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[This corrects the article DOI: 10.3389/fimmu.2023.1043631.].
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Effective secondary response to antigen is a hallmark of immunological memory. However, the extent of memory CD8 T cell response to secondary boost varies at different times after a primary response. Considering the central role of memory CD8 T cells in long-lived protection against viral infections and tumors, a better understanding of the molecular mechanisms underlying the changing responsiveness of these cells to antigenic challenge would be beneficial. We examined here primed CD8 T cell response to boost in a BALB/c mouse model of intramuscular vaccination by priming with HIV-1 gag-encoding Chimpanzee adenovector, and boosting with HIV-1 gag-encoding Modified Vaccinia virus Ankara. We found that boost was more effective at day(d)100 than at d30 post-prime, as evaluated at d45 post-boost by multi-lymphoid organ assessment of gag-specific CD8 T cell frequency, CD62L-expression (as a guide to memory status) and in vivo killing. RNA-sequencing of splenic gag-primed CD8 T cells at d100 revealed a quiescent, but highly responsive signature, that trended toward a central memory (CD62L+) phenotype. Interestingly, gag-specific CD8 T cell frequency selectively diminished in the blood at d100, relative to the spleen, lymph nodes and bone marrow. These results open the possibility to modify prime/boost intervals to achieve an improved memory CD8 T cell secondary response.
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Linfócitos T CD8-Positivos , Imunização Secundária , Células de Memória Imunológica , Vacinas , Animais , Camundongos , Linfócitos T CD8-Positivos/imunologia , Divisão Celular , Camundongos Endogâmicos BALB C , Vacinação , Células de Memória Imunológica/imunologiaRESUMO
Here we consider how high-content flow cytometric methodology at appropriate scale and throughput rapidly provided meaningful biological data in our recent studies of COVID-19, which we discuss in the context of other similar investigations. In our work, high-throughput flow cytometry was instrumental to identify a consensus immune signature in COVID-19 patients, and to investigate the impact of SARS-CoV-2 exposure on patients with either solid or hematological cancers. We provide here some examples of our 'holistic' approach, in which flow cytometry data generated by lymphocyte and myelomonocyte panels were integrated with other analytical metrics, including SARS-CoV-2-specific serum antibody titers, plasma cytokine/chemokine levels, and in-depth clinical annotation. We report how selective differences between T cell subsets were revealed by a newly described flow cytometric TDS assay to distinguish actively cycling T cells in the peripheral blood. By such approaches, our and others' high-content flow cytometry studies collectively identified overt abnormalities and subtle but critical changes that discriminate the immuno-signature of COVID-19 patients from those of healthy donors and patients with non-COVID respiratory infections. Thereby, these studies offered several meaningful biomarkers of COVID-19 severity that have the potential to improve the management of patients and of hospital resources. In sum, flow cytometry provides an important means for rapidly obtaining data that can guide clinical decision-making without requiring highly expensive, sophisticated equipment, and/or "-omics" capabilities. We consider how this approach might be further developed.
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COVID-19 , Humanos , SARS-CoV-2 , Citometria de Fluxo , Citocinas , Subpopulações de Linfócitos TRESUMO
Facing the COVID-19 pandemic, anti-SARS-CoV-2 vaccines were developed at unprecedented pace, productively exploiting contemporary fundamental research and prior art. Large-scale use of anti-SARS-CoV-2 vaccines has greatly limited severe morbidity and mortality. Protection has been correlated with high serum titres of neutralizing antibodies capable of blocking the interaction between the viral surface protein spike and the host SARS-CoV-2 receptor, ACE-2. Yet, vaccine-induced protection subsides over time, and breakthrough infections are commonly observed, mostly reflecting the decay of neutralizing antibodies and the emergence of variant viruses with mutant spike proteins. Memory CD8 T cells are a potent weapon against viruses, as they are against tumour cells. Anti-SARS-CoV-2 memory CD8 T cells are induced by either natural infection or vaccination and can be potentially exploited against spike-mutated viruses. We offer here an overview of current research about the induction of anti-SARS-CoV-2 memory CD8 T cells by vaccination, in the context of prior knowledge on vaccines and on fundamental mechanisms of immunological memory. We focus particularly on how vaccination by two doses (prime/boost) or more (boosters) promotes differentiation of memory CD8 T cells, and on how the time-length of inter-dose intervals may influence the magnitude and persistence of CD8 T cell memory.
