obtained from the individual survey of the China Longitudinal Aging Social Survey (CLASS) offered by the National Survey Research Center at Renmin University of China. CLASS is a nationwide social survey project conducted in 2016 and collects social, psychosocial, and economic data about Chinese older adults (aged 60 and above) on individual and community levels regularly and systematically. A total of 462 neighborhoods in urban areas and villages in rural areas were randomly selected from 28 of China’s 31 provinces for CLASS. A total of 25 households with older adults were randomly chosen from each selected village or neighborhood. Researchers approached the sampled households, read questions of the survey to one of the older adults in each household, and filled out the survey based on respondents’ answers.The CLASS datasets have been popularly used by a number of published studies ,56,57. TThe retirement policy in China implements a compulsory retirement age: 60 for men, 55 for female cadres/professionals , and 50 for the rest of the female workers . Given tp < 0.001) from the Center for Epidemiologic Studies–Depression scale (CES-D) on a 3-point Likert-type scale  adapted from Silverstein et al. (2006) [p < 0.001) were adopted from Chen (2006) [Depression was assessed by nine items  . Of the n (2006)  and Chenn (2006)  Social An (2006) ,66. Sampn (2006) . Gender http://www.afhayes.com, accessed on 31 January 2023) was executed in SPSS to examine the proposed moderating effect [The data analysis process consisted of three steps. Descriptive analysis was performed first to demonstrate characteristics of the study samples with the means and standard deviations of the study variables. Second, a moderated regression model with adaptation ability as a moderator was tested to examine the impact of post-retirement work on depression. The Hayes (2017) SPSS macro PROCESS  and adaptation ability  of the working group are significantly higher than non-working group. The depression level of working group is significantly lower than non-working group . p < 0.01), supporting H1, and adaptation ability as an independent variable does not influence depression . The significant interaction effect of post-retirement work and adaptation ability  indicates that adaptation ability significantly moderates the impact of post-retirement work on depression.p < 0.01). Post-retirement work significantly decreases depression of older adults who are less adaptive  and significantly increases depression of older adults who are more adaptative . The results of the robustness check are consistent with the main study results. Additionally, we used the bootstrap technique to estimate the coefficients with multiple random samples and obtained similar results.In order to verify the robustness of the study results, we created a new dataset by randomly selecting another sample of 1433 non-working cases and ran the regression. With the new randomly selected non-working sample, the adaptation ability also significantly moderates the impact of post-retirement work on depression  that may benefit further investigation of the topic are not available. Future researchers collecting first-hand data may include these important variables in their study design to answer the related questions. Fourth, the study only examined adaptation ability as a conditional factor for the impact of post-retirement work. Future researchers are encouraged to investigate more possible conditional factors including factors relating to needs–supplies fit and value congruence to further reveal the deep relationship between post-retirement work and mental health.


Transitional hospital-to-home care programs support safe and timely transition from acute care settings back into the community. Case-mix systems that classify transitional care clients into groups based on their resource utilization can assist with care planning, calculating reimbursement rates in bundled care funding models, and predicting health human resource needs. This study evaluated the fit and relevance of the Resource Utilization Groups version III for Home Care (RUG-III/HC) case-mix classification system in transitional care programs in Ontario, Canada.n = 1,680 care episodes) in transitional home care programs in Ontario. We classified care episodes into established RUG-III/HC groups based on clients’ clinical and functional characteristics and calculated four case-mix indices to describe care relative resource utilization in the study sample. Using these indices in linear regression models, we evaluated the degree to which the RUG-III/HC system can be used to predict care resource utilization.We conducted a retrospective analysis of clinical assessment data and administrative billing records from a cohort of clients  and having Reduced physical functions (37.8%). The RUG-III/HC groups that account for the largest share of clients are those with the lowest hierarchical ranking, indicating low Activities of Daily Living limitations but a range of Instrumental Activities of Daily Living limitations. There is notable heterogeneity in the distribution of clients in RUG-III/HC groups across transitional care programs. The case-mix indices reflect decreasing hierarchical resource use within but not across RUG-III/HC categories. The RUG-III/HC predicts 23.34% of the variance in resource utilization of combined paid and unpaid care time.The distribution of clients across RUG-III/HC groups in transitional home care programs is remarkably different from clients in long-stay home care settings. Transitional care programs have a higher proportion of Clinically complex clients and a lower proportion of clients with Reduced physical function. This study contributes to the development of a case-mix system for clients in transitional home care programs which can be used by care managers to inform planning, costing, and resource allocation in these programs.The online version contains supplementary material available at 10.1186/s12913-023-10150-1. Transitional hospital-to-home care programs have emerged as a promising approach to improve care trajectories for older adults following hospitalization in Canada . These pA key feature of transitional care programs is the integration of care across a broad spectrum of services, which fosters coordination and continuity of care . TransitThe complexity and variability of client needs in transitional care programs requires a robust reimbursement model to enable appropriate care delivery and resource allocation . Long-stCase-mix classification systems aim to predict resource needs and utilization within clinical populations by classifying home care clients into defined groups based on key predictive characteristics. Ideal case-mix systems are grounded in clinically meaningful descriptions of clients in a specific care setting and reflect resource utilization over a relevant time period  . These sThe Resource Utilization Groups version III for Home Care (RUG-III/HC) case-mix classification system is widely used in home care settings in Canada , 23. ThiThe RUG-III/HC leverages routinely collected data from client assessments which facilitates its implementation and relevance for care planning and evaluation. The classification algorithm was originally designed using data from the Resident Assessment Instrument – Home Care (RAI-HC), a standardized client assessment instrument completed by clinicians to inform home care planning and delivery. An updated assessment instrument, the interRAI Home Care (interRAI-HC), was published in 2012 and has been widely implemented in long-stay home care settings in Canada . The intThe RUG-III/HC case-mix classification system has significant potential to inform care planning and resource allocation in transitional hospital-to-home care programs. However, clients receiving care in transitional home care programs may be substantially different from those in long-stay home care. The landscape of home care in Canada has also evolved significantly since the RUG-III/HC was initially validated , 28. FurOur objective was to examine the applicability of the existing RUG-III/HC case-mix classification system to clients in transitional hospital-to-home care programs in Canada. We evaluated the distribution of clients across RUG-III/HC groups in transitional care programs and examined the reliability of the RUG-III/HC case-mix groups for predicting care resource utilization.We conducted a retrospective analysis of a cohort of clients from a convenience sample of eight 10–16-week transitional hospital-to-home care programs operated by one home care provider organization in the province of Ontario. These programs were selected for inclusion in consultation with an advisory group of program managers. They represent a large proportion of the transitional home care programs currently in operation in the province and are all relatively homogeneous in their scope, referral patterns, intended length, and time-frame. The full study sample includes data from 1,680 completed care episodes with complete assessments from 2019 to 2022.Given the diverse population of clients in transitional home care programs, principles of person-centered care often require deviations from an idealized care pathway to respond to individual needs and circumstances. As such, the full study sample includes some clients with unusually short or long care episodes. The inclusion of these atypical cases in the analysis has significant implications for the interpretation of the RUG-III/HC case-mix groupings . At the These two samples facilitate complementary utilizations of the RUG-III/HC case-mix system. The full sample reflects actual care utilization patterns in transitional care programs, which is most useful for individual care planning and cross-program comparisons. In contrast, the operational sample reflects care utilization patterns of clients whose care episodes are more closely aligned with the intended scope and length of the transitional care programs, which facilitates program planning and calculating prospective payment rates.Information on each client’s clinical characteristics at the beginning of their care episode was obtained from interRAI-HC assessments completed upon admission to the transitional care program.Information on paid care resource utilization throughout each care episode was drawn from administrative billing records. These data define the start and end date of the care episode and include the total number of hours that each client received care services from a registered practical nurse (RPN), registered nurse (RN), occupational therapist (OT), physical therapist (PT), speech-language pathologist (SLP), social worker (SW), registered dietician (RD), physical therapy assistant (PTA), and personal support worker (PSW) throughout their care episode. Complete billing records were available for 1,251 care episodes in the full sample and 1,184 care episodes in the operational sample.Information on unpaid caregiving throughout each