To delineate the minimum signals required to activate and expand antigen-specific memory CD8+ T cells, we used immunotherapeutics termed Immuno-STAT (IST) that we designed to selectively engage and activate antigen-specific T-cell receptors alone or combined with a defined co-stimulatory signal to recapitulate the discrete activation signals delivered by antigen-presenting cells to human CD8+ T cells. Transcriptome analysis of highly purified CD8+ cytomegalovirus (CMV)-specific memory T cells after delivering defined antigen-specific TCR signals alone or with co-stimulatory signals revealed that the combination of TCR signaling and CD28 or 4-1BB co-stimulatory signals significantly altered the transcriptome compared to activation by TCR signaling alone. Nevertheless, IST-delivered CMV-specific or HIV-specific TCR signaling alone in the presence of IL-2 was sufficient to induce robust recall activation and expansion of CMV (NLV)-specific or HIV (SL9)-specific memory CD8+ T cells, respectively. This response contrasts with naïve antigen-specific CD8+ T cells and TCR-engineered T cells, which required CD28 co-stimulation in addition to TCR stimulation for robust antigen-specific activation and expansion. These results have important implications by indicating that immunotherapeutics that deliver antigen-specific TCR signals alone in the presence of adequate environmental cytokine support should be sufficient for immune-based strategies designed to expand antigen-specific memory CD8+ T cells to eliminate cancerous or infected cells. In contrast, strategies that aim to optimally stimulate and expand virus or cancer-specific naïve or TCR-engineered T-cell responses would benefit from the codelivery of TCR and CD28 signals.

Soon after HIV acquisition, circulating HIV-infected monocytes cross the blood-brain barrier (BBB) and infect resident brain microglia and other susceptible cells, establishing a potential viral reservoir. Despite suppressive antiretroviral therapy, these HIV-infected cells mediate a neuroinflammatory process causing HIV-associated neurocognitive deficits (HAND) in at least 20% of people with HIV. Continued migration of HIV-infected monocytes into the brain may further exacerbate neuroinflammation and replenish viral reservoirs. To ascertain how HIV infection facilitates monocyte passage across the BBB in vivo, we developed a novel mouse model to quantify circulating monocytes supporting HIV production that migrated into the brain. We demonstrate that significantly more monocytes from HIV-transgenic mice, capable of supporting HIV production, crossed the BBB compared to control transgenic mouse monocytes. This difference was particularly pronounced after recipient mice were treated with lipopolysaccharide (LPS). To explore the underlying mechanism, we compared the transcriptomes of HIV transgenic mouse monocytes and control mouse monocytes and identified multiple differentially expressed genes linked to mononuclear leukocyte trafficking, including several associated with monocyte chemotaxis. We also evaluated the effect of substance use in combination with HIV infection on monocyte migration across the BBB into the brain by treating HIV transgenic mice with either morphine or methamphetamine. Short-term exposure to either drug did not significantly alter the migration of HIV-transgenic monocytes across the BBB.IMPORTANCEOver 20% of people with HIV (PWH) develop cognitive and neurological deficits despite antiretroviral therapy. While the blood-brain barrier (BBB) normally prevents brain entry of circulating monocytes, HIV enables infected monocytes to traverse the BBB, establish viral production within the brain, and subsequently infect microglia and other cells, which drives neuroinflammation, neuronal injury, and neurocognitive impairment. To investigate how HIV stimulates in vivo migration of circulating monocytes across the BBB, we developed a novel mouse model utilizing HIV-transgenic mouse monocytes that support HIV production. After intravenous injection, a significantly higher number of HIV-transgenic monocytes migrated into the brains of wild-type mice compared to control transgenic monocytes, particularly after lipopolysaccharide (LPS) treatment. We identified multiple genes differentially expressed in HIV-transgenic monocytes associated with mononuclear leukocyte trafficking linked to HIV-mediated induction of monocyte transmigration across the BBB. These genes may represent therapeutic targets to prevent HIV-infected monocyte migration into the brain.