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COVID-19 , SARS-CoV-2 , Humanos , Pandemias , COVID-19/prevenção & controle , Linfócitos T CD8-Positivos , Vacinação , Anticorpos NeutralizantesRESUMO
Dendritic cells (DCs) are innate immune cells with a central role in immunity and tolerance. Under steady-state, DCs are scattered in tissues as resting cells. Upon infection or injury, DCs get activated and acquire the full capacity to prime antigen-specific CD4+ and CD8+ T cells, thus bridging innate and adaptive immunity. By secreting different sets of cytokines and chemokines, DCs orchestrate diverse types of immune responses, from a classical proinflammatory to an alternative pro-repair one. DCs are highly heterogeneous, and physiological differences in tissue microenvironments greatly contribute to variations in DC phenotype. Oxygen tension is normally low in some lymphoid areas, including bone marrow (BM) hematopoietic niches; nevertheless, the possible impact of tissue hypoxia on DC physiology has been poorly investigated. We assessed whether DCs are hypoxic in BM and spleen, by staining for hypoxia-inducible-factor-1α subunit (HIF-1α), the master regulator of hypoxia-induced response, and pimonidazole (PIM), a hypoxic marker, and by flow cytometric analysis. Indeed, we observed that mouse DCs have a hypoxic phenotype in spleen and BM, and showed some remarkable differences between DC subsets. Notably, DCs expressing membrane c-kit, the receptor for stem cell factor (SCF), had a higher PIM median fluorescence intensity (MFI) than c-kit- DCs, both in the spleen and in the BM. To determine whether SCF (a.k.a. kit ligand) has a role in DC hypoxia, we evaluated molecular pathways activated by SCF in c-kit+ BM-derived DCs cultured in hypoxic conditions. Gene expression microarrays and gene set enrichment analysis supported the hypothesis that SCF had an impact on hypoxia response and inhibited autophagy-related gene sets. Our results suggest that hypoxic response and autophagy, and their modulation by SCF, can play a role in DC homeostasis at the steady state, in agreement with our previous findings on SCF's role in DC survival.
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Linfócitos T CD8-Positivos , Fator de Células-Tronco , Animais , Autofagia , Hipóxia Celular , Células Cultivadas , Citocinas/metabolismo , Células Dendríticas , Hipóxia/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Oxigênio/metabolismo , Fator de Células-Tronco/metabolismoRESUMO
Remarkable progress has been made in the field of anti-tumor immunity, nevertheless many questions are still open. Thus, even though memory T cells have been implicated in long-term anti-tumor protection, particularly in prevention of cancer recurrence, the bases of their variable effectiveness in tumor patients are poorly understood. Two types of memory T cells have been described according to their traffic pathways: recirculating and tissue-resident memory T cells. Recirculating tumor-specific memory T cells are found in the cell infiltrate of solid tumors, in the lymph and in the peripheral blood, and they constantly migrate in and out of lymph nodes, spleen, and bone marrow. Tissue-resident tumor-specific memory T cells (TRM) permanently reside in the tumor, providing local protection. Anti-PD-1/PD-L1, a type of immune checkpoint blockade (ICB) therapy, can considerably re-invigorate T cell response and lead to successful tumor control, even in patients at advanced stages. Indeed, ICB has led to unprecedented successes against many types of cancers, starting a ground-breaking revolution in tumor therapy. Unfortunately, not all patients are responsive to such treatment, thus further improvements are urgently needed. The mechanisms underlying resistance to ICB are still largely unknown. A better knowledge of the dynamics of the immune response driven by the two types of memory T cells before and after anti-PD-1/PD-L1 would provide important insights on the variability of the outcomes. This would be instrumental to design new treatments to overcome resistance. Here we provide an overview of T cell contribution to immunity against solid tumors, focusing on memory T cells. We summarize recent evidence on the involvement of recirculating memory T cells and TRM in anti-PD-1/PD-L1-elicited antitumor immunity, outline the open questions in the field, and propose that a synergic action of the two types of memory T cells is required to achieve a full response. We argue that a T-centric vision focused on the specific roles and the possible interplay between TRM and recirculating memory T cells will lead to a better understanding of anti-PD-1/PD-L1 mechanism of action, and provide new tools for improving ICB therapeutic strategy.