care episode was drawn from interRAI-HC assessment data, in which clients are asked to self-report the number of hours of unpaid care they received in the preceding 3 days. This data was retrieved from assessments completed at admission as well as discharge.We first classified each care episode into one of the existing 23 RUG-III/HC groups using the established hierarchical classification algorithm developed for long-stay home care settings [To describe trends in resource utilization of clients admitted to transitional care programs, we derived four case-mix indices (CMIs) which represent the mean paid care time and cost as well as combined paid and unpaid care time and cost for clients in each RUG-III/HC group relative to the overall population. Although the RUG-III/HC is primarily used to inform care planning and resource allocation in paid care settings, the availability of unpaid caregiving has significant implications for home care clients’ health and quality of life which can, in turn, influence their utilization of paid care resources . The incThe number of hours of paid care during each care episode was calculating by aggregating the hours billed by each type of healthcare professional from billing records. To calculate the overall cost of paid care throughout a care episode, hourly billing rates were obtained for each type of healthcare professional. These represent the fair market value of their labour including their hourly wage and indirect costs relating to their employment . We standardized these billing rates by the hourly rate of a PSW to create a set of standardized cost weights. PSWs were selected as the reference group for this calculation as they provide the largest proportion of paid care to clients in home care settings . We calcWe lacked data on the exact number of hours of unpaid caregiving that each client received throughout their care episode. Total hours of unpaid caregiving were therefore estimated by calculating the mean hours of unpaid care reported by clients on interRAI-HC assessments at admission and at discharge and multiplying by the length of the care episode. The cost of unpaid caregiving must also be estimated based on assumptions about the economic value of unpaid care . Using aUsing these data, we calculated the mean and standard deviation of four resource utilization measures within each RUG-III/HC group: 1) paid care time, 2) paid care cost, 3) paid and unpaid care time, 4) paid and unpaid care cost. Four corresponding CMIs were derived by dividing each group’s mean by the overall population mean, representing relative resource utilization across RUG-III/HC groups.We examined within-group homogeneity of care resource utilization by calculating the coefficient of variation (CV) associated with the four CMIs in each RUG-III/HC group and in the overall study population.R2) of each model [To evaluate the relevance of the RUG-III/HC groups for predicting resource utilization in transitional care programs, we modelled each client’s care resource utilization as a function of the four CMIs using linear regression and extracted the variance explained  to 0.3% in the highest Special care group (SSB) and second highest Extensive services group (SE2) – excluding three groups  into which no clients were classified. Across almost all RUG-III/HC categories, the groups that account for the largest proportion of clients are those with lower hierarchical ranking, indicating low ADL limitations but a range of IADL limitations. This trend is particularly evident in the two largest RUG-III/HC categories: Clinically complex and Reduced physical functions, in which a majority of clients are classified into groups with low ADL limitations but mild to severe IADL limitations (CA2: 20.1% and PA2: 16.1%) and groups with low ADL limitations and no IADL limitations (CA1: 13.8% and PA1: 14.2%). Estimates for groups into which fewer than 10 clients have been classified are likely unstable and should be interpreted with caution.There were notable differences in the distribution of clients in RUG-III/HC groups across transitional home care programs in the study sample Fig. . There aThe four CMIs for each of the RUG-III/HC groups in the operational sample are presented in Table The four CMIs and their corresponding CVs in the full sample are nearly identical  is summarized in Table The variance in care resource utilization that is explained by the RUG-III/HC case-mix classifications R is summaTransitional hospital-to-home home care programs are designed to support clients following a hospital discharge by promoting the recovery or maintenance of functional independence. This analysis examined the applicability of the RUG-III/HC case-mix system, which was developed for long-stay home care settings, to clients in transitional home care. The results suggest that clients in transitional care programs differ from those in traditional long-stay home care. A majority of clients in transitional care were classified in the Clinically complex (41.6%) and Reduced physical functions (37.8%) RUG-III/HC groups. Although these same two groups represent the largest share of clients in long-stay home care as reported by Poss et al. in their validation of the RUG-III/HC in the Canadian home care context in 2008, their relative prevalence is reversed as more than half of long-stay home care clients were classified as having Reduced physical functions (56.1%) and the proportion of clients classified as Clinically complex was less than half that in transitional care programs (21.2%) . This fiAnother notable difference is that transitional home care programs have fewer clients with Impaired cognition (2.0%) compared to long-stay home care (12.8%) . This maWithin each of the RUG-III/HC categories, transitional home care clients were more likely to be classified into the two lowest hierarchical groups indicating low ADL limitations but a range of IADL limitations. In contrast, long-stay home care clients were more likely to be classified into the lowest hierarchical group which indicates both low ADL and IADL limitations. Given their post-acute status, clients in transitional care may experience a wider range of IADL limitations. Functional status has been found to be a significant driver of variance explained in case-mix models in long-term care settings, which may influence the applicability of the RUG-III/HC in transitional care .The predictive power of case-mix systems in home care settings is highly variable and reported variance explained in other studies has ranged from 14% in to 54.3% . The RUGAlthough it was originally developed and validated for long-stay home care settings, the RUG-III/HC case-mix system has the potential to support informed decision-making in transitional home care. By leveraging routinely-collected interRAI-HC assessment data, the RUG-III/HC can be used to guide care and program planning and evaluation. The results from the full and operational study samples can inform complementary uses of the RUG-III/HC. The full sample contains data from all completed care episodes regardless of their length, which renders the results most useful to individual care planning as well as program evaluation. The operational sample reflects an idealized client population, which is most useful for prospective program planning. However, there is an opportunity to refine the RUG-III/HC case-mix algorithm to improve its relevance for predicting care resource utilization in transitional programs. This may be achieved by modifying the classification structure and hierarchy to more accurately reflect the care delivered in transitional programs or by incorporating additional variables in the classification algorithm that may be important for explaining care resource utilization in transitional care, such as specific diagnoses  or health events , measures of mobility and self-management, and social factors that influence unpaid caregiving.The results of this analysis should be considered in the context of its limitations. First, 124 care episodes were excluded from the study sample due to missing interRAI-HC assessment data and missing billing data for 429 care episodes resulted in their exclusion from the CMI calculation. If this missing data is non-random with respect to their clinical characteristics and resource utilization, the RUG-III/HC distribution and CMI calculations may be biased. Similarly, the possibility of misclassification due to errors or bias in the interRAI-HC assessment data cannot be eliminated, even if it is unlikely , 38. SimThe contractual structure of transitional care programs as well as the timing of interRAI-HC assessments on admission to a program may also influence the classification of clients into RUG-III/HC groups. The transitional care programs examined in this study were designed to deliver a high degree of rehabilitation services and nursing care which contribute to RUG-III/HC classification and CMI calculation, respectively. In addition, several questions on the interRAI-HC require assessors to base themselves on retrospective answers, including the number of hours of rehabilitation minutes received in the past 7 days, ADL/IADL performance in the past 3 days, and unpaid caregiving in the past 3 days. Transitional care programs require interRAI-HC assessments to be completed soon after hospital discharge and admission to the transition program. However, if admission interRAI-HC assessments are completed too soon after hospital discharge, they may miss some elements of care that are relevant for RUG-III/HC classification.As healthcare systems continue to evolve and the Canadian population ages, there is a growing need to provide care for people in their homes and communities. The results of this analysis show that the profile of clients receiving post-acute care in transitional hospital-to-home care programs is remarkably different from our understanding of the profile of clients in long-stay home care settings. Transitional home care clients have a greater degree of medical complexity and a wider range of IADL limitations.Despite the differences in client population, the RUG-III/HC case-mix classification system has the potential to inform individual care delivery, program planning and evaluation, prospective calculation of bundled care pricing, and strategies to optimally allocate human resources in transitional care programs. With a better understanding of the distribution of care needs in transitional care, these programs will be better equipped to deliver appropriate person-centered care. These findings contribute to the ongoing evaluation of the RUG-III/HC classification system and open the potential opportunity to improve the system for different and newer forms of home care programs. Such classification systems can and should be used to inform the development of evidence-based policies and practices for transitional hospital-to-home care in Canada.Additional file 1. Case-mix indices (CMI) and coefficients of variation (CV) of full sample.


