Better safe than sorry: Naive T-cell dynamics in healthy ageing.

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.

Memories that last: Dynamics of memory T cells throughout the body.

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.

Human neutrophil kinetics: a call to revisit old evidence.

The half-life of human neutrophils is still controversial, with estimates ranging from 7-9 h to 3.75 days. This debate should be settled to understand neutrophil production in the bone marrow (BM) and the potential and limitations of emergency neutropoiesis following infection or trauma. Furthermore, cellular lifespan greatly influences the potential effect(s) neutrophils have on the adaptive immune response. We posit that blood neutrophils are in exchange with different tissues, but particularly the BM, as it contains the largest pool of mature neutrophils. Furthermore, we propose that the oldest neutrophils are the first to die following a so-called conveyor belt model. These guiding principles shed new light on our interpretation of existing neutrophil lifespan data and offer recommendations for future research.

Turnover of Murine Cytomegalovirus-Expanded CD8+ T Cells Is Similar to That of Memory Phenotype T Cells and Independent of the Magnitude of the Response.

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.

Quantification of T-cell dynamics during latent cytomegalovirus infection in humans.

Cytomegalovirus (CMV) infection has a major impact on the T-cell pool, which is thought to be associated with ageing of the immune system. The effect on the T-cell pool has been interpreted as an effect of CMV on non-CMV specific T-cells. However, it remains unclear whether the effect of CMV could simply be explained by the presence of large, immunodominant, CMV-specific memory CD8+ T-cell populations. These have been suggested to establish through gradual accumulation of long-lived cells. However, little is known about their maintenance. We investigated the effect of CMV infection on T-cell dynamics in healthy older adults, and aimed to unravel the mechanisms of maintenance of large numbers of CMV-specific CD8+ T-cells. We studied the expression of senescence, proliferation, and apoptosis markers and quantified the in vivo dynamics of CMV-specific and other memory T-cell populations using in vivo deuterium labelling. Increased expression of late-stage differentiation markers by CD8+ T-cells of CMV+ versus CMV- individuals was not solely explained by the presence of large, immunodominant CMV-specific CD8+ T-cell populations. The lifespans of circulating CMV-specific CD8+ T-cells did not differ significantly from those of bulk memory CD8+ T-cells, and the lifespans of bulk memory CD8+ T-cells did not differ significantly between CMV- and CMV+ individuals. Memory CD4+ T-cells of CMV+ individuals showed increased expression of late-stage differentiation markers and decreased Ki-67 expression. Overall, the expression of senescence markers on T-cell populations correlated positively with their expected in vivo lifespan. Together, this work suggests that i) large, immunodominant CMV-specific CD8+ T-cell populations do not explain the phenotypical differences between CMV+ and CMV- individuals, ii) CMV infection hardly affects the dynamics of the T-cell pool, and iii) large numbers of CMV-specific CD8+ T-cells are not due to longer lifespans of these cells.

Current best estimates for the average lifespans of mouse and human leukocytes: reviewing two decades of deuterium-labeling experiments.

Deuterium is a non-toxic, stable isotope that can safely be administered to humans and mice to study their cellular turnover rates in vivo. It is incorporated into newly synthesized DNA strands during cell division, without interference with the kinetics of cells, and the accumulation and loss of deuterium in the DNA of sorted (sub-)populations of leukocytes can be used to estimate their cellular production rates and lifespans. In the past two decades, this powerful technology has been used to estimate the turnover rates of various types of leukocytes. Although it is the most reliable technique currently available to study leukocyte turnover, there are remarkable differences between the cellular turnover rates estimated by some of these studies. We have recently established that part of this variation is due to (a) difficulties in estimating deuterium availability in some deuterium-labeling studies, and (b) assumptions made by the mathematical models employed to fit the data. Being aware of these two problems, we here aim to approach a consensus on the life expectancies of different types of T cells, B cells, monocytes, and neutrophils in mice and men. We address remaining outstanding problems whenever appropriate and discuss for which immune subpopulations we currently have too little information to draw firm conclusions about their turnover.

