Pembrolizumab and low-dose, single-fraction radiotherapy for patients with relapsed or refractory multiple myeloma: a prospective, single-centre, single-group, open-label, phase 2 pilot trial in the USA.

BACKGROUND: Currently, the use of radiotherapy alone for people with multiple myeloma is limited to palliation of pain, pending fracture, and control of spinal-cord compression. Single immune-checkpoint inhibitors, such as anti-programmed death-1 (anti-PD1), have not been successful. We aimed to evaluate the activity and safety of the combination of pembrolizumab and low-dose, single-fraction, hypofractionated radiotherapy to treat patients with relapsed or refractory multiple myeloma. METHODS: For this prospective, single-centre, single-group, open-label, phase 2 trial, we recruited patients with relapsed or refractory multiple myeloma from the Winship Cancer Institute (Emory University, Atlanta, GA, USA). Key inclusion criteria were aged 18 years or older, Eastern Cooperative Oncology Group (ECOG) performance score of 0 or 1, relapsed or refractory multiple myeloma as indicated by progression under International Myeloma Working Group (IMWG) criteria, and adequate candidacy for both pembrolizumab and radiotherapy. Baseline and post-treatment assessments were serial bone-marrow biopsy, peripheral blood collections, staging, serial serum and urine paraprotein analysis, serial PET-CT imaging, and a physical examination. On day 1, patients received hypofractionated 8 gray in 1 fraction (8 Gy/1 fx) radiotherapy to either symptomatic or progressing extra-osseous or osseous myeloma sites. Patients also received pembrolizumab (200 mg/kg intravenously) on day 2 or 3, then once every 3 weeks (±7 days) for 2 years or until progressive disease, unacceptable toxicity, withdrawal of consent, loss to follow-up, or death. Dose reduction and interruptions were not allowed. The primary outcome was acute toxicity defined as grade 3 or worse toxicity at 3 months within the radiated site when used in combination with pembrolizumab. All patients were analysed per protocol and included in safety analyses. This trial is registered on ClinicalTrials.gov (NCT03267888); it is completed and closed to accrual. FINDINGS: 32 patients were screened between June 1, 2018, and Sept 2, 2022, and 25 were enrolled in the trial and treated on protocol. Of the 25 treated patients, 11 (44%) were female and 14 (56%) were male. 19 (76%) patients were White and six (24%) were Black or African American. Toxicity, as the primary outcome, was deemed to be acceptable as no grade 4 or 5 adverse events were observed. At 3-month follow-up, eight (32%) of 25 patients had treatment benefit (one had stable disease, three had partial response, two had very good partial response, and two had complete response). There was no grade 3 or worse radiation-related toxicity within irradiated volumes. One (4%) patient of the 25 who received combination treatment had a grade 3 pembrolizumab-related adverse event. There were no treatment-related deaths. INTERPRETATION: Combination treatment of low-dose, single-fraction radiotherapy with pembrolizumab was safe, with early promise of response activity. Our approach could be an option for patients with relapsed or refractory multiple myeloma who have not responded to previous treatment. Larger trials to substantiate our findings are needed. FUNDING: Merck Sharp & Dohme.

Intensity-Modulated Reirradiation Therapy With Nivolumab in Recurrent or Second Primary Head and Neck Squamous Cell Carcinoma: A Nonrandomized Controlled Trial.

