RESUMEN
In response to excessive DNA damage, human cells can activate p53 to induce apoptosis. Cells lacking p53 can still undergo apoptosis upon DNA damage, yet the responsible pathways are unknown. We observed that p53-independent apoptosis in response to DNA damage coincided with translation inhibition, which was characterized by ribosome stalling on rare leucine-encoding UUA codons and globally curtailed translation initiation. A genetic screen identified the transfer RNAse SLFN11 and the kinase GCN2 as factors required for UUA stalling and global translation inhibition, respectively. Stalled ribosomes activated a ribotoxic stress signal conveyed by the ribosome sensor ZAKα to the apoptosis machinery. These results provide an explanation for the frequent inactivation of SLFN11 in chemotherapy-unresponsive tumors and highlight ribosome stalling as a signaling event affecting cell fate in response to DNA damage.
Asunto(s)
Apoptosis , Daño del ADN , Biosíntesis de Proteínas , Ribosomas , Proteína p53 Supresora de Tumor , Humanos , Línea Celular Tumoral , Codón/genética , Leucina/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Ribosomas/metabolismo , Transducción de Señal , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/genética , Quinasas Quinasa Quinasa PAM/genética , Quinasas Quinasa Quinasa PAM/metabolismoRESUMEN
Tumor-specific CD8+ T cells are key effectors of antitumor immunity but are often rendered dysfunctional in the tumor microenvironment. Immune-checkpoint blockade can restore antitumor T-cell function in some patients; however, most do not respond to this therapy, often despite T-cell infiltration in their tumors. We here explored a CD8-targeted IL2 fusion molecule (CD8-IL2) to selectively reactivate intratumoral CD8+ T cells in patient-derived tumor fragments. Treatment with CD8-IL2 broadly armed intratumoral CD8+ T cells with enhanced effector capacity, thereby specifically enabling reinvigoration of the dysfunctional T-cell pool to elicit potent immune activity. Notably, the revival of dysfunctional T cells to mediate effector activity by CD8-IL2 depended on simultaneous antigen recognition and was quantitatively and qualitatively superior to that achieved by PD-1 blockade. Finally, CD8-IL2 was able to functionally reinvigorate T cells in tumors resistant to anti-PD-1, underscoring its potential as a novel treatment strategy for patients with cancer. Significance: Reinvigorating T cells is crucial for response to checkpoint blockade therapy. However, emerging evidence suggests that the PD-1/PD-L1 axis is not the sole impediment for activating T cells within tumors. Selectively targeting cytokines toward specific T-cell subsets might overcome these barriers and stimulate T cells within resistant tumors. See related article by Moynihan et al., p. 1206 (32).
Asunto(s)
Linfocitos T CD8-positivos , Interleucina-2 , Neoplasias , Humanos , Linfocitos T CD8-positivos/inmunología , Neoplasias/inmunología , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Microambiente Tumoral/inmunología , Ratones , Animales , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Inhibidores de Puntos de Control Inmunológico/farmacología , Antígenos CD8/metabolismoRESUMEN
IL2 signals pleiotropically on diverse cell types, some of which contribute to therapeutic activity against tumors, whereas others drive undesired activity, such as immunosuppression or toxicity. We explored the theory that targeting of IL2 to CD8+ T cells, which are key antitumor effectors, could enhance its therapeutic index. To this aim, we developed AB248, a CD8 cis-targeted IL2 that demonstrates over 500-fold preference for CD8+ T cells over natural killer and regulatory T cells (Tregs), which may contribute to toxicity and immunosuppression, respectively. AB248 recapitulated IL2's effects on CD8+ T cells in vitro and induced selective expansion of CD8+T cells in primates. In mice, an AB248 surrogate demonstrated superior antitumor activity and enhanced tolerability as compared with an untargeted IL2Rßγ agonist. Efficacy was associated with the expansion and phenotypic enhancement of tumor-infiltrating CD8+ T cells, including the emergence of a "better effector" population. These data support the potential utility of AB248 in clinical settings. Significance: The full potential of IL2 therapy remains to be unlocked. We demonstrate that toxicity can be decoupled from antitumor activity in preclinical models by limiting IL2 signaling to CD8+ T cells, supporting the development of CD8+ T cell-selective IL2 for the treatment of cancer. See related article by Kaptein et al. p. 1226.