Maintenance of peripheral naive T cells is sustained by thymus output in mice but not humans.

Parallels between T cell kinetics in mice and men have fueled the idea that a young mouse is a good model system for a young human, and an old mouse, for an elderly human. By combining in vivo kinetic labeling using deuterated water, thymectomy experiments, analysis of T cell receptor excision circles and CD31 expression, and mathematical modeling, we have quantified the contribution of thymus output and peripheral naive T cell division to the maintenance of T cells in mice and men. Aging affected naive T cell maintenance fundamentally differently in mice and men. Whereas the naive T cell pool in mice was almost exclusively sustained by thymus output throughout their lifetime, the maintenance of the adult human naive T cell pool occurred almost exclusively through peripheral T cell division. These findings put constraints on the extrapolation of insights into T cell dynamics from mouse to man and vice versa.

Human CD62Ldim neutrophils identified as a separate subset by proteome profiling and in vivo pulse-chase labeling.

During acute inflammation, 3 neutrophil subsets are found in the blood: neutrophils with a conventional segmented nucleus, neutrophils with a banded nucleus, and T-cell-suppressing CD62Ldim neutrophils with a high number of nuclear lobes. In this study, we compared the in vivo kinetics and proteomes of banded, mature, and hypersegmented neutrophils to determine whether these cell types represent truly different neutrophil subsets or reflect changes induced by lipopolysaccharide (LPS) activation. Using in vivo pulse-chase labeling of neutrophil DNA with 6,6-2H2-glucose, we found that 2H-labeled banded neutrophils appeared much earlier in blood than labeled CD62Ldim and segmented neutrophils, which shared similar label kinetics. Comparison of the proteomes by cluster analysis revealed that CD62Ldim neutrophils were clearly separate from conventional segmented neutrophils despite having similar kinetics in peripheral blood. Interestingly, the conventional segmented cells were more related at a proteome level to banded cells despite a 2-day difference in maturation time. The differences between CD62Ldim and mature neutrophils are unlikely to have been a direct result of LPS-induced activation, because of the extremely low transcriptional capacity of CD62Ldim neutrophils and the fact that neutrophils do not directly respond to the low dose of LPS used in the study (2 ng/kg body weight). Therefore, we propose CD62Ldim neutrophils are a truly separate neutrophil subset that is recruited to the bloodstream in response to acute inflammation. This trial was registered at www.clinicaltrials.gov as #NCT01766414.

Human neonatal thymectomy induces altered B-cell responses and autoreactivity.

An association between T-cell lymphopenia and autoimmunity has long been proposed, but it remains to be elucidated whether T-cell lymphopenia affects B-cell responses to autoantigens. Human neonatal thymectomy (Tx) results in a decrease in T-cell numbers and we used this model to study the development of autoreactivity. Two cohorts of neonatally thymectomized individuals were examined, a cohort of young (1-5 years post-Tx, n = 10-27) and older children (>10 years, n = 26), and compared to healthy age-matched controls. T-cell and B-cell subsets were assessed and autoantibody profiling performed. Early post-Tx, a decrease in T-cell numbers (2.75 × 109 /L vs. 0.71 × 109 /L) and an increased proportion of memory T cells (19.72 vs. 57.43%) were observed. The presence of autoantibodies was correlated with an increased proportion of memory T cells in thymectomized children. No differences were seen in percentages of different B-cell subsets between the groups. The autoantigen microarray showed a skewed autoantibody response after Tx. In the cohort of older individuals, autoantibodies were present in 62% of the thymectomized children, while they were found in only 33% of the healthy controls. Overall, our data suggest that neonatal Tx skews the autoantibody profile. Preferential expansion and preservation of Treg (regulatory T) cell stability and function, may contribute to preventing autoimmune disease development after Tx.

Reconciling Estimates of Cell Proliferation from Stable Isotope Labeling Experiments.