Importance: Intensity-modulated radiation therapy (IMRT) reirradiation of nonmetastatic recurrent or second primary head and neck squamous cell carcinoma (HNSCC) results in poor progression-free survival (PFS) and overall survival (OS). Objective: To investigate the tolerability, PFS, OS, and patient-reported outcomes with nivolumab (approved standard of care for patients with HNSCC) during and after IMRT reirradiation. Design, Setting, and Participants: In this multicenter nonrandomized phase 2 single-arm trial, the treatment outcomes of patients with recurrent or second primary HNSCC who satisfied recursive partitioning analysis class 1 and 2 definitions were evaluated. Between July 11, 2018, and August 12, 2021, 62 patients were consented and screened. Data were evaluated between June and December 2023. Intervention: Sixty- to 66-Gy IMRT in 30 to 33 daily fractions over 6 to 6.5 weeks with nivolumab, 240 mg, intravenously 2 weeks prior and every 2 weeks for 5 cycles during IMRT, then nivolumab, 480 mg, intravenously every 4 weeks for a total nivolumab duration of 52 weeks. Main Outcomes and Measures: The primary end point was PFS. Secondary end points included OS, incidence, and types of toxic effects, including long-term treatment-related toxic effects, patient-reported outcomes, and correlatives of tissue and blood biomarkers. Results: A total of 62 patients were screened, and 51 were evaluable (median [range] age was 62 [56-67] years; 42 [82%] were male; 6 [12%] had p16+ disease; 38 [75%] had salvage surgery; and 36 [71%.] had neck dissection). With a median follow-up of 24.5 months (95% CI, 19.0-25.0), the estimated 1-year PFS was 61.7% (95% CI, 49.2%-77.4%), rejecting the null hypothesis of 1-year PFS rate of less than 43.8% with 1-arm log-rank test P = .002 within a 1-year timeframe. The most common treatment-related grade 3 or higher adverse event (6 [12%]) was lymphopenia with 2 patients (4%) and 1 patient each (2%) exhibiting colitis, diarrhea, myositis, nausea, mucositis, and myasthenia gravis. Functional Assessment of Cancer Therapy-General and Functional Assessment of Cancer Therapy-Head and Neck Questionnaire quality of life scores remained stable and consistent across all time points. A hypothesis-generating trend favoring worsening PFS and OS in patients with an increase in blood PD1+, KI67+, and CD4+ T cells was observed. Conclusions and Relevance: This multicenter nonrandomized phase 2 trial of IMRT reirradiation therapy and nivolumab suggested a promising improvement in PFS over historical controls. The treatment was well tolerated and deserves further evaluation. Trial Registration: ClinicalTrials.gov Identifier: NCT03521570.

Severe disease during both primary and secondary dengue virus infections in pediatric populations.

Dengue is a global epidemic causing over 100 million cases annually. The clinical symptoms range from mild fever to severe hemorrhage and shock, including some fatalities. The current paradigm is that these severe dengue cases occur mostly during secondary infections due to antibody-dependent enhancement after infection with a different dengue virus serotype. India has the highest dengue burden worldwide, but little is known about disease severity and its association with primary and secondary dengue infections. To address this issue, we examined 619 children with febrile dengue-confirmed infection from three hospitals in different regions of India. We classified primary and secondary infections based on IgM:IgG ratios using a dengue-specific enzyme-linked immunosorbent assay according to the World Health Organization guidelines. We found that primary dengue infections accounted for more than half of total clinical cases (344 of 619), severe dengue cases (112 of 202) and fatalities (5 of 7). Consistent with the classification based on binding antibody data, dengue neutralizing antibody titers were also significantly lower in primary infections compared to secondary infections (P ≤ 0.0001). Our findings question the currently widely held belief that severe dengue is associated predominantly with secondary infections and emphasizes the importance of developing vaccines or treatments to protect dengue-naive populations.

Reversible, tunable epigenetic silencing of TCF1 generates flexibility in the T cell memory decision.

The immune system encodes information about the severity of a pathogenic threat in the quantity and type of memory cells it forms. This encoding emerges from lymphocyte decisions to maintain or lose self-renewal and memory potential during a challenge. By tracking CD8+ T cells at the single-cell and clonal lineage level using time-resolved transcriptomics, quantitative live imaging, and an acute infection model, we find that T cells will maintain or lose memory potential early after antigen recognition. However, following pathogen clearance, T cells may regain memory potential if initially lost. Mechanistically, this flexibility is implemented by a stochastic cis-epigenetic switch that tunably and reversibly silences the memory regulator, TCF1, in response to stimulation. Mathematical modeling shows how this flexibility allows memory T cell numbers to scale robustly with pathogen virulence and immune response magnitudes. We propose that flexibility and stochasticity in cellular decisions ensure optimal immune responses against diverse threats.

Early generation of a precursor CD8 T cell that can adapt to acute or chronic viral infection.