Chimeric antigen receptor T cell (CAR-T cell) therapy targeting and eliminating HIV-infected cells offers a promising approach to provide people living with HIV (PLWH) with a functional cure by preventing the recurrence of viremia caused by reactivation of latent HIV-1-infected cells. We previously described a bispecific CAR-T cell targeting two highly conserved gp120 epitopes (duoCAR-T cell) with potent anti-HIV-1 activity that is currently in clinical trials. However, elevated levels of transforming growth factor β (TGF-β) present in many PLWH may hinder the activity of both infused HIV-1-specific CAR-T cells, such as duoCAR-T cells and endogenous HIV-1-specific CD8+ T cells, thereby limiting their effectiveness to achieve a functional HIV-1 cure. We hypothesized that HCW9218, a novel bifunctional immunomodulatory protein composed of TGF-βRII and IL-15/IL-15Rα, would enhance anti-HIV-1 immunity by TGF-βRII binding and neutralizing TGF-β, while IL-15/IL-15Rα would stimulate effector cells and reactivate latent HIV-1-infected cells. We used duoCAR-T cells generated from CD4+ and CD8+ T cells from people without HIV (PWoH) and PLWH donors to demonstrate the in vitro capacity of HCW9218 to block TGF-β activity, and enhance duoCAR-T cell proliferation, cytotoxicity, and anti-HIV-1 activity. HCW9218 also functioned as a latency-reversing agent, stimulating HIV-1 production by CD4+ T cells from ART-suppressed PLWH. Production of HIV-1 by HCW9218-treated CD4+ T cells from ART-suppressed PLWH donors was suppressed by co-culture with autologous duoCAR-T cells. Together, these findings highlight the potential of HCW9218 to augment T cell and CAR-T-based therapies and contribute to strategies aimed at achieving a functional cure for HIV-1.IMPORTANCEThe persistence of HIV-1 reservoirs remains the primary barrier to an HIV-1 cure because antiretroviral therapy (ART) suppresses viral replication but does not eliminate latent HIV-1-infected cells. Treatment with anti-HIV-1 duoCAR-T cells is a potential strategy to target and eliminate HIV-1-infected cells, but their activity may be impaired by the immunosuppressive environment in lymphoid tissues of people living with HIV (PLWH). Transforming growth factor β (TGF-β), a pleiotropic cytokine elevated in PLWH, is a key mediator of this immunosuppression. Here, we show that HCW9218, a bifunctional fusion protein with TGF-β-neutralizing activity and IL-15 superagonist activity, preserves duoCAR-T cell function in the presence of TGF-β and reactivates HIV-1 production by latent HIV-1-infected cells in ART-suppressed CD4+ T cells from PLWH. These findings highlight HCW9218 as a unique dual-function immunotherapy that may enhance the efficacy of duoCAR-T cells while facilitating clearance of the HIV reservoir.

Functional persistence of chimeric antigen receptor T cells (CAR T cells) is limited by conventional CAR T cell manufacturing using anti-CD3/CD28 (αCD3/28) stimulation, which generates terminally differentiated and shorter-lived CAR T cells. We demonstrated that HCW9206, a unique protein scaffold linking interleukin-7 (IL-7), an IL-15/IL-15 receptor α (IL-15Rα) complex, and IL-21, generates CAR T cells without requiring αCD3/28 activation, which are highly enriched in long-lived T memory stem cells (TSCM cells) (>50%) and display potent activity across distinct disease models, HIV-1 or B cell leukemia. In a humanized mouse HIV infection model, HCW9206-generated anti-HIV duoCAR T cells suppressed viremia more effectively than αCD3/28-generated anti-HIV duoCAR T cells. In a xenograft leukemia mouse model, a recall proliferative response and complete clearance of leukemia rechallenge were displayed by HCW9206-generated but not by αCD3/28-generated anti-CD19 CAR T cells. HCW9206, a first-in-class cytokine scaffold-based platform, enables production of more potent CAR T cell-based immunotherapies by generating a CAR T cell population, which is highly functional and also markedly enriched for long-lived TSCM cells. This strategy is broadly applicable to increase persistence and functionality of CAR T cells, enhancing their efficacy for treating infectious disease and cancer.