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Inibidores de Checkpoint Imunológico/imunologia , Memória Imunológica/imunologia , Neoplasias/imunologia , Linfócitos T/imunologia , Animais , Humanos , Neoplasias/tratamento farmacológicoRESUMO
The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer-reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state-of-the-art handbook for basic and clinical researchers.
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Doenças Autoimunes/imunologia , Citometria de Fluxo , Infecções/imunologia , Neoplasias/imunologia , Animais , Doença Crônica , Humanos , Camundongos , Guias de Prática Clínica como AssuntoRESUMO
A multicolor flow cytometry panel was designed and optimized to define the following nine mouse T cell subsets: Treg (CD3+ CD4+ CD8- FoxP3+ ), CD4+ T naïve (CD3+ CD4+ CD8- FoxP3- CD44int/low CD62L+ ), CD4+ T central memory (CD3+ CD4+ CD8- FoxP3- CD44high CD62L+ ), CD4+ T effector memory (CD3+ CD4+ CD8- FoxP3- CD44high CD62L- ), CD4+ T EMRA (CD3+ CD4+ CD8- FoxP3- CD44int/low CD62L- ), CD8+ T naïve (CD3+ CD8+ CD4- CD44int/low CD62L+ ), CD8+ T central memory (CD3+ CD8+ CD4- CD44high CD62L+ ), CD8+ T effector memory (CD3+ CD8+ CD4- CD44high CD62L- ), and CD8+ T EMRA (CD3+ CD8+ CD4- CD44int/low CD62L- ). In each T cell subset, a dual staining for Ki-67 expression and DNA content was employed to distinguish the following cell cycle phases: G0 (Ki67- , with 2n DNA), G1 (Ki67+ , with 2n DNA), and S-G2 /M (Ki67+ , with 2n < DNA ≤ 4n). This panel was established for the analysis of mouse (C57BL/6J) spleen.
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Baço , Linfócitos T Reguladores , Animais , Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Ciclo Celular , Memória Imunológica , Selectina L , Células T de Memória , Camundongos , Camundongos Endogâmicos C57BL , Subpopulações de Linfócitos TRESUMO
[This corrects the article DOI: 10.3389/fimmu.2021.653974.].
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Drug-induced liver injury (DILI) is a challenging clinical event in medicine, particularly because of its ability to present with a variety of phenotypes including that of autoimmune hepatitis or other immune mediated liver injuries. Limited diagnostic and therapeutic tools are available, mostly because its pathogenesis has remained poorly understood for decades. The recent scientific and technological advancements in genomics and immunology are paving the way for a better understanding of the molecular aspects of DILI. This review provides an updated overview of the genetic predisposition and immunological mechanisms behind the pathogenesis of DILI and presents the state-of-the-art experimental models to study DILI at the pre-clinical level.
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Doença Hepática Induzida por Substâncias e Drogas/genética , Doença Hepática Induzida por Substâncias e Drogas/imunologia , Doença Hepática Induzida por Substâncias e Drogas/patologia , Predisposição Genética para Doença/genética , Hepatite Autoimune/imunologia , Humanos , Imunogenética/métodos , Fígado/patologia , Modelos Teóricos , Fenótipo , Fatores de RiscoRESUMO
BACKGROUND: The efficacy and safety profiles of vaccines against SARS-CoV-2 in patients with cancer is unknown. We aimed to assess the safety and immunogenicity of the BNT162b2 (Pfizer-BioNTech) vaccine in patients with cancer. METHODS: For this prospective observational study, we recruited patients with cancer and healthy controls (mostly health-care workers) from three London hospitals between Dec 8, 2020, and Feb 18, 2021. Participants who were vaccinated between Dec 8 and Dec 29, 2020, received two 30 µg doses of BNT162b2 administered intramuscularly 21 days apart; patients vaccinated after this date received only one 30 µg dose with a planned follow-up boost at 12 weeks. Blood samples were taken before vaccination and at 3 weeks and 5 weeks after the first vaccination. Where possible, serial nasopharyngeal real-time RT-PCR (rRT-PCR) swab tests were done every 10 days or in cases of symptomatic COVID-19. The coprimary endpoints were seroconversion to SARS-CoV-2 spike (S) protein in patients with cancer following the first vaccination with the BNT162b2 vaccine and the effect of vaccine boosting after 21 days on seroconversion. All participants with available data were included in the safety and immunogenicity analyses. Ongoing follow-up is underway for further blood sampling after the delayed (12-week) vaccine boost. This study is registered with the NHS Health Research Authority and Health and Care Research Wales (REC ID 20/HRA/2031). FINDINGS: 151 patients with cancer (95 patients with solid cancer and 56 patients with haematological cancer) and 54 healthy controls were enrolled. For this interim data analysis of the safety and immunogenicity of vaccinated patients with cancer, samples and data obtained up to March 19, 2021, were analysed. After exclusion of 17 patients who had been exposed to SARS-CoV-2 (detected by either antibody seroconversion or a positive rRT-PCR COVID-19 swab test) from the immunogenicity analysis, the proportion of positive anti-S IgG titres at approximately 21 days following a single vaccine inoculum across the three cohorts were 32 (94%; 95% CI 81-98) of 34 healthy controls; 21 (38%; 26-51) of 56 patients with solid cancer, and eight (18%; 10-32) of 44 patients with haematological cancer. 16 healthy controls, 25 patients with solid cancer, and six patients with haematological cancer received a second dose on day 21. Of the patients with available blood samples 2 weeks following a 21-day vaccine boost, and excluding 17 participants with evidence of previous natural SARS-CoV-2 exposure, 18 (95%; 95% CI 75-99) of 19 patients with solid cancer, 12 (100%; 76-100) of 12 healthy controls, and three (60%; 23-88) of five patients with haematological cancers were seropositive, compared with ten (30%; 17-47) of 33, 18 (86%; 65-95) of 21, and four (11%; 4-25) of 36, respectively, who did not receive a boost. The vaccine was well tolerated; no toxicities were reported in 75 (54%) of 140 patients with cancer following the first dose of BNT162b2, and in 22 (71%) of 31 patients with cancer following the second dose. Similarly, no toxicities were reported in 15 (38%) of 40 healthy controls after the first dose and in five (31%) of 16 after the second dose. Injection-site pain within 7 days following the first dose was the most commonly reported local reaction (23 [35%] of 65 patients with cancer; 12 [48%] of 25 healthy controls). No vaccine-related deaths were reported. INTERPRETATION: In patients with cancer, one dose of the BNT162b2 vaccine yields poor efficacy. Immunogenicity increased significantly in patients with solid cancer within 2 weeks of a vaccine boost at day 21 after the first dose. These data support prioritisation of patients with cancer for an early (day 21) second dose of the BNT162b2 vaccine. FUNDING: King's College London, Cancer Research UK, Wellcome Trust, Rosetrees Trust, and Francis Crick Institute.
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Vacinas contra COVID-19/uso terapêutico , COVID-19/imunologia , Neoplasias/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Antivirais/sangue , Vacina BNT162 , COVID-19/sangue , COVID-19/complicações , COVID-19/virologia , Vacinas contra COVID-19/imunologia , Relação Dose-Resposta Imunológica , Feminino , Humanos , Imunogenicidade da Vacina/imunologia , Londres/epidemiologia , Masculino , Pessoa de Meia-Idade , Neoplasias/sangue , Neoplasias/complicações , Neoplasias/virologia , Estudos Prospectivos , SARS-CoV-2 , País de GalesRESUMO
This study discusses substantive advances in T cell proliferation analysis, with the aim to provoke a re-evaluation of the generally-held view that Ki-67 is a reliable proliferation marker per se, and to offer a more sensitive and effective method for T cell cycle analysis, with informative examples in mouse and human settings. We summarize recent experimental work from our labs showing that, by Ki-67/DNA dual staining and refined flow cytometric methods, we were able to identify T cells in the S-G2/M phases of the cell-cycle in the peripheral blood (collectively termed "T Double S" for T cells in S-phase in Sanguine: in short "TDS" cells). Without our refinement, such cells may be excluded from conventional lymphocyte analyses. Specifically, we analyzed clonal expansion of antigen-specific CD8 T cells in vaccinated mice, and demonstrated the potential of TDS cells to reflect immune dynamics in human blood samples from healthy donors, and patients with type 1 diabetes, infectious mononucleosis, and COVID-19. The Ki-67/DNA dual staining, or TDS assay, provides a reliable approach by which human peripheral blood can be used to reflect the dynamics of human lymphocytes, rather than providing mere steady-state phenotypic snapshots. The method does not require highly sophisticated "-omics" capabilities, so it should be widely-applicable to health care in diverse settings. Furthermore, our results argue that the TDS assay can provide a window on immune dynamics in extra-lymphoid tissues, a long-sought potential of peripheral blood monitoring, for example in relation to organ-specific autoimmune diseases and infections, and cancer immunotherapy.
