Objectives: To investigate the short- and long-term dynamics of intact and defective proviral HIV DNA during ART. Design: We evaluated viral reservoir dynamics in a cohort of nine individuals with chronic HIV-1 subtype B infection who initiated first-line ART and were followed for 20 years while continuing ART. Methods: PBMCs were obtained before ART (n = 5), during the first year, and after 8.5 and 20 years of treatment. T cell subsets (naive, central-memory, transitional-memory and effector-memory) were sorted at 8.5 and 20 years. DNA was isolated and analyzed using the intact proviral DNA assay (IPDA). Deep-sequencing of the viral env region enabled analysis of viral evolution and cellular mechanisms underlying HIV persistence. Results: Initially, defective and intact proviral DNA in PBMCs declined with half-lives of 3.6 and 5.4 weeks, respectively. Over the following 8.5 years, the intact reservoir continued to decrease, with a half-life of 18.8 months in PBMCs, while defective proviral DNA levels stabilized. After 8.5 and 20 years of ART, the intact reservoir showed no further decline, with most intact proviral DNA residing in memory T cell subsets. Phylogenetic analysis revealed no signs of viral evolution over time, both within and between T cell subsets. Conclusions: PBMCs containing intact proviral DNA are selectively lost during the first decade of suppressive ART, followed by a decade of stabilization of this reservoir in the memory T cell subsets. In the absence of clear signs of viral evolution and massive clonal expansion, homeostatic proliferation might be an important driver of HIV persistence during long-term ART.

Estimating production and loss rates of cell populations is essential but difficult. The current state-of-the-art method to estimate these rates involves mathematical modelling of deuterium labelling experiments. Current models typically assume that the labelling in the precursors of the population of interest (POI) is proportional to the deuterium enrichment in body water/glucose. This assumption is not always true and it is known that this can have a significant effect on the rates estimated from labelling experiments. Here we quantify the effect that different turnover (replacement) rates of the precursors can have on the estimated proliferation and loss rates of a POI by explicitly modelling the dynamics of the precursors. We first confirm earlier results that the labelling curve of the POI only reflects its own turnover rate if either the turnover rate of the precursors is sufficiently fast, or the contribution from the precursors is sufficiently small. Next, we describe three realistic scenarios with a slowly turning over precursor population, and show how this changes the interpretation of the different parameter estimates. Our analyses underpin that uniquely identifying the turnover rate of a POI requires measurements (or knowledge) on the turnover of its immediate precursors.

Background Psoriasis is a systemic inflammatory disease for which there is currently no cure, in part due to an incomplete understanding of its pathophysiology. Methods To better understand the immune response in psoriasis, we performed single-cell RNA sequencing (scRNA-seq) on peripheral blood mononuclear cells (PBMCs) and on lesional and non-lesional skin samples from a cohort of 11 psoriasis patients and 8 healthy controls. Additionally, we conducted flow cytometry on PBMCs from a separate cohort of 13 psoriasis patients and 11 ankylosing spondylitis. Findings Our study revealed altered immune signatures of specific myeloid and lymphocyte subsets in blood and skin, both in terms of cell numbers and gene expression. Specifically, we discovered elevated proportions of circulating CD14++ monocytes, increased expression of major histocompatibility complex (MHC) class II molecule by circulating CD16+ monocytes, as well as increased expression of genes related to skin homing and to pro-inflammatory responses in psoriasis by circulating plasmacytoid dendritic cells (pDCs). Circulating CD8+ T effector memory cells in psoriasis patients exhibited reduced abundance but increased skin-homing potential. In psoriatic lesions, we observed a hyperinflammatory myeloid-cell state and enrichment of IL17-producing cells with a tissue-resident memory T-cell signature. Interpretation The changes in immune cell numbers and gene expression indicate a significant alteration in the immune landscape of psoriasis patients. This suggests that the immune system in psoriasis is reprogrammed, affecting both innate and adaptive branches. These findings provide new insights into the aberrant immune-cell signatures in the circulation and skin lesions in psoriasis, and thereby help to understand its pathophysiology. Funding This study was financially supported by the National Natural Science Foundation of China (U23A6012), Science and Technology Planning Project of Guangzhou (2024A03J0055, 202206080005), Innovation Team and Talents Cultivation Program of National Administration of Traditional Chinese Medicine (ZYYCXTD-C-202204). ### Competing Interest Statement The authors have declared no competing interest.