Stable isotope labeling is the state of the art technique for in vivo quantification of lymphocyte kinetics in humans. It has been central to a number of seminal studies, particularly in the context of HIV-1 and leukemia. However, there is a significant discrepancy between lymphocyte proliferation rates estimated in different studies. Notably, deuterated (2)H2-glucose (D2-glucose) labeling studies consistently yield higher estimates of proliferation than deuterated water (D2O) labeling studies. This hampers our understanding of immune function and undermines our confidence in this important technique. Whether these differences are caused by fundamental biochemical differences between the two compounds and/or by methodological differences in the studies is unknown. D2-glucose and D2O labeling experiments have never been performed by the same group under the same experimental conditions; consequently a direct comparison of these two techniques has not been possible. We sought to address this problem. We performed both in vitro and murine in vivo labeling experiments using identical protocols with both D2-glucose and D2O. This showed that intrinsic differences between the two compounds do not cause differences in the proliferation rate estimates, but that estimates made using D2-glucose in vivo were susceptible to difficulties in normalization due to highly variable blood glucose enrichment. Analysis of three published human studies made using D2-glucose and D2O confirmed this problem, particularly in the case of short term D2-glucose labeling. Correcting for these inaccuracies in normalization decreased proliferation rate estimates made using D2-glucose and slightly increased estimates made using D2O; thus bringing the estimates from the two methods significantly closer and highlighting the importance of reliable normalization when using this technique.

Novel mutations in TNFRSF7/CD27: Clinical, immunologic, and genetic characterization of human CD27 deficiency.

BACKGROUND: The clinical and immunologic features of CD27 deficiency remain obscure because only a few patients have been identified to date. OBJECTIVE: We sought to identify novel mutations in TNFRSF7/CD27 and to provide an overview of clinical, immunologic, and laboratory phenotypes in patients with CD27 deficiency. METHODS: Review of the medical records and molecular, genetic, and flow cytometric analyses of the patients and family members were performed. Treatment outcomes of previously described patients were followed up. RESULTS: In addition to the previously reported homozygous mutations c.G24A/p.W8X (n = 2) and c.G158A/p.C53Y (n = 8), 4 novel mutations were identified: homozygous missense c.G287A/p.C96Y (n = 4), homozygous missense c.C232T/p.R78W (n = 1), heterozygous nonsense c.C30A/p.C10X (n = 1), and compound heterozygous c.C319T/p.R107C-c.G24A/p.W8X (n = 1). EBV-associated lymphoproliferative disease/hemophagocytic lymphohistiocytosis, Hodgkin lymphoma, uveitis, and recurrent infections were the predominant clinical features. Expression of cell-surface and soluble CD27 was significantly reduced in patients and heterozygous family members. Immunoglobulin substitution therapy was administered in 5 of the newly diagnosed cases. CONCLUSION: CD27 deficiency is potentially fatal and should be excluded in all cases of severe EBV infections to minimize diagnostic delay. Flow cytometric immunophenotyping offers a reliable initial test for CD27 deficiency. Determining the precise role of CD27 in immunity against EBV might provide a framework for new therapeutic concepts.

Defects in neutrophil granule mobilization and bactericidal activity in familial hemophagocytic lymphohistiocytosis type 5 (FHL-5) syndrome caused by STXBP2/Munc18-2 mutations.

Familial hemophagocytic lymphohistiocytosis (FHL) is caused by genetic defects in cytotoxic granule components or their fusion machinery, leading to impaired natural killer cell and/or T lymphocyte degranulation and/or cytotoxicity. This may accumulate into a life-threatening condition known as macrophage activation syndrome. STXBP2, also known as MUNC18-2, has recently been identified as the disease-causing gene in FHL type 5 (FHL-5). A role for STXBP2 in neutrophils, and for neutrophils in FHL in general, has not been documented thus far. Here, we report that FHL-5 neutrophils have a profound defect in granule mobilization, resulting in inadequate bacterial killing, in particular, of gram-negative Escherichia coli, but not of Staphylococcus aureus, which rather depends on intact reduced NAD phosphate oxidase activity. This impairment of bacterial killing may contribute to the apparent susceptibility to gastrointestinal tract inflammation in patients with FHL-5.