Mutations and the glycosylation of epitopes can convert immunogenic epitopes into non-immunogenic ones via natural selection or evolutionary pressure, thereby decreasing their sensitivity to neutralizing antibodies. Based on Thomas Francis’s theory, memory B and T cells induced during primary infections or vaccination will freeze the new mutated epitopes specific to naïve B and T cells from the repertoire. On this basis, some researchers argue that the current vaccines derived from the previous strains of the SARS-CoV-2 virus do not increase immunity and may also prevent the immune response against new epitopes. However, evidence shows that even if the binding affinity is reduced, the previous antibodies or T cell receptors (TCRs) can still bind to this new epitope of the Beta, Gamma, and Delta variant if their concentration is high enough (from a booster injection) and neutralize the virus. This paper presents some convincing immunological reasons that may challenge this theory and argue for the continuation of universal vaccination to prevent further mutations of the SARS-CoV-2 virus. Simultaneously, the information presented can be used to develop vaccines that target novel epitopes or create new recombinant drugs that do not lose their effectiveness when the virus mutates. One crucial aspect of SARS-CoV-2 is the ability of the virus to rapidly mutate and create antigenically distinct strains. Changes in the amino acids and glycosylation or deglycosylation of sites create new epitopes by changing the previous epitopes. These established new epitopes form novel N-glycan shields that can mediate other contemporaneous SARS-CoV-2-neutralizing antibodies.The Original antigenic sin (OAS) theory, described in 1960 by Thomas Francis, states that the immune system preferentially uses immunological memory based on a previous infection when encountering a second, slightly different version of that foreign pathogen. This leaves the immune system “trapped” by its first response to each antigen and unable to mount potentially more effective responses during subsequent infections. Based on this theory, memory B and T cells induced during infections or vaccinations with the primary variant of the pathogen will freeze the new mutated epitopes’ specific naïve B and T cells (cross-reactive memory against specific naïve B or T cells) from the repertoire . GarritySuppose a primary antigen with several epitopes enters the body. The epitopes are divided into several categories: exposed immunogenic epitopes, hidden immunogenic epitopes, exposed non-immunogenic epitopes, and hidden non-immunogenic epitopes. The exposed immunogenic epitopes  are expected to naturally stimulate the cellular and humoral immune system and induce T and B cell memories. Similarly, the hidden immunogenic linear epitopes will stimulate the humoral and cellular immune systems by producing antigen-specific antibodies (Ab1) during the first immune reaction following exposure to the antigen. In the case of SARS-CoV-2, mutations might have several consequences: the exposed immunogenic epitopes may change so that the antibodies produced against these epitopes no longer can neutralize the virus, and the virus will remain pathogenic.Furthermore, the production of anti-idiotype antibodies (Ab2) can be induced, specifically targeting Ab1 to inhibit its action by forming immune complexes. Paratopes of Ab2 can mimic antigens by binding to Ab1. Moreover, their structural similarities enable them to bind to the specific receptor of the original antigen and induce agonist or antagonist cell signaling in the cells targeted by the virus. This mechanism mimics the pathological reaction and triggers a long-term response after the first contact. Characterizing the action of Ab2 would help us to understand several adverse effects during SARS-CoV-2 infection or vaccination .After mutation due to changes in amino acids or glycosylation (or deglycosylation), new epitopes are created by changing the previous epitopes. Even if the binding affinity is reduced, the previous antibodies or T cell receptors (TCRs) can still bind to this new epitope if their concentration is high enough (from a booster injection) to neutralize the virus. This has been demonstrated based on the neutralization of new variants of the SARS-CoV-2 virus  through convalescent and post-vaccination sera with high titers . On the Another argument is that mutations and glycosylation can convert immunogenic epitopes into non-immunogenic ones via natural selection or evolutionary pressure, decreasing their sensitivity to neutralizing antibodies . Unlike Glycans disrupt the immune response and promote immune escape by inhibiting recognition by antibodies and immune cells. In influenza, the severity of the disease depends on the glycosylation profile of surface proteins such as hemagglutinin (HA) from past exposure. Infection with a highly glycosylated variant induces a deficient adaptive response with a lack of neutralizing antibodies and a robust T response. In the event of reinfection with a low-glycosylated variant, antibody-mediated neutralization is significantly reduced, and the disease is more severe. This explains why hosts previously infected with highly glycosylated seasonal H1N1 viruses develop severe lower respiratory tract disease due to the pandemic H1N1 strain .Furthermore, some evidence indicates that some new glycosylation on the SARS-CoV-2 spike glycoprotein not only shields the immunogenic epitope but also, at the same time, may change the S binding affinity to ACE2 B. Zhao eThe professional antigen-presenting cells (APC), B lymphocytes, also present an opportunity to refute the OAS theory. Any antigen it recognizes can present its different epitopes and the epitope that specifically binds to its receptor, can bind to T helper lymphocytes in the MHC class II cleft C. The glAfter mutation, the change in the epitopes mentioned above may lead to a situation where the previous vaccine-induced antibody’s binding to their adjacent epitopes increases their binding affinity to ACE2. As Liu et al. reported, several antibodies bind to parts of the SARS-CoV-2 N-terminal domain (NTD), increasing its binding to the ACE2 receptor . This isMattiuzzi and Lippi  conducteThe SARS-CoV-2 S glycoprotein (SGP) is a trimeric class I fusion protein that comprises two subunits, including the S1, the receptor-binding subunit attached to the S2, the fusion-mediating subunit, such that the complete entity is a trimer of S1–S2 heterodimers . The S2 Conversely, post-fusion or other aberrant protein conformations induce primarily non-neutralizing antibodies (non-NAbs) with no or limited protective capacity. In other cases, non-NAbs can even be harmful. When expressed as a recombinant protein, the SARS-CoV-2 SGP trimer can undergo spontaneous conformational changes and decay into its post-fusion form, inducing non-NAbs. These non-NAbs can be categorized as Fc-antibody-dependent infection enhancement antibodies produced by the previous vaccination, and the induction of such non-NAbs results in immune complex (IC) deposition and complement activation. These IC deposits have been linked to COVID-19 disease severity and many of the long-term consequences of SARS-CoV-2 infection such as arthritis and vasculitis . This viThe most utilized and internationally accepted SARS-CoV-2 vaccines are nucleic acid vaccines such as the Pfizer-BioNTech and the Moderna Spikevax. For RNA-based mRNA vaccines, the mRNA is transcribed in vitro from DNA before the RNA is encapsulated in the nanoparticles prior to vaccine administration, so no DNA is present, and no transcription is needed in the nucleus ,31,32,33While these vaccines are some of the most efficacious available, there is potential for host glycosylation to negatively impact their success. Previous studies have found that host protein glycosylation impairs immune responses against bacteria even after using nucleic acid vaccines . SimilarA new approach introduces specific glycosylation sites onto the RBD to counteract this issue and thus elicit more robust immune responses to both previous and mutated variants. A novel study by Carnell et al. proposed a modified RBD spike subunit that triggers more broad neutralizing immune responses as a potentially improved booster vaccine . Their mMoreover, data suggests that the N-glycosylation and O-glycosylation of proteins can occur . This glFrom the SARS-CoV-2 pandemic, we can learn a lot about vaccine design and efficacy. It is evident that glycosylation plays an essential and contrasting role in immunogenicity; therefore, it has been suggested that removing the glycosylation sites around vulnerable NAb epitopes may be an effective strategy. The ACE2 receptor binding domain of the SARS-CoV-2 spike protein, which is required for viral anchoring to enable cell entry, is not glycosylated to increase the fitness of the virus . HoweverAnother method that may improve the efficacy of these nucleic acid vaccines is to create proper glycosylation matches. This can be accomplished by ensuring that the same cell types infected during viral infection express the nucleic acid vaccines. The rationale for this approach is based on the fact that the surface glycoproteins of enveloped viruses use the host glycosylation machinery, and the glycosylation of the viral proteins must be highly similar to the glycosylation of the mRNA vaccines . AdoptinWhile the COVID-19 pandemic may seem to be a part of history, it presents a great learning opportunity for preparing for a potential future pandemic of the exact same nature. If vaccines are produced and used for the new Omicron variants, how will we know that future mutated variants will not cause the same problem again? Since the probability of such a problem occurring is very low, evidence based on the existing data indicates that the cross-reactivity of antibodies is a phenomenal concept that can either be advantageous or detrimental to the host . For exaM.R.S. reports patents 55983477-9CN and CAN-DMS-150056368.1 on the compositions and methods for the treatment of coronavirus infection and respiratory compromise. M.R.S. reports having sought approval from Health Canada to test the therapeutic treatment reported in the patents in a human clinical trial.


To simplify black-blood late gadolinium enhancement (BL-LGE) cardiac imaging in clinical practice using an image-based algorithm for automated inversion time (TI) selection.The algorithm selects from BL-LGE TI scout images, the TI corresponding to the image with the highest number of sub-threshold pixels within a region of interest (ROI) encompassing the blood-pool and myocardium. The threshold value corresponds to the most recurrent pixel intensity of all scout images within the ROI. ROI dimensions were optimized in 40 patients’ scans. The algorithm was validated retrospectively (80 patients) versus two experts and tested prospectively (5 patients) on a 1.5 T clinical scanner.Automated TI selection took ~ 40 ms per dataset . Fleiss’ kappa coefficient for automated-manual, intra-observer and inter-observer agreements were Thanks to its good performance and simplicity of implementation, the proposed algorithm is a good candidate for automated BL-LGE imaging in clinical practice.The online version contains supplementary material available at 10.1007/s10334-023-01101-2. Bright-Blood Late Gadolinium Enhancement (BR-LGE) cardiovascular magnetic resonance (CMR) imaging is the reference technique for the assessment of regional scar formation and myocardial fibrosis –3. In BRIn clinical routine, timing parameter selection is performed by MR operators by selecting the optimal times from a series of 2D short-axis images acquired with different timing parameters. This manual process increases the complexity of the BL-LGE acquisition while adding to the workload of the MR operator. Moreover, like with any manual process, it can be prone to both inter- and intra-observer variability. Automation of timing parameter selection could therefore be beneficial to accelerate and standardize the LGE workflow, increase the exam reproducibility, while reducing the MR operator workload. To the best of our knowledge, no algorithms have been proposed for automated BL-LGE parameters selection.Automation of contrast selection is a small, yet necessary, incremental improvement toward a more accurate, effortless, and time efficient procedure for myocardial scar characterization. We propose an image-based algorithm for automated contrast selection in magnetization-prepared BL-LGE . This BLOptimal TI selection was performed by detecting the scout image with the highest number of low-intensity pixels within a ROI located inside the heart and containing parts of the ventricular blood pool and myocardium. The algorithm was implemented in C+ + without external libraries to enable its seamless incorporation to any MRI scanner. A retrospective study was conducted in 120 patients and a prospective study was conducted in 5 patients. All patients underwent CMR for known or suspected structural heart disease. The study was approved by the Biomedical Research Ethics Committee and all participants provided informed consent for participation.Acquisitions were performed on a 1.5 T clinical scanner , using an 18-element body coil and a 32-element spine coil. A prototype electrocardiogram-triggered 2D single-shot, balanced steady-state free-precession BL-LGE scout was performed prior to a fast free-breathing motion-compensated T1ρ-prepared BL-LGE sequence . A schemFor the sake of simplicity and explainability, the proposed algorithm purposefully mimics the manual selection process which consists of selecting, from the series of scout images, the image with the highest scar-blood and scar-viable myocardium contrast. In practice, this image has the lowest signal intensity within the ventricular blood pool and the healthy myocardium. To do so, the automated algorithm operates on the TI scout images in two distinct steps. Step 1: Extraction of a region of interest (ROI), containing relevant information about the ventricular blood pool and myocardium. Step 2: Selection of the image with the highest number of low intensity pixels within this ROI. The corresponding TI is then used for the subsequent whole-heart BL-LGE acquisition. The overall process is depicted in Fig. Accurate localization of the myocardium and ventricular blood pools would lead to optimal ROI selection. However, because our T1-rho pulse has been thoroughly optimized using extended phase graph simulation to cancel out blood and healthy myocardium signals at the same TI, the optimization can be performed equally well on a unique ROI rather than on two individual ones. Therefore, this problem can be greatly simplified by selecting a coarse ROI that includes pixels from the blood pools only or from both the blood pools and the myocardium.ROI extraction is performed in two steps: (A) Coarse detection of the heart within the image and (B) Selection of a ROI within the heart. Acquisition of 2D CMR scout images requires the positioning of a rectangular shim box centered on the heart. This information can be exploited to easily obtain the coarse detection of the heart within the image (Online Resource 2).offline retrospective study section.Optimization of the shape and dimension of the ROI within the heart is detailed in the N corresponds to the number of TI scout images, i of a given image n, max PI is the maximum pixel intensity, n, and α is the index of the image with the highest histogram’s peak:The selection is performed by detecting the image with the highest number of low-intensity pixels within the extracted ROI. To do so, for each scout image, intensity histograms of bin size 1 are calculated on the pixels within the ROI. Histogram peaks are detected and the intensity value corresponding to the global maximum over all images is selected as a common threshold value The proposed algorithm was first implemented offline and tested on TI scouts retrospectively collected on 120 patients  were visually inspected for qualitative assessment. The time for manual TI selection was recorded for one expert.The automated BL-LGE sequence was integrated on a 1.5 T clinical scanner  and was tested prospectively on 5 patients with known or suspected structural heart disease  were good. The overall automated-manual Fleiss’ kappa agreement  and the assurance of finding the optimal TI. However, the proposed algorithm can be used with any range and number of TIs. The optimal ROI size was derived from 40 patient scans. Rectangular ROI of size During TI selection, two main problems can arise that require an additional acquisition. (1) The selection of a sub-optimal TI range, with a minimum TI too high or a maximum TI too low for optimal nulling of both healthy myocardium and blood-pools signals that can occur for an acquisition timing outside the expected time frame of 12–15 min post-gadolinium injection. (2) The presence of artifacts that could affect the TI selection process. Examples of TI scouts presenting these issues are visible in Figure Online Resource 6. Quality feedback is therefore needed to decide whether the whole-heart BL-LGE sequence can be performed with the selected TI or if a new scout acquisition is required. The problem of sub-optimal TI range can be easily addressed as it corresponds to the cases where either the first or the last image of the TI scout is selected. Acquiring new images using, respectively smaller or larger TIs until the selected TI is no longer the smallest or the largest value of the chosen range would address this issue. Examples of such cases and detection by the automated algorithm are visible in Figure Online Resource 7. The presence of artifacts in the ROI will result in a high number of hyperintense pixels and conversely in a smaller proportion of low intensity pixels. Artifact detection could be implemented by comparing the percentage of sub-thresholds pixels within the ROI of the selected image with the median percentage of sub-threshold values computed from a validation set. This quality control and re-acquisition remain to be implemented inline to enable robust automated BL-LGE.The BL-LGE sequence employed in this study makes use of a T1ρ preparation module to generate a black-blood contrast, as recently validated by Sridi et al.  and MuscFinally, while providing good results, the robustness of the technique and especially the sensitivity of the method to deviation in shim positioning also need to be further quantified.We present a new algorithm for a reproducible and time efficient myocardial scar characterization using automated BL-LGE. Automated TI selection demonstrated to be fast and accurate with a performance slightly better than the manual variability observed between two experts. The algorithm’s simplicity enabled a straightforward on-site implementation while it’s explainability foster usage confidence and clinical acceptance. Moreover, the straightforward, yet efficient, design of the proposed automated algorithm provides several opportunities for other BL-LGE techniques. Inline testing in larger group of patients is now warranted.Supplementary file1 (DOCX 3882 KB)Below is the link to the electronic supplementary material.


Nth order stub-loaded coupled-line ultra-wideband bandpass filter. A unit element of a proposed filter topology consists of two short-circuited stubs loaded at the edges of coupled lines. A distributed equivalent circuit model of a proposed topology is extracted and used to acquire a generalized filtering function. The extracted filtering function is of rational form. The denominator of the filtering function causes a mismatch with Chebyshev type-I polynomials. For conventional narrowband filters, the denominator term can be neglected because of the close vicinity of band-edge frequencies; however, for the ultra-wideband filter response, the factor in the denominator cannot be neglected and hence requires a new mathematical procedure to compensate for the effect of the frequency-dependent term in the denominator. The electrical parameters are calculated using the proposed synthesis and used to design an ideal filter topology on ADS. To validate the proposed design procedure, fabrication is performed on a high-frequency substrate. The proposed filter is miniaturized in size and has good out-of-band performance. The simulated and measured results provide good agreement.This paper presents a novel synthesis of a quasi-Chebyshev  The increasing demand for miniaturized, cost-effective, and sophisticated wireless systems has driven the development of highly integrated multi-functional microwave circuits, particularly emphasizing passive microwave components. In response to this evolving landscape, there is a compelling need for the creation of a filter characterized by two key attributes: high selectivity and an expansive stopband. Such a filter not only ensures the reliable transmission of signals but also effectively mitigates the distortions induced by electromagnetic interference. Recent advancements have introduced several distinct methodologies for designing wideband bandpass filters. In the work delineated in , a widebThe synthesis method in this paper involves the development of a novel ultra-wideband (UWB) bandpass filter. This synthesis offers numerical accuracy, an optimal solution, and a comprehensive understanding of the filter. By obtaining the generalized filtering function through the equivalent circuit of a filter, this synthesis approximates the filtering function as a quasi-Chebyshev function. This approach enables the determination of parameters to achieve increased selectivity and equal ripple response, addressing the limitations associated with previous design methods. In light of the constraints and challenges posed by existing methodologies, the pursuit of accurate synthesis theory for UWB bandpass filters is now more critical than ever before in the context of the evolving wireless communication landscape.Nth order ultra-wideband bandpass filter with its distributive equivalent circuit model. Quarter wavelength coupled lines with loaded short-circuit stubs are used to design the proposed filter. The even and odd-mode characteristics impedance of coupled lines is Equation  shows thAfter determining the transfer function using Equation , the traion G4(θThe filter coefficients Equation (4) delineates the filtering function associated with the topology depicted in In pursuit of rectifying this deviation, a pivotal step involves a meticulous normalization process applied to the filtering function. This normalization step serves to curtail the influence of frequency variations, thereby restoring the characteristic equal ripple behavior. The resultant ripple factor, an outcome of this normalization, serves as a critical metric. Subsequently, leveraging this ripple factor, a synthesized rendition of the filtering function is systematically engineered, enabling the identification of an optimal solution that harmonizes with the desired filtering criteria.The return loss is correlated with the ripple factor By using the transfer function, the value of the parameters Equation .(16)S21H.This complies with the following characteristics: UWB bandwidth (4.5–10 GHz), four transmission poles within the passband (for better selectivity), equal ripple (approximating Chebyshev frequency response), adjustable ripple level by ripple factor Equation  is equalBy using Equation  and applThe disappearance of To ensure that the transfer function has four poles, it is essential thatTo address the disparitiesIn Equations  and 22)22), the quations  and (22)Here, Equation –12) γ i γ iBW rez = 28.57, The impedance values for various bandwidths are calculated in By altering the electrical length, it is evident that one can exercise control over the center frequency (CF). As depicted in This section introduces a comprehensive configuration aimed at substantiating the proposed synthesis approach for a seventh-order bandpass filter. The structure in question entails a pair of quarter-wavelength coupled lines, each adorned with three short-circuited stubs characterized by an impedance value denoted as Z and an electrical length parameter In this case, Equation  demonstrEquation  to deterTo validate the novel synthesis of the fourth-order ultra-wideband bandpass filter, microstrip transmission lines are used to design the proposed UWB-BPF filter. The electrical parameters are determined by the proposed synthesis procedure. For the passband with the bandwidth of 5.5 GHz centered at 7.25 GHz, the values of quations –13), we, weσ andThe current distribution at The physical parameters This paper introduces an innovative approach for the synthesis of the proposed ultra-wideband (UWB) bandpass filter, showcasing its distinctive characteristics. The underpinning methodology leverages a distributed equivalent circuit model, instrumental in attaining the sought-after quasi-Chebyshev filtering response. By skillfully mapping this quasi-Chebyshev-type filtering function onto the established Chebyshev type-I filtering response, the essential filter parameters are rigorously ascertained. The resulting UWB bandpass filter design stands out due to its compact form factor, effectively addressing size constraints. To substantiate the efficacy of the design procedure proposed herein, the envisioned filter is realized through fabrication. The subsequent comparative analysis of the simulated and measured outcomes underscores a noteworthy alignment, affirming the robustness and viability of the presented design methodology.