Asunto(s)
Linfocitos T CD8-positivos , Interleucina-2 , Animales , Linfocitos T CD8-positivos/inmunología , Interleucina-2/farmacología , Ratones , Humanos , Línea Celular Tumoral , Ensayos Antitumor por Modelo de Xenoinjerto , Femenino , Neoplasias/inmunología , Neoplasias/tratamiento farmacológicoRESUMEN
The dysregulated expression of immune checkpoint molecules enables cancer cells to evade immune destruction. While blockade of inhibitory immune checkpoints like PD-L1 forms the basis of current cancer immunotherapies, a deficiency in costimulatory signals can render these therapies futile. CD58, a costimulatory ligand, plays a crucial role in antitumor immune responses, but the mechanisms controlling its expression remain unclear. Using two systematic approaches, we reveal that CMTM6 positively regulates CD58 expression. Notably, CMTM6 interacts with both CD58 and PD-L1, maintaining the expression of these two immune checkpoint ligands with opposing functions. Functionally, the presence of CMTM6 and CD58 on tumor cells significantly affects T cell-tumor interactions and response to PD-L1-PD-1 blockade. Collectively, these findings provide fundamental insights into CD58 regulation, uncover a shared regulator of stimulatory and inhibitory immune checkpoints, and highlight the importance of tumor-intrinsic CMTM6 and CD58 expression in antitumor immune responses.
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Antígeno B7-H1 , Proteínas con Dominio MARVEL , Proteínas de la Mielina , Neoplasias , Linfocitos T , Humanos , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Inmunidad , Inmunoterapia , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , Linfocitos T/inmunología , Proteínas de la Mielina/metabolismo , Proteínas con Dominio MARVEL/metabolismoRESUMEN
To dissect the effect of neoadjuvant PD-1 and CTLA4 blockade on intratumoral T cells in treatment-naive head and neck squamous cell carcinoma, we analyzed primary tumor immune infiltrates from responding and nonresponding patients. At baseline, a higher ratio between active (4-1BB/OX40+) and inactive regulatory CD4+ T cells was associated with immunotherapy response. Furthermore, upon therapy, this active regulatory T-cell (Treg) population showed a profound decrease in responding patients. In an analogous process, intratumoral dysfunctional CD8+ T cells displayed decreased expression of activity and dysfunction-related genes in responding patients, whereas in clinical nonresponders, natural killer cells showed an increased cytotoxic profile early upon treatment. These data reveal immunologic changes in response to dual PD-1/CTLA4 blockade, including a parallel remodeling of presumed tumor-reactive Treg and CD8+ T-cell compartments in responding patients, and indicate that the presence of activated Tregs at baseline may be associated with response. SIGNIFICANCE: In head and neck squamous cell carcinoma, neoadjuvant PD-1/CTLA4 blockade has shown substantial response rates (20%-35%). As recognition of tumor antigens by T cells appears to be a critical driver of therapy response, a better understanding of alterations in T-cell state that are associated with response and resistance is of importance. This article is featured in Selected Articles from This Issue, p. 2109.
RESUMEN
Tumor-specific cytotoxic T cells unleashed by the blockade of immune checkpoints have to overcome a hostile tumor microenvironment (TME). They start from very small numbers of T cells with tumor antigen specificity and, despite expansion, likely remain at a numerical disadvantage to the tumor cells they target. To overcome these obstacles, we propose that T cells need to change the TME to make it permissive for their antitumor effects by altering the phenotype of cells beyond the cancer cells they are in physical contact with. In this process, IFNγ secreted by tumor-specific T cells plays a critical role, as it changes the expression of hundreds of genes in cancer cells and other immune cells in the TME up to 40 layers of cells away from their location, effectively turning these cells into enablers of the antitumor immune response. In this perspective, we postulate that the clinical activity of cancer immunotherapy with immune-checkpoint blocking antibodies and adoptively transferred T cells requires that cancer cells facilitate the antitumor immune response. IFNγ effectively changes the balance of power in the TME to enable the antitumor activity of tumor antigen-specific cytotoxic T cells.