Virus specific PD-1+ TCF-1+ TOX+ stem-like CD8+ T cells are essential for maintaining T cell responses during chronic infection and are also critical for PD-1 directed immunotherapy. In this study we have used the mouse model of chronic LCMV infection to examine when these virus specific stem-like CD8+ T cells are generated during the course of chronic infection and what is the role of antigen in maintaining the stem-like program. We found that these stem-like CD8+ T cells are generated early (day 5) during chronic infection and that antigen is essential for maintaining their stem-like program. This early generation of stem-like CD8+ T cells suggested that the fate commitment to this cell population was agnostic to the eventual outcome of infection and the immune system prepares a priori for a potential chronic infection. Indeed, we found that an identical virus specific stem-cell like CD8+ T cell population was also generated during acute LCMV infection but these cells were lost once the virus was cleared. To determine the fate of these early PD-1+TCF-1+TOX+ stem-like CD8+ T cells that are generated during both acute and chronic LCMV infection we set up two reciprocal adoptive transfer experiments. In the first experiment we transferred day 5 stem-like CD8+ T cells from chronically infected into acutely infected mice and examined their differentiation after viral clearance. We found that these early stem-like CD8+ T cells downregulated canonical markers of the chronic stem-like CD8+ T cells and expressed markers (CD127 and CD62L) associated with central memory CD8+ T cells. In the second experiment, we transferred day 5 stem-like cells from acutely infected mice into chronically infected mice and found that these CD8+ T cells could function like resource cells after transfer into a chronic environment by generating effector CD8+ T cells in both lymphoid and non-lymphoid tissues while also maintaining the number of stem-like CD8+ T cells. These findings provide insight into the generation and maintenance of virus specific stem-like CD8+ T cells that play a critical role in chronic viral infection. In particular, our study highlights the early generation of stem-like CD8+ T cells and their ability to adapt to either an acute or chronic infection. These findings are of broad significance since these novel stem-like CD8+ T cells play an important role in not only viral infections but also in cancer and autoimmunity.

Functional impairment of "helpless" CD8 + memory T cells is transient and driven by prolonged but finite cognate antigen presentation.

Generation of functional CD8 + T cell memory typically requires engagement of CD4 + T cells. However, in certain scenarios, such as acutely-resolving viral infections, effector (T E ) and subsequent memory (T M ) CD8 + T cell formation appear impervious to a lack of CD4 + T cell help during priming. Nonetheless, such "helpless" CD8 + T M respond poorly to pathogen rechallenge. At present, the origin and long-term evolution of helpless CD8 + T cell memory remain incompletely understood. Here, we demonstrate that helpless CD8 + T E differentiation is largely normal but a multiplicity of helpless CD8 T M defects, consistent with impaired memory maturation, emerge as a consequence of prolonged yet finite exposure to cognate antigen. Importantly, these defects resolve over time leading to full restoration of CD8 + T M potential and recall capacity. Our findings provide a unified explanation for helpless CD8 + T cell memory and emphasize an unexpected CD8 + T M plasticity with implications for vaccination strategies and beyond.

Harnessing CD8 T cell responses using PD-1-IL-2 combination therapy.

There is considerable interest in developing more effective programmed cell death (PD)-1 combination therapies against cancer. One major obstacle to these efforts is a dysfunctional/exhausted state of CD8 T cells, which PD-1 monotherapy is not able to overcome. Recent studies have highlighted that PD-1+ T cell factor (TCF)-1+ stem-like CD8 T cells are not fate locked into the exhaustion program and their differentiation trajectory can be changed by interleukin (IL)-2 signals. Modifying the CD8 T cell exhaustion program and generating better effectors from stem-like CD8 T cells by IL-2 form the fundamental immunological basis for combining IL-2 with PD-1 therapy. Many versions of IL-2-based products are being tested and each product should be carefully evaluated for its ability to modulate dysfunctional states of anti-tumor CD8 T cells.

Mitochondrial metabolic flexibility is critical for CD8+ T cell antitumor immunity.

Mitochondria use different substrates for energy production and intermediatory metabolism according to the availability of nutrients and oxygen levels. The role of mitochondrial metabolic flexibility for CD8+ T cell immune response is poorly understood. Here, we report that the deletion or pharmacological inhibition of protein tyrosine phosphatase, mitochondrial 1 (PTPMT1) significantly decreased CD8+ effector T cell development and clonal expansion. In addition, PTPMT1 deletion impaired stem-like CD8+ T cell maintenance and accelerated CD8+ T cell exhaustion/dysfunction, leading to aggravated tumor growth. Mechanistically, the loss of PTPMT1 critically altered mitochondrial fuel selection-the utilization of pyruvate, a major mitochondrial substrate derived from glucose-was inhibited, whereas fatty acid utilization was enhanced. Persistent mitochondrial substrate shift and metabolic inflexibility induced oxidative stress, DNA damage, and apoptosis in PTPMT1 knockout cells. Collectively, this study reveals an important role of PTPMT1 in facilitating mitochondrial utilization of carbohydrates and that mitochondrial flexibility in energy source selection is critical for CD8+ T cell antitumor immunity.