T cells are an essential part of the immune system with crucial roles in adaptive response and the maintenance of tissue homeostasis. Depending on their microenvironment, T cells can be differentiated into multiple states with distinct functions. This myriad of cellular activities have prompted the development of numerous smart probes, ranging from small molecule fluorophores to nanoconstructs with variable molecular architectures and fluorescence emission mechanisms. In this Tutorial Review, we summarize recent efforts in the design, synthesis and application of smart probes for imaging T cells in tumors and inflammation sites by targeting metabolic and enzymatic biomarkers as well as specific surface receptors. Finally, we briefly review current strategies for how smart probes are employed to monitor the response of T cells to anti-cancer immunotherapies. We hope that this Review may help chemists, biologists and immunologists to design the next generation of molecular imaging probes for T cells and anti-cancer immunotherapies. T cells are an essential component of the immune system and display multiple biological functions. Smart probes range from small fluorophores to nanoconstructs, and can target metabolic and enzymatic biomarkers as well as cell-surface receptors. Furthermore, T cell-mediated responses play a critical role in anti-cancer immunotherapies, with immune checkpoint inhibitors and chimeric antigen receptor T cells (CAR T cells) being promising therapeutic strategies to stimulate or reprogram their ability to kill cancer cells.22,23 In the recent years, smart probes have been harnessed to directly evaluate the efficacy of such treatments without the need for invasive techniques  and also been adapted to high-throughput screening platforms to optimize combination therapies.T lymphocytes are key players of the adaptive immune system and essential for the homeostasis of both cellular and humoral immunity . Cytotox24 instead, we will focus on the molecular design of fluorescent conjugates combining optical reporters and biomolecular units  that target multiple aspects of T cell biology . Furthermore, the review will also cover examples of how T cell-targeted smart probes have been applied to monitor the efficacy of anti-cancer immunotherapies, including a variety of optical imaging modalities and preclinical models.In this review, we will present the latest advances in the chemical design and applications of fluorescent smart probes for imaging the activity of T cells. With a few exceptions, our article does not primarily cover the design and application of genetically encoded reporters, because these have been reviewed elsewhere;2.25–27 Therefore, the design of imaging agents that can monitor the migration, biodistribution and activity of T cells is needed to optimize the efficacy of cell transfer therapies. Current methods to characterize T cell subtypes at inflammation sites typically rely on antibody-based methods to trace the expression of cell surface proteins and/or cytokine production;28 however, antibodies have limited permeability and tissue accessibility as well as slow kinetics, which hamper real-time imaging of intact live cells and tissues. Therefore, there is a demand for alternative strategies using smaller fluorescent probes that can be used to phenotype the functional state of T cells in clinical samples in real time. Smart fluorescent probes can also be chemically fine-tuned to selectively image subsets of T cells with enhanced sensitivity. In this section, we will discuss the chemical design of fluorescent probes to image the migration, proliferation and metabolic reprogramming of T cells.The migration of T cells to tumor sites is crucial to achieve effective immune responses against cancer cells. In the complex tumor microenvironment (TME), specific cell–cell interactions and environmental factors define the proliferation, metabolism and function of T cells.2.1.i.e., VT680) bearing an amine reactive N-hydroxysuccinimide (NHS) group that could enter T cells and covalently bind to cellular components without leaking into adjacent cells.29 In experiments with HA-bearing tumor mouse models of adoptive transfer immunotherapy, VT680-labeled HA-specific cytotoxic T lymphocytes (CTLs) were injected into mice to monitor their biodistribution and accumulation in tumors and lymph nodes. The fluorescence signals of VT680-labeled T cells were quantified at single-cell level and for several days post injection. Similarly, Foster et al. reported the NIR IRDye800CW as another NHS-containing reactive fluorophore for monitoring the migration of CD3-activated human T cells in mice bearing subcutaneous tumors.30 In these experiments, tumors were modified to express the chemokine C–C Chemokine Ligand 5 (CCL5), which is a potent chemoattractant for immune cells. Increased fluorescence signals from IRDye800CW confirmed stronger infiltration of T cells at the tumor site when CCL5 was present in the TME when compared to control tumors. On the other hand, the authors also observed a rapid decrease in the signal from dividing cells, which limited its application for long-term imaging to assess proliferation or division indexes by fluorescence. This could be due to the negatively charged structure of IRDye800CW NHS, which favored the reaction with extracellular amino groups found on surface proteins, as shown by the fluorescence emission found around the cell membranes. In contrast, the cell permeable VT680 probe accumulated not only in the cell membrane but also in the cytoplasm and nucleus of T cells.Some of the first chemical designs for imaging T cell in inflamed tissues were based on non-specific, cell-permeable fluorophores. For example, the group of Weissleder prepared a far-red fluorescent probe  for up to 7 days, outperforming previous NIR dyes .Considering the unmet need for chemical tools to monitor the outcome of immunotherapies, more sophisticated and versatile constructs have been designed in the last few years. The group of Fu and Nie reported the agent NIR-797 isothiocyanate for both fluorescent and photoacoustic monitoring of T cells, allowing much deeper penetration depth (up to 9.5 mm) for long-term imaging.ine dyes .32 In a 2.2.et al. reported Forster resonance energy transfer (FRET) probes targeting conformational changes of lymphocyte function–associated antigen 1 (LFA-1), an important integrin for the communication between immune cells and other antigen-presenting cells  unit as a donor and a lipid acceptor probe. With this design, T cell activation led to the extension of LFA-1 conformation and unquenching of the FRET signal. Alternatively, FRET probes have been also exploited to monitor payload release after cellular uptake for the modulation of T cell activity. Wiemer et al. combined a coumarin-Dabcyl FRET pair with a phosphonate-cleavable moiety to release an agonist ligand of the transmembrane protein BTN3A1 upon metabolic activation of γδ T cells . In another example of phosphonate-mediated cleavage, Wiemer et al. reported a similar coumarin diphosphate analogue to release a prodrug upon internalization to stimulate the immune response of human Vg9Vd2 T cells.34 Again, K562 cells pre-loaded with the probe were able to trigger Vg9Vd2 T cell-mediated killing (EC50: 180 nM).Recent efforts on T cell imaging have been aimed at not only detecting the presence of T lymphocytes but also their functional state. Chigaev ng cells .33 These T cells .34 Notab35,36 In this context, Siska et al. reported a cystine-FITC probe to study the upregulation of the cystine transporter xCT (SLC7a11) in activated CD4+ and CD8+ human T cells.37 Although not very significant, higher cystine-FITC intracellular signals were detected in stimulated CD8+ T cells relative to CD4+ T cells, enabling to differentiate cystine uptake across different lymphocyte subpopulations. Fatty acids can also regulate the functional state of T cells in the TME. Whereas energy production on activated T cells is predominantly based on aerobic glycolysis, regulatory T cells (Tregs) rely on fatty acid oxidation pathways. Lesniak and co-workers reported quantum dot conjugates with BODIPY fluorophores and fatty acids to monitor the uptake of lipids in T cells in vivo.38 The authors found that fatty acids were predominantly taken up by CD4+ Foxp3+ Tregs when compared to effector T cell subsets in tumors, thus validating the role of fatty acid metabolism in Tregs. Nitric oxide (NO) is an important signaling intermediate in T cell activation known to regulate mitochondrial and cytoplasmic Ca2+ signals.39 In another example of cellular uptake, Vorob’ev et al. described the preparation of an acridone-based probe for sensing NO in human Jurkat T cells.40 In this case, the reaction between the smart probe and NO yielded a triazole derivative with increased fluorescence -due to blockage of photoinduced electron transfer (PeT)- and an hypsochromic shift in the emission wavelength . Metabolism-associated enzymes are also attractive targets for imaging T cells. Wang and colleagues reported the red-emissive cell-permeable DDPB probe with turn-on fluorescence response upon reaction with carboxylesterase-2 (CES2), which is associated with higher CD8+ T cell infiltration in the TME.41,42 The fluorescence activation mechanism relied on the enzymatic hydrolysis by CES2 to release a benzoyl group that suppressed the intramolecular charge transfer (ICT) quenching. The authors evaluated the smart probe in inactive and active T cells and tumor cells and showed that the fluorescence signals were higher in active T cells and correlated with the distribution of the enzyme.After activation, T cells experience multiple metabolic changes to allow survival, proliferation, and effector function. Some of these changes involve the modulation of signaling pathways and metabolite uptake. For instance, cysteine incorporation to regulate glutathione levels is known to be increased in activated T cells to remove the excess of reactive oxygen species (ROS).43 MTY was identified from a high-throughput screening approach, where immune cells from peripheral blood were incubated with a diversity-oriented fluorescence library. Interestingly, the same authors had previously reported a BODIPY probe bearing a long fatty acid chain to preferentially accommodate in the membrane of B cells over T cells due to higher membrane flexibility   for the isolation of CD8+ T cells with fatty acid oxidation-based metabolism by means of their distinct MMP  .44 In thinct MMP .45 The a46 Recently, Goun et al. reported a bioluminescent probe (BiNR) for in vivo monitoring of nicotinamide riboside uptake (47 BiNR employed a two-step process based on (1) the addition of a caged luciferin and (2) a subsequent Staudinger ligation with an azido-labeled metabolite to uncage the luciferin moiety. Using this approach, the bioluminescence emission after reaction with luciferase was directly proportional to the uptake of azido-modified metabolites. Notably, the authors observed that nicotinamide riboside uptake was significantly increased upon T cell activation, with 3-fold higher BiNR signals in activated human CD3+ T cells compared to metabolically less active naïve CD3+ T cells. Moreover, both helper CD4+ and cytotoxic CD8+ T cells exhibited 6-fold increase of nicotinamide riboside uptake on day 2-post activation relative to day 11, in line with a decreased stimulation effect. In another example, the group of Walsh and Skala48 used the fluorescence lifetime signals of the metabolic co-enzymes NAD(P)H and flavin adenine dinucleotide (FAD) as direct indicators of the redox and activation states of T cells. The authors used machine learning models to classify T cells according to their optical redox ratios H over NAD(P)H plus FAD) and found that active T cells displayed higher ratios than quiescent T cells.Nicotinamide adenine dinucleotide (NAD) is an important metabolic co-enzyme, with low levels of NAD being linked to different pathologies.e uptake .47 BiNR 3.e.g., CD28) and the mutually exclusive T cell surface receptors CD8 and CD4. T cell inhibitory type receptors targeted by immune checkpoint inhibitors are other key contributors in regulating the immune response. Importantly, the upregulation of such receptors during chronic antigen stimulation is a known factor associated with T cell exhaustion. This section reviews fluorescent smart probes targeting a variety of T cell receptors, including G protein-coupled receptors (GPCRs) and immune checkpoint proteins as well as the composition and dynamics of the cell membrane to monitor and modulate antitumor immunity.The interactions between T cells and other cells and signaling molecules that regulate T cell function is orchestrated by a plethora of cell-surface receptors . Thus, T3.1.49–52 In particular, a variety of GPCRs expressed in T cells have been found to play key roles in regulating migration, activation and differentiation.53,54 Thus, dynamic changes in the GPCR expression levels on T cells are tightly linked to their biological roles, exhibiting different expression patterns depending on the activation state.55 Among these, the chemokine receptor CXCR4 is expressed in most leukocytes  and its presence with its cognate ligand CXCL12 has also been found in metastatic tumors, regulating the infiltration of T cells. In early studies from Hatse et al., the authors used an AlexaFluor647 (AF647)-CXCL12 conjugate to study CXCR4 expression in different subpopulations of lymphocytes, showing high probe signals in both T and B cell subsets.56 In a different approach, Khan et al. described a copper(ii) complex of a rhodamine-azamacrocycle antagonist to bind CXCR4 in Jurkat T cells.57 Copper ions were reported to enhance binding by interaction with extracellular aspartate residues in CXCR4 followed by probe uptake into the cell cytoplasm.GPCRs are the largest family of cell surface proteins, and transduce signaling pathways related to cell homeostasis and function.58,59 In this context, Diaz et al.60 developed a nitrobenzoxadiazole (NBD) probe containing the CB2 inhibitor 6-methoxyisatin to target CB2 receptors in CD4+ T cells with a Ki of 387 nM and good selectivity over CB1 receptors. Interestingly, the NBD fluorophore was rationally embedded into the probe to fit in a lipophilic pocket within the CB2 receptor binding cavity. On a related project, Palomares et al. reported HU210-AlexaFluor488 as the first small molecule probe for CB1 receptors in immune cells.61 The probe was built by conjugation of the cannabinoid agonist HU210 and the AlexaFluor488 fluorophore through a 6-aminohexanoyl linker. The resulting conjugate exhibited high binding affinity for CB1 receptors over CB2 receptors, and allowed their quantification in B cells, T helper cells and cytotoxic T cells. Furthermore, the authors employed the probes to monitor the internalization of CB1 receptors in peripheral blood mononuclear cells (PBMCs) after stimulation with cannabinoid agonists.The cannabinoid receptors 1 and 2 (CB1 and CB2) are other GPCRs commonly found in immune cells. Both CB1 and CB2 play an important immunomodulatory role, regulating cytokine production and cell migration during inflammation.62,63 In this regard, the group of López-Rodríguez and Ortega-Gutiérrez described the preparation of a BODIPY probe for the visualization of serotonin 5-HT1A receptors in immune cells  or phorbol 12-myristate 13-acetate (PMA), whereas use of the anti-inflammatory drug dexamethasone led to reduced fluorescent signals.Serotonin receptors are GPCRs with key roles in both innate and adaptive immune responses.ne cells .61 The bvia their cognate chemokines CCL19 and CCL21.64,65 Recently, Legler and co-workers described an innovative method for the semi-automated production of fluorescent CCL19 and CCL21 recombinant chemokines bearing C-terminal S6-tags for site-specific labeling with fluorophores.66 The fluorescent chemokines  bound to CCR7-expressing cells, and CCL19-S6Dy649P1 was used to monitor receptor-mediated endocytosis in CD3+ T cell subpopulations isolated from PBMCs and splenocytes.The chemokine receptor CCR7 guides immune cell migration and homing to lymphoid organs 3.2.67 The activation of T lymphocytes is also linked to membrane rearrangements at both the T cell receptor and immunological synapse levels.68 In early studies of lipid membrane composition, the solvatochromic dye Laurdan naphthalene) was used to visualize the formation of lipid ordered domains in the vicinity of T cell receptor activation sites and immunological synapses.69 Given its lipophilic character, Laurdan inserted into the phospholipid membranes and displayed a shift in its emission wavelength when found in condensed membranes . In experiments with Jurkat T cells, a remarkable increase of condensed membrane was detected upon activation of T cells with anti-CD3–coated beads. This effect was further extended by co-stimulation of CD3 and CD28 receptors. Subsequent reports rendered C-Laurdan as a derivative with improved photostability and sensitivity.70 With this analogue, Parmryd et al. could analyze the differences between the order of the plasma membrane and internal membranes in Jurkat T cells.71The signaling of GPCRs is known to be mediated by lipid–receptor, protein–receptor and receptor–receptor interactions that direct their spatial organization and clustering within the cell membrane.72,73 In this context, Zhang et al.74 developed an aggregation-induced emission (AIE)-based probe bearing a cholesterol anchor and a tetraphenylethene (TPE) dye (COOH-TPE-PEG-Chol) for the detection of T cell activation in real-time  and acceptor (Atto 647N) fluorophores on each strand and cholesterol attached at the other end of each strand. This probe enabled monitoring of the dynamic changes of membrane order in Jurkat T cells during anti-CD3/anti-CD28-stimulated T cell receptor signaling or by after incubation with the lipid disrupting agent methyl-β-cyclodextrin. In another approach, the group of Gaus reported a FRET probe (CliF) to detect intermolecular associations of neighbouring proteins during T cell receptor clustering in real-time.78 CliF combined a single-chain FRET pair between the yellow fluorescent protein Venus with the red fluorescent protein mCherry, a membrane anchor and the cytoplasmic domain of the subunit CD3ζ. CliF also contained the light-sensitive photoreceptor cryptochrome 2, which oligomerizes upon blue light irradiation at 488 nm, to control self-association. The authors further used CliF in Jurkat T cells to observe differences in the remodeling of CD3 clustering densities depending on the T cell stimulation method. Furthermore, the CliF technology proved also compatible with FLIM, displaying distinct lifetimes before and after clustering .The plasma membrane is a dynamic structure where signaling events can be influenced by the interactions between protein and lipid structures.al range .77 The pe.g., LFA-1).79 Back in 2007, Kaizuka, Vale and colleagues80 provided a model to study the formation of immunological synapses in Jurkat T cells transfected with GFP-actin and a glass-supported lipid bilayer containing fluorescently labeled stimulatory molecules  as mimics of antigen-presenting cells. This model was used to study the segregation of T cell receptors and adhesion proteins at earliest stages of synapse formation. Mechanical forces are also implicated in the discrimination of antigen peptides on ligand-engaged T cell receptors during antigen recognition.81,82 The group of Schütz described a FRET probe bearing a TCR-reactive antibody fragment or a peptide-loaded MHC to quantify the forces associated with T cell receptor signaling at a molecular level  approach , cytotoxic T-lymphocyte antigen 4 (CTLA-4), T cell immunoglobulin and mucin-domain containing-3 (TIM-3)).85,86 In particular, the CTLA-4 signaling pathway is an important regulator of the functions of Tregs.87 Examples of CTLA-4-targeting probes include the detection of CTLA-4+ T cell subsets in peripheral blood and tumor tissues by means of fluorescent quantum dots coupled to CTLA-4-specific nanobodies (QDs-Nb36).88 Upon incubation with QDs-Nb36, polyhydroxyalkanoate-stimulated T cells displayed stronger fluorescence signals compared to untreated T cells. In another example, Kobayashi et al.89 reported an antibody-photoabsorber conjugate bearing the NIR IRDye700 fluorophore (anti-CTL-4-IRDye700) to study the local depletion of intratumoral CTLA-4 + Foxp3+ Tregs by means of photoimmunotherapy with NIR light. Blocking of CTLA4-axis led to an increase in activation and infiltration of CD8+ T cells within the TME. CD25 is a component of the IL-2 receptor known to mediate the activity of effector T cells.90 In another strategy, a CD25-targeted NIR photoimmunotherapy was reported to selectively deplete CD4 + CD25 + Foxp3+ Tregs within the TME, with concomitant activation of CD8+ T and NK cytotoxic effects.91 The authors observed >85% depletion of CD25+ Tregs using irradiation regimes that were compatible with in vivo experiments. Moreover, the depletion of intratumoral Tregs lasted for up to 4 days and this effect was extended to distant tumors. Despite these recent contributions, the use of fluorescent smart probes to target T cell exhaustion has been underexplored and will likely emerge in the coming years due to its significance in tumor immunology.The persistent exposure of infiltrating CD8+ T cells to tumor microenvironmental factors induces a dynamic differentiation process, so called exhaustion, characterized by the upregulation of cell membrane inhibitory receptors  makes them attractive scaffolds for the construction of activatable probes that can report the functional state of enzymes. Among the different families of enzymes found in lymphocytes, granzymes are serine proteases that are primarily produced and secreted by cytotoxic T cells and natural killer (NK) cells as part of the immune response to viral infections and cancer cells.92 Granzymes play a key role in programmed cell death by cleaving specific proteins in the target cells  and initiating a cascade of events that results in cell death.93 In view of this, granzyme-targeting smart fluorescent probes have been considered as an effective approach to detect and visualize T cell and NK cell activity under physiological conditions.Enzymes are among the most common biological targets of fluorescent smart probes. The chemical adaptability of enzyme substrates, which can range from small molecules to peptides or larger proteins, to