Transforming growth factor-ß (TGF- ß) is a critical immunoregulatory cytokine that modulates immune homeostasis and suppresses T cell cytotoxic function. Elevated TGF- ß levels, often observed in people with HIV (PWH) even under antiretroviral therapy (ART), are associated with systemic inflammation and immune suppression. We previously developed a duoCAR-T cell therapy targeting HIV-1 gp120, combining two CARs: one recognizing a CD4-induced gp120 epitope and another includes a single domain from CD4, to both block HIV infection and target HIV-1-infected cells for elimination. We hypothesized that inhibiting TGF- ß while stimulating IL-15 signaling would increase duoCAR-T cell anti-HIV activity. To test this, we used a bifunctional protein, HCW9218 composed of TGF- ß RII and IL-15/IL-15a to simultaneously neutralize TGF- ß and stimulate NK and CD8 T cells. Control variants with inactive IL-15 or TGF- ß RII domains were included. Functional activity of the TGF- ß RII domains in HCW9218 was demonstrated by its inhibition of TGF- ß -induced SMAD2/3 phosphorylation, and of the IL-15 domain by its stimulation of STAT5 phosphorylation and duoCAR-T cell proliferation. duoCAR-T cells treated with HCW9218, but not control variants, showed increased cytotoxicity and HIV suppression under TGF- ß conditions. The HCW9218 cytokine-based scaffold holds promise as an adjunctive therapy to enhance CAR-T efficacy, offering potential for a more durable anti-HIV-1 immune response. Vaccines and Immunotherapy (VAC)

Adoptive cell transfer (ACT), a promising immunotherapeutic approach, treats viral infections or cancer by ex vivo expansion and infusion of antigen-specific CD8+ T cells, respectively. However, its wider use is limited by logistical challenges associated with the conventional method of using patient-derived dendritic cells (DCs) loaded with peptides for ex vivo antigen-specific CD8+ T cell expansion. To overcome these limitations, we developed Immuno-STAT (IST), a dimeric protein scaffold that delivers peptide-specific T cell receptor (TCR) activation with or without CD28 costimulatory signals to expand CD8+ T cells specific for defined viral or cancer epitopes. In this proof-of-concept study, we demonstrate that anti-CD28-IST can selectively activate and expand polyfunctional cytotoxic CD8+ T cells from the naive repertoire, targeting the HIV-associated SL9 or melanoma-associated MART-1 epitopes. Naive MART-1-specific CD8+ T cells were reliably expanded by both peptide-loaded DCs and IST. In contrast, naive SL9-specific CD8+ T cells were expanded only by SL9-specific IST and not by conventional DC-based approaches, underscoring a unique ability of IST to stimulate some naive HIV-specific T cell responses. IST-derived SL9-specific CD8+ T cells exhibited potent cytotoxicity, diverse TCR clonotypes, and memory-differentiated phenotypes, marking a significant advance in generating antigen-specific T cells against HIV. The modular IST platform provides a scalable modality to stimulate naive CD8+ T cells to potentially mobilize preemptive CD8+ T cell responses against predicted immune escape variants, as well as subdominant conserved HIV epitopes to empower the development of innovative ACT, vaccine, and other immune strategies to advance treatments for HIV, other persistent viral infections, and cancer. IMPORTANCE:Adoptive transfer of ex vivo-expanded T cells with potent and broad anti-HIV activity may control HIV replication in people with HIV in the absence of antiretroviral therapy. To selectively activate and expand naive CD8+ cells targeting defined viral or cancer epitopes, we developed a unique protein architecture, termed Immuno-STAT, which delivers cognate peptide-specific T cell receptor (TCR) activation alone or in combination with CD28 costimulation. We demonstrated that polyfunctional cytotoxic CD8+ T cells specific for the HIV-associated SL9 or melanoma-associated MART-1 epitopes were expanded by αCD28-Immuno-STAT delivering peptide-specific TCR and CD28 signals, but not peptide-specific TCR signals alone. αCD28-Immuno-STAT-generated SL9-specific CD8+ T cells exhibited diverse TCR clonotypes, polyfunctionality, and potent SL9-specific cytotoxicity. Adoptive transfer of αCD28-Immuno-STAT-generated CD8+ T cells specific for defined HIV epitopes may provide the broad yet targeted responses specific for conserved HIV epitopes and predicted immune escape variants required to control HIV replication and provide a functional HIV cure.