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Linfócitos T CD8-Positivos/imunologia , COVID-19/imunologia , Ciclo Celular/imunologia , Diabetes Mellitus Tipo 1/imunologia , Antígeno Ki-67/imunologia , Neoplasias/imunologia , SARS-CoV-2/imunologia , Animais , Linfócitos T CD8-Positivos/patologia , COVID-19/patologia , COVID-19/prevenção & controle , Vacinas contra COVID-19/imunologia , Vacinas contra COVID-19/uso terapêutico , Diabetes Mellitus Tipo 1/patologia , Diabetes Mellitus Tipo 1/terapia , Humanos , Camundongos , Neoplasias/patologia , Neoplasias/terapiaRESUMO
The cell cycle of antigen-specific T cells in vivo has been examined by using a few methods, all of which possess some limitations. Bromodeoxyuridine (BrdU) marks cells that are in or recently completed S-phase, and carboxyfluorescein succinimidyl ester (CFSE) detects daughter cells after division. However, these dyes do not allow identification of the cell cycle phase at the time of analysis. An alternative approach is to exploit Ki67, a marker that is highly expressed by cells in all phases of the cell cycle except the quiescent phase G0. Unfortunately, Ki67 does not allow further differentiation as it does not separate cells in S-phase that are committed to mitosis from those in G1 that can remain in this phase, proceed into cycling, or move into G0. Here, we describe a flow cytometric method for capturing a "snapshot" of T cells in different cell cycle phases in mouse secondary lymphoid organs. The method combines Ki67 and DNA staining with major histocompatibility complex (MHC)-peptide-multimer staining and an innovative gating strategy, allowing us to successfully differentiate between antigen-specific CD8 T cells in G0, in G1 and in S-G2/M phases of the cell cycle in the spleen and draining lymph nodes of mice after vaccination with viral vectors carrying the model antigen gag of human immunodeficiency virus (HIV)-1. Critical steps of the method were the choice of the DNA dye and the gating strategy to increase the assay sensitivity and to include highly activated/proliferating antigen-specific T cells that would have been missed by current criteria of analysis. The DNA dye, Hoechst 33342, enabled us to obtain a high-quality discrimination of the G0/G1 and G2/M DNA peaks, while preserving membrane and intracellular staining. The method has great potential to increase knowledge about T cell response in vivo and to improve immuno-monitoring analysis.
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Linfócitos T CD8-Positivos/imunologia , Ciclo Celular , DNA/metabolismo , Epitopos/imunologia , Citometria de Fluxo/métodos , Antígeno Ki-67/metabolismo , Vacinação , Animais , Células da Medula Óssea/citologia , Análise de Dados , Feminino , Humanos , Linfonodos/citologia , Camundongos Endogâmicos BALB C , Baço/citologia , Coloração e RotulagemRESUMO
Immune checkpoints are inhibitory receptor/ligand pairs regulating immunity that are exploited as key targets of anti-cancer therapy. Although the PD-1/PD-L1 pair is one of the most studied immune checkpoints, several aspects of its biology remain to be clarified. It has been established that PD-1 is an inhibitory receptor up-regulated by activated T, B, and NK lymphocytes and that its ligand PD-L1 mediates a negative feedback of lymphocyte activation, contributing to the restoration of the steady state condition after acute immune responses. This loop might become detrimental in the presence of either a chronic infection or a growing tumor. PD-L1 expression in tumors is currently used as a biomarker to orient therapeutic decisions; nevertheless, our knowledge about the regulation of PD-L1 expression is limited. The present review discusses how NF-κB, a master transcription factor of inflammation and immunity, is emerging as a key positive regulator of PD-L1 expression in cancer. NF-κB directly induces PD-L1 gene transcription by binding to its promoter, and it can also regulate PD-L1 post-transcriptionally through indirect pathways. These processes, which under conditions of cellular stress and acute inflammation drive tissue homeostasis and promote tissue healing, are largely dysregulated in tumors. Up-regulation of PD-L1 in cancer cells is controlled via NF-κB downstream of several signals, including oncogene- and stress-induced pathways, inflammatory cytokines, and chemotherapeutic drugs. Notably, a shared signaling pathway in epithelial cancers induces both PD-L1 expression and epithelial-mesenchymal transition, suggesting that PD-L1 is part of the tissue remodeling program. Furthermore, PD-L1 expression by tumor infiltrating myeloid cells can contribute to the immune suppressive features of the tumor environment. A better understanding of the interplay between NF-κB signaling and PD-L1 expression is highly relevant to cancer biology and therapy.