different activation mechanisms , with granzyme B being the most targeted of all. Differential levels of granzyme B (GzmB) have been associated with multiple disorders, from cancer to infection or auto-immune diseases. Notably, GzmB was found to be overexpressed in biological fluids of patients affected by chronic inflammatory diseases, including synovial fluid in rheumatoid arthritis and broncho-alveolar lavage from asthma patients. GzmB is also known to play a role in regulating inflammation and the host immune response, therefore being a potential biomarker of disease progression and response to therapy.94There are 5 granzymes in humans  with a diphenyl phosphonate warhead led to highly specific inhibitors. These probes were exploited to gain insight on granzyme biology and helped in determining the roles of GzmA and GzmB in NK cell-mediated target cell lysis.97 In addition to their potential use as therapeutics, the discovery of selective GzmB inhibitors opened avenues to the molecular design of radiolabeled agents for PET imaging of T cell activity. In this case, the covalent binding of radiotracers to the active site results in preferential accumulation of the probe in tissues with high GzmB activity. Several examples of such inhibitor-based molecular probes have been described in immuno-oncology studies to predict response to checkpoint inhibitors in mouse preclinical models of cancer.98–100 For instance, Craik and colleagues designed a smart PET probe based on a restricted interaction peptide in which a radiolabeled antimicrobial peptide was connected to a caged sequence by means of a GzmB cleavable linker.101 Upon cleavage of the sequence mediated by GzmB, the radiolabeled antimicrobial peptide was liberated and able to accumulate in adjacent cell membranes. This smart probe was successfully used to monitor GzmB activity in three different syngeneic mouse models of cancer and also in a model of pulmonary inflammation.The proteolytic activity of granzymes prompted the discovery of peptide sequences that could be specifically recognised by the different enzyme subtypes. Seminal work in this field was reported by the groups of Nicholson and Craik, who investigated the substrate specificity of GzmA and GzmB to prepare peptide-based inhibitors.4.2.102 In this case, the contrast would be generated by the release of a unique optical fingerprint  after the smart probe would have reacted with the active form of the enzyme. Some of the first designs considered the construction of smart nanomaterials that incorporated GzmB-reactive sequences. For instance, Libby and co-workers reported a GzmB-targeting nanoprobe using a poly-lysine graft copolymer scaffold.103 Briefly, the authors modified the murine-specific sequence GIEFDSGGC with a Cy5.5 fluorophore and generated a self-quenched fluorogenic polymer that released bright emission after cleavage by the enzyme. Their nanoprobe displayed ∼4-fold fluorescence increase upon reaction with mouse GzmB and was used in vivo to image cytotoxic CD8+ T cells in the heart of mice undergoing lymphocyte-mediated myocarditis via fluorescence molecular tomography.In parallel, the discovery of selective sequences for GzmB also accelerated the design of smart probes that could generate a detectable optical readouts after enzymatic cleavage.104 In this case, the authors conjugated a NIR-labeled GzmB peptide substrate  to PEGylated iron oxide nanoparticles for efficient biodistribution and extended circulation. The smart nanoprobes were administered systemically in mouse models of skin graft rejection, where their accumulation in allograft tissues and subsequent cleavage by GzmB resulted in the release of the fluorescent peptide to the urine.A similar self-quenched nanoprobe was reported by the group of Kwong for non-invasive detection of acute transplant rejection, where recipient T cells secrete high levels of GzmB during the onset of rejection.105 The cleavage of the peptides led to reduced fluorescence and photoacoustic signals but unchanged signals from the semiconducting polymer, thus producing a direct ratiometric readout of GzmB activity.Self-quenching mechanisms can be also integrated into polymers that generate ratiometric signals in a GzmB-dependent manner. A good example of this approach was reported by the group of Pu, who exploited the dual fluorescence and photoacoustic properties of IRDye800CW to synthesize self-assembled semiconducting polymers containing NIR-labeled peptide substrates.et al. recently described smart nanoprobes where FRET-paired peptide substrates  were conjugated to a poly scaffold and subsequently coupled to anti-PD-L1.106 The inhibition of PD-1/PD-L1 induced T cell-mediated GzmB release and enabled real-time imaging of immunotherapy response in tumor-bearing mice, being able to distinguish between highly responsive and poorly responsive tumors.In addition to self-quenching nanomaterials, smart nanoprobes for monitoring GzmB activity can be also generated using alternative reporting mechanisms, such as FRET pairs. Kulkarni via the preparation of AND-gate or dual-locked probes, which require two or more sequential degrees of activation to produce detectable signals.107 The group of Liang built on this concept to prepare dual-quenched nanoparticles for imaging T cell activity in vivo.108 In this work, the authors designed a Cy5.5-labeled GzmB-reactive sequence with a 2-cyanobenzothiazole tag that could form a cyclic dimeric product and spontaneously self-assemble into silent GzmB-responsive nanoparticles where the fluorescence signals were quenched through (1) intermolecular hydrophobic interaction and (2) π–π stacking. The authors demonstrated that GzmB cleavage led to nanoparticle disassembly and turn-on fluorescence emission, allowing in vivo imaging of active GzmB in tumor-bearing mice. Interestingly, nanoprobe self-assembly can be also used as a chemical strategy to turn-on fluorescence emission. An excellent example was recently reported by the team of Rao, who designed G-SNAT as a GzmB-activatable self-assembly smart probe for the assessment of cytotoxic T lymphocyte-mediated cancer cell killing.109 G-SNAT incorporated a 2-cyanopyrimidine group in close proximity to a GzmB-responsive IEFD peptide so that intramolecular cyclization occurred after (1) GzmB cleavage and (2) reduction of the disulfide bond by intracellular glutathione  and IEPD (human) to a hemicyanine reporter  and norvaline (Nva) led to GzmB-selective probes with kcat/KM values in the 105 M−1 s−1 range. Furthermore, once optimal sequences were identified, the authors prepared diphenylphosphonate inhibitor-based probes -for cell lysate analysis- and FRET-based smart probes -for live cell microscopy- and employed them to image the localization and activity of GzmB in a range of NK cells.A critical aspect to optimize in GzmB-activatable probes is their selectivity over other enzymes that cleave after an aspartic acid residue, such as caspases. Kasperkiewicz 117 In the last years, the field of chemiluminescence imaging has been boosted by the seminal discovery of Shabat and co-workers with biocompatible 1,2-dioxetanes.118 These structures are modular and versatile scaffolds for the construction of smart probes, including enzyme-activatable probes.119 Together with Kitamura and Shabat, our group reported the first GzmB-activatable chemiluminescent probe.120 Briefly, the probe consisted of the reactive IEPD sequence conjugated to a 1,2-dioxetane chemiluminophore via a self-immolative linker , which were exploited to image the anti-cancer activity of human NK cells in co-cultures and in mouse xenografts. The group of Rao reported an analogous design to prepare GBLI-2 as the first bioluminescent smart probe for mouse GzmB detection.121 In this case, the authors conjugated the IEFD sequence to luciferin with the same p-aminobenzyl alcohol linker. GBLI-2 was used to predict the efficacy of immunotherapies by longitudinal imaging of CD8+ T cell activity in mouse models in vivo.Whereas most optical probes to study GzmB activity in T cells and NK cells are based on fluorescent structures, recent studies have reported the preparation of activatable chemi- and bioluminescent probes. Chemi- and bioluminophores are advantageous in that, unlike fluorophores, they do not require excitation with an external light source, which can result in lower background signals and increased turn-on ratios.e linker . Notably4.4.122 Although its biological function remains controversial, GzmA is reported to cause the death of the target cell by inducing reactive oxygen species (ROS) production and by activating different pro-apoptotic proteins compared to GzmB.123 Initial approaches to detect GzmA activity have exploited some of the molecular designs that had been previously successful for GzmB targeting. For instance, our group recently reported the first NIR fluorogenic probes for monitoring GzmA activity.124 This family of probes consisted of GzmA-reactive tetrapeptides  and a fluorogenic hemicyanine that turned on upon selective enzymatic cleavage. The probes displayed good selectivity over granzymes and proteases, as shown in experiments with tissue lysates from GzmA knock-out mice. The fast reactivity of the probes also enabled real-time imaging of antigen-driven recognition of mouse cancer cells by cytotoxic T lymphocytes where active GzmA and active GzmB were visualized simultaneously by combining the NIR GzmA probe and the previously reported green fluorescent H5 smart probe for GzmB. Future efforts in this area will involve the design of more reactive and selective chemical structures over other members of the tryptase-like family of proteases. To this end, the work by Kasperkiewicz and co-workers with the screening of novel peptide sequences to identify more selective GzmA substrates125 will open avenues to generate the next generation of fluorescent smart probes to study T cell function under physiological conditions.In addition to GzmB, recent efforts to generate T-cell targeted enzymatic smart probes have been focused on the protein GzmA. GzmA is another important member of the granzyme family present in the cytolytic granules from CTLs and NK cells.5.126 Despite several successful examples, anti-cancer immunotherapies do not benefit all patients and better ways to optimize regimes  and dosages are urgently needed. Given that the efficacy of anti-cancer immunotherapies is closely related to the infiltration of cytotoxic cells , multiple approaches to track their in vivo localization and function have been described.The advent of immunotherapy has revolutionized the treatment of cancer, providing a level of effectiveness that was rarely achievable with traditional chemotherapeutic agents. Nowadays, many first-line treatments of neoplastic diseases are based on immunotherapies with good clinical efficacy.in vivo imaging of therapeutic response is the use of transgenic mouse models containing optical reporter genes  in specific cells. The incorporation of such targeting vectors enables targeted imaging at multiple levels, from fluorescence microscopy cell-based assays for drug screens to intravital and whole-body imaging in intact organisms that correlate T cell location and therapy outcome. The main advantage of optical reporter