New strategies to efficiently generate virus- and cancer-specific CD8 T cells are needed to increase antigenic breadth, efficacy and use of adoptive T-cell therapies for infectious diseases and cancer. We developed VirTac (VT), a modular lentivirus (LV)-based non-infectious immunotherapeutic platform to mimic features of APCs by presenting membrane proteins that elicit the selective expansion of antigen-specific CD8 T cells. We demonstrate that VT bearing single chain HLA-A*0201 MHC constructs presenting SL9 (HIV), NLV (CMV) or YLQ (CoV-2) peptides, either alone or combined with an agonist αCD28 scFv, can selectively modulate antigen-specific CD8 T cells. In vitro treatment of human donor PBMCs (n = 2-5) with SL9-, NLV- or YLQ-VT markedly expanded CD8 T cell populations specific for HIV (>20%), CMV (>87%) or CoV2 (>67%), respectively. Intravenous infusion of VT into NSG mice expanded HIV- and CMV-specific T cells by ∼30-fold; this response was enhanced in some of these donors by inclusion of the agonist αCD28 scFv in the VT. Antigen-specific T cells expanded with VT displayed potent antigen-specific cytotoxic activity, IFNγ and TNFα production and suppression of in vitro HIV or CMV infections. VT represents a highly modular platform that drives robust and selective ex vivo and in vivo expansion of antigen-specific CD8 T cells and enables diverse applications for enhancing adoptive cellular therapies to treat infectious diseases, autoimmune diseases and oncology indications. NIH R01 AI145024; NIH R01AI172607; NIH P30 AI124414 Vaccines and Immunotherapy (VAC)

Hypermutated proviruses, which arise in a single HIV replication cycle when host antiviral APOBEC3 proteins introduce extensive G-to-A mutations throughout the viral genome, persist in all people living with HIV receiving antiretroviral therapy (ART). But, the within-host evolutionary origins of hypermutated sequences are incompletely understood because phylogenetic inference algorithms, which assume that mutations gradually accumulate over generations, incorrectly reconstruct their ancestor-descendant relationships. Using >1400 longitudinal single-genome-amplified HIV env-gp120 sequences isolated from six women over a median 18 years of follow-up − including plasma HIV RNA sequences collected over a median 9 years between seroconversion and ART initiation, and >500 proviruses isolated over a median 9 years on ART − we evaluated three approaches for removing hypermutation from nucleotide alignments. Our goals were to 1) reconstruct accurate phylogenies that can be used for molecular dating and 2) phylogenetically infer the integration dates of hypermutated proviruses persisting during ART. Two of the tested approaches (stripping all positions containing putative APOBEC3 mutations from the alignment, or replacing individual putative APOBEC3 mutations in hypermutated sequences with the ambiguous base R) consistently normalized tree topologies, eliminated erroneous clustering of hypermutated proviruses, and brought env-intact and hypermutated proviruses into comparable ranges with respect to multiple tree-based metrics. Importantly, these corrected trees produced integration date estimates for env-intact proviruses that were highly concordant with those from benchmark trees that excluded hypermutated sequences, indicating that the corrected trees can be used for molecular dating. Use of these trees to infer the integration dates of hypermutated proviruses persisting during ART revealed that these spanned a wide age range, with the oldest ones dating to shortly after infection. This indicates that hypermutated proviruses, like other provirus types, begin to be seeded into the proviral pool immediately following infection, and can persist for decades. In two of the six participants, hypermutated proviruses differed from env-intact ones in terms of their age distributions, suggesting that different provirus types decay at heterogeneous rates in some hosts. These simple approaches to reconstruct hypermutated provirus' evolutionary histories, allow insights into their in vivo origins and longevity, towards a more comprehensive understanding of HIV persistence during ART.