Asunto(s)
Neoplasias , Microambiente Tumoral , Anticuerpos Bloqueadores , Antígenos de Neoplasias , Humanos , Inmunidad , Inmunoterapia , Neoplasias/genética , Neoplasias/terapiaRESUMEN
Resident memory CD8+ T (Trm) cells permanently reside in nonlymphoid tissues where they act as a first line of defense against recurrent pathogens. How and when antigen-inexperienced CD8+ T cells differentiate into Trm has been a topic of major interest, as knowledge on how to steer this process may be exploited in the development of vaccines and anticancer therapies. Here, we first review the current understanding of the early signals that CD8+ T cells receive before they have entered the tissue and that govern their capacity to develop into tissue-resident memory T cells. Subsequently, we discuss the tissue-derived factors that promote Trm maturation in situ. Combined, these data sketch a model in which a subset of responding T cells develops a heightened capacity to respond to local cues present in the tissue microenvironment, which thereby imprints their ability to contribute to the tissue-resident memory CD8+ T-cell pool that provide local control against pathogens.
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Linfocitos T CD8-positivos/fisiología , Memoria Inmunológica , Células T de Memoria/fisiología , Animales , Diferenciación Celular , HumanosRESUMEN
BACKGROUND: The profound disparity in response to immune checkpoint blockade (ICB) by cutaneous melanoma (CM) and uveal melanoma (UM) patients is not well understood. Therefore, we characterized metastases of CM and UM from the same metastatic site (liver), in order to dissect the potential underlying mechanism in differential response on ICB. METHODS: Tumor liver samples from CM (n=38) and UM (n=28) patients were analyzed at the genomic (whole exome sequencing), transcriptional (RNA sequencing) and protein (immunohistochemistry and GeoMx Digital Spatial Profiling) level. RESULTS: Comparison of CM and UM metastases from the same metastatic site revealed that, although originating from the same melanocyte lineage, CM and UM differed in somatic mutation profile, copy number profile, tumor mutational burden (TMB) and consequently predicted neoantigens. A higher melanin content and higher expression of the melanoma differentiation antigen MelanA was observed in liver metastases of UM patients. No difference in B2M and human leukocyte antigen-DR (HLA-DR) expression was observed. A higher expression of programmed cell death ligand 1 (PD-L1) was found in CM compared with UM liver metastases, although the majority of CM and UM liver metastases lacked PD-L1 expression. There was no difference in the extent of immune infiltration observed between CM and UM metastases, with the exception of a higher expression of CD163 (p<0.0001) in CM liver samples. While the extent of immune infiltration was similar for CM and UM metastases, the ratio of exhausted CD8 T cells to cytotoxic T cells, to total CD8 T cells and to Th1 cells, was significantly higher in UM metastases. CONCLUSIONS: While TMB was different between CM and UM metastases, tumor immune infiltration was similar. The greater dependency on PD-L1 as an immune checkpoint in CM and the identification of higher exhaustion ratios in UM may both serve as explanations for the difference in response to ICB. Consequently, in order to improve current treatment for metastatic UM, reversal of T cell exhaustion beyond programmed cell death 1 blockade should be considered.
Asunto(s)
Melanoma/complicaciones , Neoplasias Cutáneas/complicaciones , Neoplasias de la Úvea/complicaciones , Femenino , Humanos , Neoplasias Hepáticas/secundario , Masculino , Melanoma/patología , Estudios Retrospectivos , Neoplasias Cutáneas/patología , Neoplasias de la Úvea/patología , Melanoma Cutáneo MalignoRESUMEN
T cell-secreted IFNγ can exert pleiotropic effects on tumor cells that include induction of immune checkpoints and antigen presentation machinery components, and inhibition of cell growth. Despite its role as key effector molecule, little is known about the spatiotemporal spreading of IFNγ secreted by activated CD8+ T cells within the tumor environment. Using multiday intravital imaging, we demonstrate that T cell recognition of a minor fraction of tumor cells leads to sensing of IFNγ by a large part of the tumor mass. Furthermore, imaging of tumors in which antigen-positive and -negative tumor cells are separated in space reveals spreading of the IFNγ response, reaching distances of >800 µm. Notably, long-range sensing of IFNγ can modify tumor behavior, as both shown by induction of PD-L1 expression and inhibition of tumor growth. Collectively, these data reveal how, through IFNγ, CD8+ T cells modulate the behavior of remote tumor cells, including antigen-loss variants.