The evolution and determinants of neutralization of potent head-binding antibodies against Ebola virus.

Monoclonal antibodies against the Ebola virus (EBOV) surface glycoprotein are effective treatments for EBOV disease. Antibodies targeting the EBOV glycoprotein (GP) head epitope have potent neutralization and Fc effector function activity and thus are of high interest as therapeutics and for vaccine design. Here we focus on the head-binding antibodies 1A2 and 1D5, which have been identified previously in a longitudinal study of survivors of EBOV infection. 1A2 and 1D5 have the same heavy- and light-chain germlines despite being isolated from different individuals and at different time points after recovery from infection. Cryoelectron microscopy analysis of each antibody in complex with the EBOV surface GP reveals key amino acid substitutions in 1A2 that contribute to greater affinity, improved neutralization potency, and enhanced breadth as well as two strategies for antibody evolution from a common site.

Platinum-based chemotherapy attenuates the effector response of CD8 T cells to concomitant PD-1 blockade.

PURPOSE: Combination of chemotherapy (CT) with programmed cell death (PD)-1 blockade is a front-line treatment for lung cancer. However, it remains unknown whether and how CT affects the response of exhausted CD8 T cells to PD-1 blockade. EXPERIMENTAL DESIGN: We used the well-established mouse model of T cell exhaustion with chronic lymphocytic choriomeningitis virus (LCMV) infection to assess the effect of CT (cisplatin+pemetrexed) on T cell response to PD-1 blockade, in the absence of the impact of CT on antigen release and presentation observed in tumor models. RESULTS: When concomitantly administered with PD-1 blockade, CT affected the differentiation path of LCMV-specific CD8 T cells from stem-like to transitory effector cells, thereby reducing their expansion and production of interferon (IFN)-γ. After combination treatment, these restrained effector responses resulted in impaired viral control, compared to PD-1 blockade alone. The sequential combination strategy, where PD-1 blockade followed CT, proved to be superior to the concomitant combination, preserving the proliferative response of exhausted CD8 T cells to PD-1 blockade. Our findings suggest that the stem-like CD8 T cells themselves are relatively unaffected by CT partly because they are quiescent and maintained by slow self-renewal at the steady state. However, upon the proliferative burst mediated by PD-1 blockade, the accelerated differentiation and self-renewal of stem-like cells may be curbed by concomitant CT, ultimately resulting in impaired overall CD8 T cell effector functions. CONCLUSIONS: In a translational context, we provide a proof-of-concept to consider optimizing the timing of chemo-immunotherapy strategies for improved CD8 T cell functions.

BA.5 bivalent booster vaccination enhances neutralization of XBB.1.5, XBB.1.16 and XBB.1.9 variants in patients with lung cancer.

This study reports that most patients with NSCLC had a significant increase in the nAb response to the currently circulating Omicron variants after bivalent booster vaccination and had Ab titers comparable to healthy participants. Interestingly, though the durability of the nAb response persisted in most of the healthy participants, patients with NSCLC had significantly reduced nAb titers after 4-6 months of vaccination. Our data highlight the importance of COVID-19 bivalent booster vaccination as the standard of care for patients with NSCLC given the evolution of new variants of concern.

Longitudinal nucleocapsid antibody testing reveals undocumented SARS-CoV-2 infections in patients with lung cancer.

Patients diagnosed with lung cancer (LC) exhibit increased susceptibility to SARS-CoV-2 infection. Rodilla et al. monitor the levels of plasma anti-nucleocapsid antibodies within a cohort of fully vaccinated LC patients and reveal that the actual infection rate is nearly twice the documented rate, indicating a significant prevalence of unreported cases.

Infants and young children generate more durable antibody responses to SARS-CoV-2 infection than adults.