We have previously argued that the antigen receptors of T and B lymphocytes evolved to be sufficiently specific to avoid massive deletion of clonotypes by negative selection. Their optimal ‘specificity’ level, i.e., probability of binding any particular epitope, was shown to be inversely related to the number of self-antigens that the cells have to be tolerant to. Experiments have demonstrated that T lymphocytes also become more specific during negative selection in the thymus, because cells expressing the most crossreactive receptors have the highest likelihood of binding a self-antigen, and hence to be tolerized (i.e., deleted, anergized, or diverted into a regulatory T cell phenotype). Thus, there are two —not mutually exclusive— explanations for the exquisite specificity of T cells, one involving evolution and the other thymic selection. To better understand the impact of both, we extend a previously developed mathematical model by allowing for T cells with very different binding probabilities in the pre-selection repertoire. We confirm that negative selection tends to tolerize the most crossreactive clonotypes. As a result, the average level of specificity in the functional post-selection repertoire depends on the number of self-antigens, even if there is no evolutionary optimization of binding probabilities. However, the evolutionary optimal range of binding probabilities in the pre-selection repertoire also depends on the number of self-antigens. Species with more self antigens need more specific pre-selection repertoires to avoid excessive loss of T cells during thymic selection, and hence mount protective immune responses. We conclude that both evolution and negative selection are responsible for the high level of specificity of lymphocytes.

To protect older adults against influenza A virus (IAV) infection, innovative strategies are imperative to overcome the decrease in protective immune response with age. One approach involves the boosting of CD8+ T cells at middle age that were previously induced by natural infection. At this stage, the immune system is still fit. Given the high conservation of T-cell epitopes within internal viral proteins, such a response may confer lasting protection against evolving influenza strains at older age, also reducing the high number of influenza immunizations currently required. However, at the time of vaccination, some individuals may have been more recently exposed to IAV than others, which could affect the T-cell response. We therefore investigated the fundamental principle of how the interval between the last infection and booster immunization during middle age influences the CD8+ T-cell response. To model this, female mice were infected at either 6 or 9 months of age and subsequently received a heterosubtypic infection booster at middle age (12 months). Before the booster infection, 6-month-primed mice displayed lower IAV-specific CD8+ T-cell responses in the spleen and lung than 9-month-primed mice. Both groups were better protected against the subsequent heterosubtypic booster infection compared to naïve mice. Notably, despite the different CD8+ T-cell levels between the 6-month- and 9-month-primed mice, we observed comparable responses after booster infection, based on IFNγ responses, and IAV-specific T-cell frequencies and repertoire diversity. Lung-derived CD8+ T cells of 6- and 9-month-primed mice expressed similar levels of tissue-resident memory-T-cell markers 30 days post booster infection. These data suggest that the IAV-specific CD8+ T-cell response after boosting is not influenced by the time post priming.

Background: Respiratory syncytial virus (RSV) is a leading cause of childhood mortality in infants below 6 months of age. In low-income and middle-income countries (LMICs), the public health burden is substantial and resources are limited. It is critical to inform decision makers about effectiveness of new interventions. Methods: We developed a mathematical model where individual RSV subtype A (RSV-A) and B (RSV-B) maternally derived neutralizing titers were predicted at time of birth after maternal vaccination with the RSV prefusion F protein-based vaccine. We estimated the subsequent duration of vaccine-induced immunity and compared this to the age at time of death distribution in the RSV GOLD Mortality Database to predict the potential impact of maternal vaccination on RSV-related childhood mortality. We used country-specific timing of antenatal care visits distributions and mortality estimates to make country-specific predictions for number of cases averted. Findings: The model predicts that on average a neonate born at 40 weeks gestational age will be protected between 6 and 7 months from RSV-A and approximately 5 months from RSV-B related mortality. We estimated the potential impact of RSV-related mortality for in-hospital and out-of-hospital cases in LMICs and predicted that in 51 GAVI-eligible countries maternal vaccination could avert between 55% and 63% of the RSV-related in-hospital mortality cases below 6 months of age. Interpretation: We show that maternal vaccination could substantially decrease RSV-A and RSV-B related in-hospital and out-of-hospital mortality in LMICs in the first 6 months of life.

Background: Psoriasis is an immune-mediated inflammatory skin disease. Psoriasis severity evaluation is important for clinicians in the assessment of disease severity and subsequent clinical decision making. However, no objective biomarker is available for accurately evaluating disease severity in psoriasis. Objective: To define and compare biomarkers of disease severity and progression in psoriatic skin. Methods: We performed proteome profiling to study the proteins circulating in the serum from patients with psoriasis, psoriatic arthritis and ankylosing spondylitis, and transcriptome sequencing to investigate the gene expression in skin from the same cohort. We then used machine learning approaches to evaluate different biomarker candidates across several independent cohorts. In order to reveal the cell-type specificity of different biomarkers, we also analyzed a single-cell dataset of skin samples. In-situ staining was applied for the validation of biomarker expression. Results: We identified that the peptidase inhibitor 3 (PI3) was significantly correlated with the corresponding local skin gene expression, and was associated with disease severity. We applied machine learning methods to confirm that PI3 was an effective psoriasis classifier, Finally, we validated PI3 as psoriasis biomarker using in-situ staining and public datasets. Single-cell data and in-situ staining indicated that PI3 was specifically highly expressed in keratinocytes from psoriatic lesions. Conclusion: Our results suggest that PI3 may be a psoriasis-specific biomarker for disease severity and hyper-keratinization. (c) 2023 The Authors. Published by Elsevier B.V. on behalf of Japanese Society for Investigative Dermatology. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Supplementary Table 1 from In vivo Dynamics of Stable Chronic Lymphocytic Leukemia Inversely Correlate with Somatic Hypermutation Levels and Suggest No Major Leukemic Turnover in Bone Marrow