Asunto(s)
Linfocitos T CD8-positivosRESUMEN
Cytotoxic CD8+ T cells can effectively kill target cells by producing cytokines, chemokines, and granzymes. Expression of these effector molecules is however highly divergent, and tools that identify and preselect CD8+ T cells with a cytotoxic expression profile are lacking. Human CD8+ T cells can be divided into IFN-γ- and IL-2-producing cells. Unbiased transcriptomics and proteomics analysis on cytokine-producing fixed CD8+ T cells revealed that IL-2+ cells produce helper cytokines, and that IFN-γ+ cells produce cytotoxic molecules. IFN-γ+ T cells expressed the surface marker CD29 already prior to stimulation. CD29 also marked T cells with cytotoxic gene expression from different tissues in single-cell RNA-sequencing data. Notably, CD29+ T cells maintained the cytotoxic phenotype during cell culture, suggesting a stable phenotype. Preselecting CD29-expressing MART1 TCR-engineered T cells potentiated the killing of target cells. We therefore propose that CD29 expression can help evaluate and select for potent therapeutic T cell products.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Citotoxicidad Inmunológica/inmunología , Integrina beta1/metabolismo , Interferón gamma/metabolismo , Interleucina-2/metabolismo , Melanoma/patología , Linfocitos T Citotóxicos/inmunología , Perfilación de la Expresión Génica , Humanos , Melanoma/inmunología , Melanoma/metabolismo , Pronóstico , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Tasa de SupervivenciaRESUMEN
The promiscuous nature of T-cell receptors (TCRs) allows T cells to recognize a large variety of pathogens, but makes it challenging to understand and control T-cell recognition. Existing technologies provide limited information about the key requirements for T-cell recognition and the ability of TCRs to cross-recognize structurally related elements. Here we present a 'one-pot' strategy for determining the interactions that govern TCR recognition of peptide-major histocompatibility complex (pMHC). We measured the relative affinities of TCRs to libraries of barcoded peptide-MHC variants and applied this knowledge to understand the recognition motif, here termed the TCR fingerprint. The TCR fingerprints of 16 different TCRs were identified and used to predict and validate cross-recognized peptides from the human proteome. The identified fingerprints differed among TCRs recognizing the same epitope, demonstrating the value of this strategy for understanding T-cell interactions and assessing potential cross-recognition before selection of TCRs for clinical development.
RESUMEN
The clinical benefit for patients with diverse types of metastatic cancers that has been observed upon blockade of the interaction between PD-1 and PD-L1 has highlighted the importance of this inhibitory axis in the suppression of tumour-specific T-cell responses. Notwithstanding the key role of PD-L1 expression by cells within the tumour micro-environment, our understanding of the regulation of the PD-L1 protein is limited. Here we identify, using a haploid genetic screen, CMTM6, a type-3 transmembrane protein of previously unknown function, as a regulator of the PD-L1 protein. Interference with CMTM6 expression results in impaired PD-L1 protein expression in all human tumour cell types tested and in primary human dendritic cells. Furthermore, through both a haploid genetic modifier screen in CMTM6-deficient cells and genetic complementation experiments, we demonstrate that this function is shared by its closest family member, CMTM4, but not by any of the other CMTM members tested. Notably, CMTM6 increases the PD-L1 protein pool without affecting PD-L1 (also known as CD274) transcription levels. Rather, we demonstrate that CMTM6 is present at the cell surface, associates with the PD-L1 protein, reduces its ubiquitination and increases PD-L1 protein half-life. Consistent with its role in PD-L1 protein regulation, CMTM6 enhances the ability of PD-L1-expressing tumour cells to inhibit T cells. Collectively, our data reveal that PD-L1 relies on CMTM6/4 to efficiently carry out its inhibitory function, and suggest potential new avenues to block this pathway.