As SARS-CoV-2 becomes endemic, it is critical to understand immunity following early-life infection. We evaluated humoral responses to SARS-CoV-2 in 23 infants/young children. Antibody responses to SARS-CoV-2 spike antigens peaked approximately 30 days after infection and were maintained up to 500 days with little apparent decay. While the magnitude of humoral responses was similar to an adult cohort recovered from mild/moderate COVID-19, both binding and neutralization titers to WT SARS-CoV-2 were more durable in infants/young children, with spike and RBD IgG antibody half-life nearly 4X as long as in adults. IgG subtype analysis revealed that while IgG1 formed the majority of the response in both groups, IgG3 was more common in adults and IgG2 in infants/young children. These findings raise important questions regarding differential regulation of humoral immunity in infants/young children and adults and could have broad implications for the timing of vaccination and booster strategies in this age group.

Characteristics and anatomic location of PD-1+TCF1+ stem-like CD8 T cells in chronic viral infection and cancer.

CD8 T cells play an essential role in antitumor immunity and chronic viral infections. Recent findings have delineated the differentiation pathway of CD8 T cells in accordance with the progenitor-progeny relationship of TCF1+ stem-like and Tim-3+TCF1- more differentiated T cells. Here, we investigated the characteristics of stem-like and differentiated CD8 T cells isolated from several murine tumor models and human lung cancer samples in terms of phenotypic and transcriptional features as well as their location compared to virus-specific CD8 T cells in the chronically lymphocytic choriomeningitis virus (LCMV)-infected mice. We found that CD8 tumor-infiltrating lymphocytes (TILs) in both murine and human tumors exhibited overall similar phenotypic and transcriptional characteristics compared to corresponding subsets in the spleen of chronically infected mice. Moreover, stem-like CD8 TILs exclusively responded and produced effector-like progeny CD8 T cells in vivo after antigenic restimulation, confirming their lineage relationship and the proliferative potential of stem-like CD8 TILs. Most importantly, similar to the preferential localization of PD-1+ stem-like CD8 T cells in T cell zones of the spleen during chronic LCMV infection, we found that the PD-1+ stem-like CD8 TILs in lung cancer samples are preferentially located not in the tumor parenchyma but in tertiary lymphoid structures (TLSs). The stem-like CD8 T cells are present in TLSs located within and at the periphery of the tumor, as well as in TLSs closely adjacent to the tumor parenchyma. These findings suggest that TLSs provide a protective niche to support the quiescence and maintenance of stem-like CD8 T cells in the tumor.

Light chain of a public SARS-CoV-2 class-3 antibody modulates neutralization against Omicron.

The pairing of antibody genes IGHV2-5/IGLV2-14 is established as a public immune response that potently cross-neutralizes SARS-CoV-2 variants, including Omicron, by targeting class-3/RBD-5 epitopes in the receptor binding domain (RBD). LY-CoV1404 (bebtelovimab) exemplifies this, displaying exceptional potency against Omicron sub-variants up to BA.5. Here, we report a human antibody, 002-S21B10, encoded by the public clonotype IGHV2-5/IGLV2-14. While 002-S21B10 neutralized key SARS-CoV-2 variants, it did not neutralize Omicron, despite sharing >92% sequence similarity with LY-CoV1404. The structure of 002-S21B10 in complex with spike trimer plus structural and sequence comparisons with LY-CoV1404 and other IGHV2-5/IGLV2-14 antibodies revealed significant variations in light-chain orientation, paratope residues, and epitope-paratope interactions that enable some antibodies to neutralize Omicron but not others. Confirming this, replacing the light chain of 002-S21B10 with the light chain of LY-CoV1404 restored 002-S21B10's binding to Omicron. Understanding such Omicron evasion from public response is vital for guiding therapeutics and vaccine design.

Reciprocal transmission of activating and inhibitory signals and cell fate in regenerating T cells.

The ability of activated progenitor T cells to self-renew while producing differentiated effector cell descendants may underlie immunological memory and persistent responses to ongoing infection. The nature of stem-like T cells responding to cancer and during treatment with immunotherapy is not clear. The subcellular organization of dividing progenitor CD8+ T cells from mice challenged with syngeneic tumors is examined here. Three-dimensional microscopy reveals an activating hub composed of polarized CD3, CD28, and phosphatidylinositol 3-kinase (PI3K) activity at the putative immunological synapse with an inhibitory hub composed of polarized PD-1 and CD73 at the opposite pole of mitotic blasts. Progenitor T cells from untreated and inhibitory checkpoint blockade-treated mice yield a differentiated TCF1- daughter cell, which inherits the PI3K activation hub, alongside a discordantly fated, self-renewing TCF1+ sister cell. Dynamic organization of opposite activating and inhibitory signaling poles in mitotic lymphocytes may account for the enigmatic durability of specific immunity.