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Antígeno B7-H1/imunologia , NF-kappa B/imunologia , Neoplasias/imunologia , Transição Epitelial-Mesenquimal/imunologia , Regulação Neoplásica da Expressão Gênica/imunologia , Humanos , Imunidade/imunologia , Inflamação/imunologia , Transdução de Sinais/imunologiaRESUMO
A Correction to this paper has been published: https://doi.org/10.1038/s41591-020-01186-5.
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An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Improved understanding and management of COVID-19, a potentially life-threatening disease, could greatly reduce the threat posed by its etiologic agent, SARS-CoV-2. Toward this end, we have identified a core peripheral blood immune signature across 63 hospital-treated patients with COVID-19 who were otherwise highly heterogeneous. The signature includes discrete changes in B and myelomonocytic cell composition, profoundly altered T cell phenotypes, selective cytokine/chemokine upregulation and SARS-CoV-2-specific antibodies. Some signature traits identify links with other settings of immunoprotection and immunopathology; others, including basophil and plasmacytoid dendritic cell depletion, correlate strongly with disease severity; while a third set of traits, including a triad of IP-10, interleukin-10 and interleukin-6, anticipate subsequent clinical progression. Hence, contingent upon independent validation in other COVID-19 cohorts, individual traits within this signature may collectively and individually guide treatment options; offer insights into COVID-19 pathogenesis; and aid early, risk-based patient stratification that is particularly beneficial in phasic diseases such as COVID-19.
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Anticorpos Antivirais/imunologia , Linfócitos B/imunologia , Infecções por Coronavirus/imunologia , Citocinas/imunologia , Células Dendríticas/imunologia , Pneumonia Viral/imunologia , Linfócitos T/imunologia , Idoso , Subpopulações de Linfócitos B/imunologia , Basófilos/imunologia , Betacoronavirus , COVID-19 , Estudos de Casos e Controles , Ciclo Celular , Quimiocina CXCL10/imunologia , Quimiocinas/imunologia , Estudos de Coortes , Infecções por Coronavirus/sangue , Progressão da Doença , Feminino , Citometria de Fluxo , Hospitalização , Humanos , Memória Imunológica , Imunofenotipagem , Interleucina-10/imunologia , Interleucina-6/imunologia , Contagem de Leucócitos , Ativação Linfocitária/imunologia , Masculino , Pessoa de Meia-Idade , Pandemias , Pneumonia Viral/sangue , Prognóstico , SARS-CoV-2 , Índice de Gravidade de Doença , Subpopulações de Linfócitos T/imunologia , Regulação para CimaRESUMO
The ready availability of human blood makes it the first choice for immuno-monitoring. However, this has been largely confined to static metrics, particularly resting T cell phenotypes. Conversely, dynamic assessments have mostly relied on cell stimulation in vitro which is subject to multiple variables. Here, immunodynamic insights from the peripheral blood are shown to be obtainable by applying a revised approach to cell-cycle analysis. Specifically, refined flow cytometric protocols were employed, assuring the reliable quantification of T cells in the S-G2/M phases of the cell-cycle (collectively termed "T Double S" for T cells in S-phase in Sanguine: in short "TDS" cells). Without protocol refinement, TDS could be either missed, as most of them layed out of the conventional lymphocyte gates, or confused with cell doublets artefactually displaying high DNA-content. To illustrate the nature of TDS cells, and their relationship to different immunodynamic scenarios, we examined them in healthy donors (HD); infectious mononucleosis (IM) patients versus asymptomatic EBV+ carriers; and recently-diagnosed T1D patients. TDS were reproducibly more abundant among CD8+ T cells and a defined subset of T-regulatory CD4+ T cells, and were substantially increased in IM and a subset of T1D patients. Of note, islet antigen-reactive TDS cell frequencies were associated with an aggressive T cell effector phenotype, suggesting that peripheral blood can reflect immune events within tissues in T1D, and possibly in other organ-specific autoimmune diseases. Our results suggest that tracking TDS cells may provide a widely applicable means of gaining insight into ongoing immune response dynamics in a variety of settings, including tissue immunopathologies where the peripheral blood has often not been considered insightful.