Asunto(s)
Antígeno B7-H1/metabolismo , Proteínas con Dominio MARVEL/metabolismo , Antígeno B7-H1/biosíntesis , Antígeno B7-H1/química , Sistemas CRISPR-Cas , Línea Celular Tumoral , Células Dendríticas/metabolismo , Prueba de Complementación Genética , Haploidia , Humanos , Proteínas con Dominio MARVEL/genética , Melanoma/genética , Melanoma/metabolismo , Unión Proteica , Estabilidad Proteica , UbiquitinaciónRESUMEN
BACKGROUND: Usual type vulvar intraepithelial neoplasia (uVIN) is caused by HPV, predominantly type 16. Several forms of HPV immunotherapy have been studied, however, clinical results could be improved. A novel intradermal administration route, termed DNA tattooing, is superior in animal models, and was tested for the first time in humans with a HPV16 E7 DNA vaccine (TTFC-E7SH). METHODS: The trial was designed to test safety, immunogenicity, and clinical response of TTFC-E7SH in twelve HPV16+ uVIN patients. Patients received six vaccinations via DNA tattooing. The first six patients received 0.2 mg TTFC-E7SH and the next six 2 mg TTFC-E7SH. Vaccine-specific T-cell immunity was evaluated by IFNγ-ELISPOT and multiparametric flow cytometry. RESULTS: Only grade I-II adverse events were observed upon TTFC-E7SH vaccination. The ELISPOT analysis showed in 4/12 patients a response to the peptide pool containing shuffled E7 peptides. Multiparametric flow cytometry showed low CD4+ and/or CD8+ T-cell responses as measured by increased expression of PD-1 (4/12 in both), CTLA-4 (2/12 and 3/12), CD107a (5/12 and 4/12), or the production of IFNγ (2/12 and 1/12), IL-2 (3/12 and 4/12), TNFα (2/12 and 1/12), and MIP1ß (3/12 and 6/12). At 3 months follow-up, no clinical response was observed in any of the twelve vaccinated patients. CONCLUSION: DNA tattoo vaccination was shown to be safe. A low vaccine-induced immune response and no clinical response were observed in uVIN patients after TTFC-E7SH DNA tattoo vaccination. Therefore, a new phase I/II trial with an improved DNA vaccine format is currently in development for patients with uVIN.
Asunto(s)
ADN/genética , Papillomavirus Humano 16/genética , Proteínas Oncogénicas Virales/inmunología , Vacunas de ADN/inmunología , Neoplasias de la Vulva/genética , Adulto , Femenino , Humanos , Persona de Mediana Edad , Neoplasias de la Vulva/terapiaRESUMEN
Adoptive T cell therapy (ATT) can achieve regression of large tumors in mice and humans; however, tumors frequently recur. High target peptide-major histocompatibility complex-I (pMHC) affinity and T cell receptor (TCR)-pMHC affinity are thought to be critical to preventing relapse. Here, we show that targeting two epitopes of the same antigen in the same cancer cells via monospecific T cells, which have similar pMHC and pMHC-TCR affinity, results in eradication of large, established tumors when targeting the apparently subdominant but not the dominant epitope. Only the escape but not the rejection epitope required postproteasomal trimming, which was regulated by IFN-γ, allowing IFN-γ-unresponsive cancer variants to evade. The data describe a novel immune escape mechanism and better define suitable target epitopes for ATT.
Asunto(s)
Epítopos de Linfocito T/inmunología , Complejo de la Endopetidasa Proteasomal/metabolismo , Escape del Tumor/inmunología , Secuencia de Aminoácidos , Animales , Afinidad de Anticuerpos , Antígenos/inmunología , Epítopos de Linfocito T/química , Antígenos de Histocompatibilidad Clase I/inmunología , Interferón gamma/metabolismo , Leucil Aminopeptidasa/metabolismo , Ratones Endogámicos C57BL , Neoplasias/inmunología , Neoplasias/patología , Péptidos/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Transducción de Señal , Linfocitos T/inmunologíaRESUMEN
BACKGROUND: Approximately 75% of objective responses to anti-programmed death 1 (PD-1) therapy in patients with melanoma are durable, lasting for years, but delayed relapses have been noted long after initial objective tumor regression despite continuous therapy. Mechanisms of immune escape in this context are unknown. METHODS: We analyzed biopsy samples from paired baseline and relapsing lesions in four patients with metastatic melanoma who had had an initial objective tumor regression in response to anti-PD-1 therapy (pembrolizumab) followed by disease progression months to years later. RESULTS: Whole-exome sequencing detected clonal selection and outgrowth of the acquired resistant tumors and, in two of the four patients, revealed resistance-associated loss-of-function mutations in the genes encoding interferon-receptor-associated Janus kinase 1 (JAK1) or Janus kinase 2 (JAK2), concurrent with deletion of the wild-type allele. A truncating mutation in the gene encoding the antigen-presenting protein beta-2-microglobulin (B2M) was identified in a third patient. JAK1 and JAK2 truncating mutations resulted in a lack of response to interferon gamma, including insensitivity to its antiproliferative effects on cancer cells. The B2M truncating mutation led to loss of surface expression of major histocompatibility complex class I. CONCLUSIONS: In this study, acquired resistance to PD-1 blockade immunotherapy in patients with melanoma was associated with defects in the pathways involved in interferon-receptor signaling and in antigen presentation. (Funded by the National Institutes of Health and others.).