The European Medicines Agency and the US Food and Drug Administration have recently approved a maternal vaccine for respiratory syncytial virus. The US Food and Drug Administration limits vaccination to later in pregnancy. Mathematical modeling demonstrates that this vaccination window may reduce the global mortality impact of the vaccine by 12%. Policymakers should carefully consider vaccine risks and benefits to safeguard vulnerable infants effectively.

T cells recognize pathogens by their highly specific T-cell receptor (TCR), which can bind small fragments of an antigen presented on the Major Histocompatibility Complex (MHC). Antigens that are provided through vaccination cause specific T cells to respond by expanding and forming specific memory to combat a future infection. Quantification of this T-cell response could improve vaccine monitoring or identify individuals with a reduced ability to respond to a vaccination. In this proof-of-concept study we use longitudinal sequencing of the TCRβ repertoire to quantify the response in the CD4+ memory T-cell pool upon pneumococcal conjugate vaccination. This comes with several challenges owing to the enormous size and diversity of the T-cell pool, the limited frequency of vaccine-specific TCRs in the total repertoire, and the variation in sample size and quality. We defined quantitative requirements to classify T-cell expansions and identified critical parameters that aid in reliable analysis of the data. In the context of pneumococcal conjugate vaccination, we were able to detect robust T-cell expansions in a minority of the donors, which suggests that the T-cell response against the conjugate in the pneumococcal vaccine is small and/or very broad. These results indicate that there is still a long way to go before TCR sequencing can be reliably used as a personal biomarker for vaccine-induced protection. Nevertheless, this study highlights the importance of having multiple samples containing sufficient T-cell numbers, which will support future studies that characterize T-cell responses using longitudinal TCR sequencing.

CD8 + T cells are promising targets for vaccination against influenza A virus (IAV) infection. Their induction via peptide vaccination is not trivial, because peptides are weakly immunogenic. One strategy to overcome this is by vaccination with chemically enhanced altered peptide ligands (CPLs), which have improved MHC-binding and immunogenicity. It remains unknown how peptide-modification affects the resulting immune response. We studied the effect of CPLs derived from the influenza M1 58–66 epitope (GILGFVFTL) on the T-cell response. In HLA-A2*0201 transgenic mice, CPL-vaccination led to higher T-cell frequencies, but only a small percentage of the induced T cells recognized the GILG-wildtype (WT) peptide. CPL-vaccination resulted in a lower richness of the GILG-WT-specific T-cell repertoire and no improved protection against IAV-infection compared to GILG-WT peptide-vaccination. One CPL even appeared to enhance pathology after IAV-challenge. CPL-vaccination thus induces T cells not targeting the original peptide, which may lead to potential unwanted side effects.

It is well-known that the functioning of the immune system gradually deteriorates with age, and we are increasingly confronted with its consequences as the life expectancy of the human population increases. Changes in the T-cell pool are among the most prominent features of the changing immune system during healthy ageing, and changes in the naive T-cell pool in particular are generally held responsible for its gradual deterioration. These changes in the naive T-cell pool are thought to be due to involution of the thymus. It is commonly believed that the gradual loss of thymic output induces compensatory mechanisms to maintain the number of naive T cells at a relatively constant level, and induces a loss of diversity in the T-cell repertoire. Here we review the studies that support or challenge this widely-held view of immune ageing and discuss the implications for vaccination strategies.

Memory T cells form an essential part of immunological memory, which can last for years or even a lifetime. Much experimental work has shown that the individual cells that make up the memory T-cell pool are in fact relatively short-lived. Memory T cells isolated from the blood of humans, or the lymph nodes and spleen of mice, live about 5-10 fold shorter than naive T cells, and much shorter than the immunological memory they convey. The commonly accepted view is, therefore, that long-term T-cell memory is maintained dynamically rather than by long-lived cells. This view is largely based on memory T cells in the circulation, identified using rather broad phenotypic markers, and on research in mice living in overly clean conditions. We wondered to what extent there may be heterogeneity in the dynamics and lifespans of memory T cells. We here review what is currently known about the dynamics of memory T cells in different memory subsets, locations in the body and conditions of microbial exposure, and discuss how this may be related to immunometabolism and how this knowledge can be used in various clinical settings.