PURPOSE OF REVIEW:Successful sustained remission of HIV infection has been achieved after CCR5Δ32/Δ32 allogeneic hematopoietic stem cell transplantation for treatment of leukemia in a small cohort of people living with HIV (PLWH). This breakthrough demonstrated that the goal of curing HIV was achievable. However, the high morbidity and mortality associated with bone marrow transplantation limits the routine application of this approach and provides a strong rationale for pursuing alternative strategies for sustained long-term antiretroviral therapy (ART)-free HIV remission. Notably, long-term immune-mediated control of HIV replication observed in elite controllers and posttreatment controllers suggests that potent HIV-specific immune responses could provide sustained ART-free remission in PLWH. The capacity of chimeric antigen receptor (CAR)-T cells engineered to target malignant cells to induce remission and cure in cancer patients made this an attractive approach to provide PLWH with a potent HIV-specific immune response. Here, we review the recent advances in the design and application of anti-HIV CAR-T-cell therapy to provide a functional HIV cure. RECENT FINDINGS:HIV reservoirs are established days after infection and persist through clonal expansion of infected cells. The continuous interaction between latently infected cells and the immune system shapes the landscape of HIV latency and likely contributes to ART-free viral control in elite controllers. CAR-T cells can exhibit superior antiviral activity as compared with native HIV-specific T cells, particularly because they can be engineered to have multiple HIV specificities, resistance to HIV infection, dual costimulatory signaling, immune checkpoint inhibitors, stem cell derivation, CMV TCR coexpression, and tissue homing ligands. These modifications can significantly improve the capacities of anti-HIV CAR-T cells to prevent viral escape, resist HIV infection, and enhance cytotoxicity, persistence, and tissue penetration. Collectively, these novel modifications of anti-HIV CAR-T cell design have increased their capacity to control HIV infection. SUMMARY:Anti-HIV CAR-T cells can be engineered to provide potent and sustained in-vitro and in-vivo antiviral function. The combination of anti-HIV CAR-T cells with other immunotherapeutics may contribute to long-term HIV remission in PLWH.

The inability of people living with HIV (PLWH) to eradicate human immunodeficiency virus (HIV) infection is due in part to the inadequate HIV-specific cellular immune response. The antiviral function of cytotoxic CD8+ T cells, which are crucial for HIV control, is impaired during chronic viral infection because of viral escape mutations, immune exhaustion, HIV antigen downregulation, inflammation, and apoptosis. In addition, some HIV-infected cells either localize to tissue sanctuaries inaccessible to CD8+ T cells or are intrinsically resistant to CD8+ T cell killing. The novel design of synthetic chimeric antigen receptors (CARs) that enable T cells to target specific antigens has led to the development of potent and effective CAR-T cell therapies. While initial clinical trials using anti-HIV CAR-T cells performed over 20 years ago showed limited anti-HIV effects, the improved CAR-T cell design, which enabled its success in treating cancer, has reinstated CAR-T cell therapy as a strategy for HIV cure with notable progress being made in the recent decade.Effective CAR-T cell therapy against HIV infection requires the generation of anti-HIV CAR-T cells with potent in vivo activity against HIV-infected cells. Preclinical evaluation of anti-HIV efficacy of CAR-T cells and their safety is fundamental for supporting the initiation of subsequent clinical trials in PLWH. For these preclinical studies, we developed a novel humanized mouse model supporting in vivo HIV infection, the development of viremia, and the evaluation of novel HIV therapeutics. Preclinical assessment of anti-HIV CAR-T cells using this mouse model involves a multistep process including peripheral blood mononuclear cells (PBMCs) harvested from human donors, T cell purification, ex vivo T cell activation, transduction with lentiviral vectors encoding an anti-HIV CAR, CAR-T cell expansion and infusion in mice intrasplenically injected with autologous PBMCs followed by the determination of CAR-T cell capacity for HIV suppression. Each of the steps described in the following protocol were optimized in the lab to maximize the quantity and quality of the final anti-HIV CAR-T cell products.

Within-host HIV populations continually diversify during untreated infection, and members of these diverse forms persist within infected cell reservoirs, even during antiretroviral therapy (ART). Characterizing the diverse viral sequences that persist during ART is critical to HIV cure efforts, but our knowledge of on-ART proviral evolutionary dynamics remains incomplete, as does our understanding of the differences between the overall pool of persisting proviral DNA (which is largely genetically defective) and the subset of intact HIV sequences capable of reactivating. Here, we reconstructed within-host HIV evolutionary histories in blood from seven participants of the Women's Interagency HIV Study (WIHS) who experienced HIV seroconversion. We measured diversity, lineage origins and ages of proviral sequences (env-gp120) sampled up to four times, up to 12 years on ART. We used the same techniques to study HIV sequences emerging from the reservoir in two participants. Proviral clonality generally increased over time on ART, with clones frequently persisting across multiple time points. The integration dates of proviruses persisting on ART generally spanned the duration of untreated infection (though were often skewed towards years immediately pre-ART), while in contrast, reservoir-origin viremia emerging in plasma was exclusively "younger" (i.e., dated to the years immediately pre-ART). The genetic and age distributions of distinct proviral sequences remained highly stable during ART in all but one participant in whom, after 12 years, there was evidence that "younger" proviruses had been preferentially eliminated. Analysis of within-host recombinant proviral sequences also suggested that HIV reservoirs can be superinfected with virus reactivated from an older era, yielding infectious viral progeny with mosaic genomes of sequences with different ages. Overall, results underscore the remarkable genetic stability of distinct proviral sequences that persist on ART, yet suggest that replication-competent HIV reservoir represents a genetically-restricted and overall "younger" subset of the overall persisting proviral pool in blood.