Asunto(s)
Resistencia a Antineoplásicos/genética , Inmunoterapia , Janus Quinasa 1/genética , Janus Quinasa 2/genética , Melanoma/genética , Mutación , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Microglobulina beta-2/genética , Anticuerpos Monoclonales Humanizados/uso terapéutico , Antineoplásicos/uso terapéutico , Biopsia , Exoma , Regulación Neoplásica de la Expresión Génica , Genes MHC Clase I , Humanos , Interferón gamma/uso terapéutico , Melanoma/tratamiento farmacológico , Melanoma/secundario , Receptor de Muerte Celular Programada 1/metabolismo , Recurrencia , Análisis de Secuencia de ADN , Transducción de SeñalRESUMEN
The therapeutic landscape of melanoma is improving rapidly. Targeted inhibitors show promising results, but drug resistance often limits durable clinical responses. There is a need for in vivo systems that allow for mechanistic drug resistance studies and (combinatorial) treatment optimization. Therefore, we established a large collection of patient-derived xenografts (PDXs), derived from BRAF(V600E), NRAS(Q61), or BRAF(WT)/NRAS(WT) melanoma metastases prior to treatment with BRAF inhibitor and after resistance had occurred. Taking advantage of PDXs as a limitless source, we screened tumor lysates for resistance mechanisms. We identified a BRAF(V600E) protein harboring a kinase domain duplication (BRAF(V600E/DK)) in â¼10% of the cases, both in PDXs and in an independent patient cohort. While BRAF(V600E/DK) depletion restored sensitivity to BRAF inhibition, a pan-RAF dimerization inhibitor effectively eliminated BRAF(V600E/DK)-expressing cells. These results illustrate the utility of this PDX platform and warrant clinical validation of BRAF dimerization inhibitors for this group of melanoma patients.
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Duplicación de Gen , Melanoma/tratamiento farmacológico , Melanoma/genética , Proteínas Proto-Oncogénicas B-raf/química , Proteínas Proto-Oncogénicas B-raf/genética , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Aberraciones Cromosómicas , Resistencia a Antineoplásicos/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Humanos , Indoles/farmacología , Indoles/uso terapéutico , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Melanoma/patología , Ratones , Mutación/genética , Metástasis de la Neoplasia , Dominios Proteicos , Multimerización de Proteína , Reproducibilidad de los Resultados , Sulfonamidas/farmacología , Sulfonamidas/uso terapéutico , VemurafenibRESUMEN
T cell responses display two key characteristics. First, a small population of epitope-specific naive T cells expands by several orders of magnitude. Second, the T cells within this proliferating population take on diverse functional and phenotypic properties that determine their ability to exert effector functions and contribute to T cell memory. Recent technological advances in lineage tracing allow us for the first time to study these processes in vivo at single-cell resolution. Here, we summarize resulting data demonstrating that although epitope-specific T cell responses are reproducibly similar at the population level, expansion potential and diversification patterns of the offspring derived from individual T cells are highly variable during both primary and recall immune responses. In spite of this stochastic response variation, individual memory T cells can serve as adult stem cells that provide robust regeneration of an epitope-specific tissue through population averaging. We discuss the relevance of these findings for T cell memory formation and clinical immunotherapy.