Supplementary Table 2 from In vivo Dynamics of Stable Chronic Lymphocytic Leukemia Inversely Correlate with Somatic Hypermutation Levels and Suggest No Major Leukemic Turnover in Bone Marrow

Lymphocyte numbers naturally change through age. Normalization functions to account for this are sparse and mostly disregard measurements from children in which these changes are most prominent. In this study, we analyze cross-sectional numbers of mainly T lymphocytes (CD3(+), CD3(+) CD4(+), and CD3(+)CD8(+)) and their subpopulations (naive and memory) from 673 healthy Dutch individuals ranging from infancy to adulthood (0-62 y). We fitted the data by a delayed exponential function and estimated parameters for each lymphocyte subset. Our modeling approach follows general laboratory measurement procedures in which absolute cell counts of T lymphocyte subsets are calculated from observed percentages within a reference population that is truly counted (typically the total lymphocyte count). Consequently, we obtain one set of parameter estimates per T cell subset representing both the trajectories of their counts and percentages. We allow for an initial time delay of half a year before the total lymphocyte counts per microliter of blood start to change exponentially, and we find that T lymphocyte trajectories tend to increase during the first half a year of life. Thus, our study provides functions describing the general trajectories of T lymphocyte counts and percentages of the Dutch population. These functions provide important references to study T lymphocyte dynamics in disease, and they allow one to quantify losses and gains in longitudinal data, such as the CD4(+) T cell decline in HIV-infected children and/or the rate of T cell recovery after the onset of treatment.

The fact that T-cell numbers remain relatively stable throughout life, and that T-cell proliferation rates increase during lymphopenia, has led to the consensus that T-cell numbers are regulated in a density-dependent manner. Competition for resources among memory T cells has been proposed to underlie this ‘homeostatic’ regulation. We first review how two classic models of resource competition affect the T-cell receptor (TCR) diversity of the memory T-cell pool. First, ‘global’ competition for cytokines leads to a skewed repertoire that tends to be dominated by the very first immune response. Second, additional ‘cognate’ competition for specific antigens results in a very diverse and stable memory T-cell pool, allowing every antigen to be remembered, which we therefore define as the ‘gold-standard’. Because there is limited evidence that memory T cells of the same specificity compete more strongly with each other than with memory T cells of different specificities, i.e., for ‘cognate’ competition, we investigate whether cellular aging could account for a similar level of TCR diversity. We define cellular aging as a declining cellular fitness due to reduced proliferation. We find that the gradual erosion of previous T-cell memories due to cellular aging allows for better establishment of novel memories and for a much higher level of TCR diversity compared to global competition. A small continual source (either from stem-cell-like memory T-cells or from naive T-cells due to repeated antigen exposure) improves the diversity of the memory T-cell pool, but remarkably, only in the cellular aging model. We further show that the presence of a source keeps the inflation of chronic memory responses in check by maintaining the immune memories to non-chronic antigens. We conclude that cellular aging along with a small source provides a novel and immunologically realistic mechanism to achieve and maintain the ‘gold-standard’ level of TCR diversity in the memory T-cell pool.

The potential of memory T cells to provide protection against reinfection is beyond question. Yet, it remains debated whether long-term T cell memory is due to long-lived memory cells. There is ample evidence that blood-derived memory phenotype CD8+ T cells maintain themselves through cell division, rather than through longevity of individual cells. It has recently been proposed, however, that there may be heterogeneity in the lifespans of memory T cells, depending on factors such as exposure to cognate Ag. CMV infection induces not only conventional, contracting T cell responses, but also inflationary CD8+ T cell responses, which are maintained at unusually high numbers, and are even thought to continue to expand over time. It has been proposed that such inflating T cell responses result from the accumulation of relatively long-lived CMV-specific memory CD8+ T cells. Using in vivo deuterium labeling and mathematical modeling, we found that the average production rates and expected lifespans of mouse CMV-specific CD8+ T cells are very similar to those of bulk memory-phenotype CD8+ T cells. Even CMV-specific inflationary CD8+ T cell responses that differ 3-fold in size were found to turn over at similar rates.

Psoriasis is a chronic inflammatory skin disorder. Although it has been studied extensively, the molecular mechanisms driving the disease remain unclear. In this study, we utilized a tree-based machine learning approach to explore the gene regulatory networks underlying psoriasis. We then validated the regulators and their networks in an independent cohort. We identified some key regulators of psoriasis, which are candidates to serve as potential drug targets and disease severity biomarkers. According to the gene regulatory network that we identified, we suggest that interferon signaling represents a key pathway of psoriatic inflammation.