PD-1 blockade increases the self-renewal of stem-like CD8 T cells to compensate for their accelerated differentiation into effectors.

PD-1+TCF-1+ stem-like CD8 T cells act as critical resource cells for maintaining T cell immunity in chronic viral infections and cancer. In addition, they provide the proliferative burst of effector CD8 T cells after programmed death protein 1 (PD-1)-directed immunotherapy. However, it is not known whether checkpoint blockade diminishes the number of these stem-like progenitor cells as effector cell differentiation increases. To investigate this, we used the mouse model of chronic lymphocytic choriomeningitis virus (LCMV) infection. Treatment of chronically infected mice with either αPD-1 or αPD-L1 antibody not only increased effector cell differentiation from the virus-specific stem-like CD8 T cells but also increased their proliferation so their numbers were maintained. The increased self-renewal of LCMV-specific stem-like CD8 T cells was mTOR dependent. We used microscopy to understand the division of these progenitor cells and found that after PD-1 blockade, an individual dividing cell could give rise to a differentiated TCF-1- daughter cell alongside a self-renewing TCF-1+ sister cell. This asymmetric division helped to preserve the number of stem-like cells. Moreover, we found that the PD-1+TCF-1+ stem-like CD8 T cells retained their transcriptional program and their in vivo functionality in terms of responding to viral infection and to repeat PD-1 blockade. Together, our results demonstrate that PD-1 blockade does not deplete the stem-like population despite increasing effector differentiation. These findings have implications for PD-1-directed immunotherapy in humans.

Mechanical force regulates ligand binding and function of PD-1.

Immune checkpoint blockade targeting PD-1 shows great success in cancer therapy. However, the mechanism of how ligand binding initiates PD-1 signaling remains unclear. As prognosis markers of multiple cancers, soluble PD-L1 is found in patient sera and can bind PD-1, but fails to suppress T cell function. This and our previous observations that T cells exert endogenous forces on PD-1-PD-L2 bonds prompt the hypothesis that mechanical force might be critical to PD-1 triggering, which is missing in the soluble ligand case due to the lack of mechanical support afforded by surface-anchored ligand. Here we show that PD-1 function is eliminated or reduced when mechanical support on ligand is removed or dampened, respectively. Force spectroscopic analysis reveals that PD-1 forms catch bonds with both PD-Ligands <7 pN where force prolongs bond lifetime, but slip bonds >8 pN where force accelerates dissociation. Steered molecular dynamics finds PD-1-PD-L2 complex very sensitive to force due to the two molecules' "side-to-side" binding via ß sheets. Pulling causes relative rotation and translation between the two molecules by stretching and aligning the complex along the force direction, yielding new atomic contacts not observed in the crystal structure. Compared to wild-type, PD-1 mutants targeting the force-induced new interactions maintain the same binding affinity but display lower rupture force, shorter bond lifetime, reduced tension, and most importantly, impaired capacity to suppress T cell activation. Our results uncover a mechanism for cells to probe the mechanical support of PD-1-PD-Ligand bonds using endogenous forces to regulate PD-1 triggering.

mRNA vaccination boosts S-specific T cell memory and promotes expansion of CD45RAint TEMRA-like CD8+ T cells in COVID-19 recovered individuals.

SARS-CoV-2 infection and mRNA vaccination both elicit spike (S)-specific T cell responses. To analyze how T cell memory from prior infection influences T cell responses to vaccination, we evaluated functional T cell responses in naive and previously infected vaccine recipients. Pre-vaccine S-specific responses are predictive of subsequent CD8+ T cell vaccine-response magnitudes. Comparing baseline with post-vaccination TCRß repertoires, we observed large clonotypic expansions correlated with the frequency of spike-specific T cells. Epitope mapping the largest CD8+ T cell responses confirms that an HLA-A∗03:01 epitope was highly immunodominant. Peptide-MHC tetramer staining together with mass cytometry and single-cell sequencing permit detailed phenotyping and clonotypic tracking of these S-specific CD8+ T cells. Our results demonstrate that infection-induced S-specific CD8+ T cell memory plays a significant role in shaping the magnitude and clonal composition of the circulating T cell repertoire after vaccination, with mRNA vaccination promoting CD8+ memory T cells to a TEMRA-like phenotype.