Although combination antiretroviral therapy (ART) has been a landmark achievement for the treatment of human immunodeficiency virus (HIV), an HIV cure has remained elusive. Elimination of latent HIV reservoirs that persist throughout HIV infection is the most challenging barrier to an HIV cure. The progressive HIV infection is marked by the increasing size and diversity of latent HIV reservoirs until an effective immune response is mobilized, which can control but not eliminate HIV infection. The stalemate between HIV replication and the immune response is manifested by the establishment of a viral set point. ART initiation during the early stage limits HIV reservoir development, preserves immune function, improves the quality of life, and may lead to ART-free viral remission in a few people living with HIV (PLWH). However, for the overwhelming majority of PLWH, early ART initiation alone does not cure HIV, and lifelong ART is needed to sustain viral suppression. A critical area of research is focused on determining whether HIV could be functionally cured if additional treatments are provided alongside early ART. Several HIV interventions including Block and Lock, Shock and Kill, broadly neutralizing antibody (bNAb) therapy, adoptive CD8+ T cell therapy, and gene therapy have demonstrated delayed viral rebound and/or viral remission in animal models and/or some PLWH. Whether or not their application during early infection can improve the success of HIV remission is less studied. Herein, we review the current state of clinical and investigative HIV interventions and discuss their potential to improve the likelihood of post-treatment remission if initiated during early infection.

Chronological aging correlates with epigenetic modifications at specific loci, calibrated to species lifespan. Such ‘epigenetic clocks’ appear conserved among mammals, but whether they are cell autonomous and restricted by maximal organismal lifespan remains unknown. We used a multilifetime murine model of repeat vaccination and memory T cell transplantation to test whether epigenetic aging tracks with cellular replication and if such clocks continue ‘counting’ beyond species lifespan. Here we found that memory T cell epigenetic clocks tick independently of host age and continue through four lifetimes. Instead of recording chronological time, T cells recorded proliferative experience through modification of cell cycle regulatory genes. Applying this epigenetic profile across a range of human T cell contexts, we found that naive T cells appeared ‘young’ regardless of organism age, while in pediatric patients, T cell acute lymphoblastic leukemia appeared to have epigenetically aged for up to 200 years. Thus, T cell epigenetic clocks measure replicative history and can continue to accumulate well-beyond organismal lifespan. Using an iterative boost and transplantation model to generate multilifetime T cells, Mi et al. show that cellular epigenetic age can be uncoupled from organism age. While naive T cells appear epigenetically young, memory T cells and T-ALL leukemia can exhibit epigenetic ages exceeding the organismal lifespan.

As the immune system ages, the protective capacity from established immunological memory wanes over time and is often referred to as immune senescence. Yet the molecular hallmarks of lymphocyte age and replicative senescence remain largely undefined. Here we report our use of a multi-lifetime murine model of repeat vaccination to define a core epigenetic signature coupled to memory T cells that evade replicative senescence. Through adoptive transfer and boosting over a period of approximately four mouse lifetimes, memory T cells became enriched for repressive DNA methylation programs at cell cycle regulators including the canonical senescence gene P19 Arf(CDKN2A/2B locus – human P14 ARF). The senescence-resistance program was progressively acquired throughout phycological and non-physiological aging of murine T cells, leading to the development of a lymphocyte epigenetic clock that can be used to accurately measure a T cell’s age. Application of this clock in physiologically aged human polyclonal and CMV-specific T cells correlated with age-estimates using established epigenetic chronologic clocks. Moreover, T-cell clock-associated senescence-resistance programs were highly enriched in human malignancies of lymphoid origin, specifically delineating known T-cell acute lymphoblastic leukemia (T-ALL) subtypes and revealing non-physiological aging of HOXA9 and MLL pediatric leukemias (estimated to be ~100 years old). Discovery of the senescence-resistance epigenetic clock provides a novel molecular hallmark for describing physiological and non-physiological lymphocyte age.