genes is that their insertion at a specific locus result in selective targeting of subpopulations of T cells and the ability to longitudinally track them over time, helping to investigate the effect of anti-cancer immunotherapies. The group of Mezzanotte recently reported a summary of transgenic mouse models for fluorescence and bioluminescence imaging of T cells.127 These models range from BLITC mice for generic monitoring of T cell localization  to Foxp3 reporter mice to detect subsets of T cells, such as T regs. On the other hand, the main shortcoming of transgenic mouse models is their limited translatability to optical imaging in humans, which has encouraged the chemical development of molecular imaging agents that can similarly target subpopulations of immune cells. For instance, our group has demonstrated that the combination of small proteins  with pH or protease-activatable fluorophores128 can be used to target defined subpopulations of immune cells that could not be monitored with a single optical reporter.129One of the most conventional methods for 5.1.130 with radiolabeled PET probes being in a more advanced translational state, as recently reviewed by Whitney and Aarntzen.131 The molecular targets for T cells are relatively diverse, from generic surface receptors  to T cell activation markers  or more recently, metabolic markers . However, one shortcoming of PET radiotracers is their limited multiplexity, which has prompted the design of multicolour optical technologies for immunohistochemistry to examine T cell phenotypes with high spatial resolution. In the context of anti-cancer immunotherapies, Vasaturo and Galon have reported methods using fluorescence imaging of fixed tumor tissue biopsies with panels of antibody-based probes to quantify the expression and co-localization of multiple biomarkers  and their adaptation to image analysis and digital pathology.28 Antibodies  conjugated to bright NIR dyes such as IRDye800CW also hold promise as imaging agents forfluorescence-guided tumor resection during surgery.132 In the future, these platforms could be integrated into existing patient management routes and accelerate the stratification of patients and the selection of immune checkpoint inhibitors in a more personalized manner.Capturing the dynamic information rising from the interactions between cancer cells and T cells requires the selection of suitable biomarkers,e.g., PD-1) can be also chemically tuned to generate smart probes with enhanced selectivity and sensitivity. One excellent example was reported by the group of Kwong with the chemical design of anti-PD-1 based sensors to monitor response to immune checkpoint blockade therapy.133 In this work, the authors conjugated  protease substrates -for GzmB and others- to anti-PD-1 antibodies so that specific proteolysis readouts from responder mice (but not from treatment-resistant subjects) was obtained. The authors included mass barcodes into the substrates that allowed simultaneous measurements of mass spectrometry readouts in urine samples  to simultaneously monitor T cell location and T cell activation in mouse models of colorectal cancer. The chemical properties of these nanoparticles were optimized to enhance detection, including long emission wavelengths for deep tissue penetration , high signal-to-noise ratios , good biocompatibility via hydrophilic coating and long fluorescence lifetimes  for multiplexed imaging in the NIR-II window. In a follow-up work, the same group further optimized the cross-linked coating on the surface of quantum dots to maximise biocompatibility and excretion after intravenous administration.136 In this case, the authors coated quantum dots with amphiphilic polymeric layers containing branched PEG and linear polyacrylic acid and modified them with anti-CD8 diabodies for T cell targeting. The good biocompatibility of these particles was exploited for NIR imaging of cytotoxic T lymphocytes in response to anti-PD-L1 immunotherapy in mice using light sheet microscopy with very high signal-to-noise ratios  and cellular resolution.Antibodies have been also conjugated to nanoparticles with optimized properties for aximized .135 The 5.2.in vivo imaging of T cell activation.137 Briefly, the nanoconstructs passively accumulated into tumors in mice, and included a caged dioxetane for chemiluminescence resonance energy transfer. Using this approach, regions of low and high superoxide levels -in principle, related to weak and strong T cell activation states- could be discerned. For instance, in areas with low levels of superoxide, only fluorescence signals from the semiconducting polymer would be detected whereas high levels of superoxide decaged the dioxetane moiety, resulting in both bright chemiluminescence and fluorescence readouts. Given the tunability and versatility of phenoxydioxetane chemiluminophores, this molecular design could represent a modular approach to construct smart probes for monitoring T cell function in response to anti-cancer immunotherapies.Alternative nanoprobe designs for imaging response to immunotherapy can generate contrast based on activatable reporters rather than antibody-based accumulation. The groups of Pu and Zhang reported semiconducting polymer nanoreporters including chemiluminophores that were sensitive to superoxide anion for e.g., NK cells) are administered intravenously to kill tumors more effectively. The groups of Liu and Song recently reported a strategy to measure the viability of adoptive transferred in NK cells using ratiometric fluorescence imaging.138 The authors prepared lanthanide-based nanoparticles with inherent NIR-II emission that were coated with the ROS-sensitive NIR-I dye IR786 and used them to pre-label adoptively transferred NK cells  improved the in vivo survival of NK cells and led to better anti-cancer outcomes in a mouse model of hepatocellular carcinoma.ROS can be also used as biomarkers for cell viability, which is an essential parameter in adoptive cell transfer immunotherapies where cancer-attacking cells  to pre-label T lymphocytes before they were administered intravenously into tumor-bearing mice.139 Because the T lymphocytes were isolated from draining lymph nodes of 4T1-sensitized mice and activated in vitro, they specifically accumulated in 4T1 tumors grown in the recipient mice. The authors performed in vivo fluorescence imaging to detect NIR emission from the labeled T cells and found that tumor accumulation peaked at 6 days and signals being detectable for up to 3 weeks after injection. In order to overcome the limited penetration depth of NIR fluorophores, recent studies have also explored photoacoustic agents, which can provide signals up to 1 cm in depth, and used them to pre-label T cells 31 and monitor their location in tumor-bearing mice. In addition to increased depth penetration, another important feature of PA imaging is the possibility to acquire label-free anatomical information from tumors in vivo,140 which can provide further insights into the efficacy of anti-cancer immunotherapies. One recent example was reported by the group of Ntziachristos, who used a combination of PA imaging modalities to visualize solid tumors receiving adoptive T cell transfer therapy and were able to monitor optical absorption changes in the tumor mass associated with blood vessel collapse during tumor rejection.141The success of adoptive T cell transfer anti-cancer therapies heavily relies on the ability of T cells to target and retain within tumors. Smaller chemical constructs, such as NIR dyes, have found good use as labeling agents for tracing the 5.3.e.g., via fluorescence and computed tomography) or theranostic probes for simultaneous detection and therapeutic delivery.142 Regarding the latter, Yoon and co-workers described nanoparticles based on heavy-atom-free BODIPY fluorophores that remained non-photoactive until disassembled by interaction with albumin.143 The authors showed the application of these constructs for both in vivo tumor detection  and photoablation  alongside its synergistic combination with immune checkpoint inhibitors. Also, the group of Dai reported phototheranostic nanoprobes with capacity for NIR-II fluorescence and PA imaging as well as photothermal therapy.144 Of note, this design considered the encapsulation of drugs , which resulted in strong T cell reinvigoration and anti-cancer immunity.Finally, some of the latest molecular constructs for the examination T cell-based anti-cancer immunotherapies have considered more sophisticated chemical designs with capabilities beyond optical imaging. Some examples include nanoparticles for tracking of T cells using multimodal imaging  against tumors. In this article, we reviewed recent advances in the chemical design of smart probes to image T cells, including different molecular architectures and numerous biological targets and applications. Initial designs were focused on tracking the migration and proliferation of T cells to sites of action , whereas recent designs have shifted towards distinguishing between different T cell subpopulations and metabolic states. For instance, the stimulation of T cells leads to changes in the lipid composition and dynamics of the plasma membrane, which can be sensed by smart probes containing environmentally sensitive fluorophores. Other important biomarkers involve the secretion of proteolytic enzymes , which are key effector molecules in cytotoxic T cells and can be used for real-time monitoring of anti-cancer immunotherapy efficacy. In parallel to the design of smart probes for stimulated T cells, there is a need for new complementary probes that can detect T cell exhaustion, a process that influences the efficacy of anti-cancer treatments in the tumor microenvironment, or that combine multiple biomarkers in one single smart probe  to target subpopulations of T cells . The development of new activatable chemical moieties will accelerate the synthesis of the next generation of smart probes for T cells. The chemical diversity in this space is large, and ranges from fluorescent amino acids and biomarker-activatable fluorophores to NIR scaffolds with emission in the NIR-II window or compatibility with photoacoustic imaging. Given the biological complexity of tissue microenvironments  and the broad range of T cell subpopulations with variable functional readouts, we envisage that fluorescent reporters coupled to T cell specific biomolecules  will produce a new toolbox of smart probes for T cells to interrogate T cell biology in multiple disease contexts. Finally, the translation of T cell probes towards the clinic holds huge potential for improving the diagnosis of inflammatory diseases and the personalized optimization of anti-cancer immunotherapies. Examples of these applications may include the integration of smart probes into existing clinical imaging modalities to track the presence of T cells directly in humans145 as well as their adaptation for the analysis of biosamples  to obtain complementary readouts to those provided by already existing procedures .Anti-cancer immunotherapies, from immune checkpoint blockade antibodies to CAR T cells, are emerging strategies to reprogram the cytotoxic activity of the host immune system (The authors declare no conflicts of interest.