Closing the gap between T-cell life span estimates from stable isotope-labeling studies in mice and humans.

Quantitative knowledge of the turnover of different leukocyte populations is a key to our understanding of immune function in health and disease. Much progress has been made thanks to the introduction of stable isotope labeling, the state-of-the-art technique for in vivo quantification of cellular life spans. Yet, even leukocyte life span estimates on the basis of stable isotope labeling can vary up to 10-fold among laboratories. We investigated whether these differences could be the result of variances in the length of the labeling period among studies. To this end, we performed deuterated water-labeling experiments in mice, in which only the length of label administration was varied. The resulting life span estimates were indeed dependent on the length of the labeling period when the data were analyzed using a commonly used single-exponential model. We show that multiexponential models provide the necessary tool to obtain life span estimates that are independent of the length of the labeling period. Use of a multiexponential model enabled us to reduce the gap between human T-cell life span estimates from 2 previously published labeling studies. This provides an important step toward unambiguous understanding of leukocyte turnover in health and disease.

Dynamics of Low-Level Viremia and Immune Activation after Switching to a Darunavir-Based Regimen.

There is an ongoing debate regarding whether low-level viremia (LLV), in particular persistent LLV, during HIV treatment with optimal adherence originates from low-level viral replication, viral production, or both. We performed an observational study in 30 individuals with LLV who switched to a boosted darunavir (DRV)-based therapy. In-depth virological analyses were used to characterize the viral population and the (activity) of the viral reservoir. Immune activation was examined using cell-bound and soluble markers. The primary outcome was defined as the effect on HIV-RNA and was categorized by responders (<50 cp/mL) or non-responders (>50 cp/mL). At week 24, 53% of the individuals were considered responders, 40% non-responders, and 7% could not be assigned. Sequencing showed no evolution or selection of drug resistance in the non-responders. Production of defective virus with mutations in either the protease (D25N) or RT active site contributed to persistent LLV in two individuals. We show that in about half of the study participants, the switch to a DRV-based regimen resulted in a viral response indicative of ongoing low-level viral replication as the cause of LLV before the switch. Our data confirm that in clinical management, high genetic barrier drugs like DRV are a safe choice, irrespective of the source of LLV.

Long-term restoration of the human T-cell compartment after thymectomy during infancy: a role for thymic regeneration?

Thymectomy during early childhood is generally thought to have serious consequences for the establishment of the T-cell compartment. In the present study, we investigated the composition of the T-cell pool in the first 3 decades after thymectomy during infancy due to cardiac surgery. In the first 5 years after thymectomy, naive and total CD4(+) and CD8(+) T-cell numbers in the blood and T-cell receptor excision circle (TREC) levels in CD4(+) T cells were significantly lower than in healthy age-matched controls. In the first years after thymectomy, plasma IL-7 levels were significantly elevated and peripheral T-cell proliferation levels were increased by ∼ 2-fold. From 5 years after thymectomy onward, naive CD4(+) and CD8(+) T-cell counts and TRECs were within the normal range. Because TREC levels are expected to decline continuously in the absence of thymic output, we investigated whether normalization of the naive T-cell pool could be due to regeneration of thymic tissue. In the majority of individuals who had been thymectomized during infancy, thymic tissue could indeed be identified on magnetic resonance imaging scans. Whereas thymectomy has severe effects on the establishment of the naive T-cell compartment during early childhood, our data suggest that functional regrowth of thymic tissue can limit its effects in subsequent years.

Decreased thymic output accounts for decreased naive T cell numbers in children with Down syndrome.

Children with Down syndrome (DS) have low numbers of naive T cells and abnormal thymus development and function. Because next to thymic production, peripheral proliferation greatly contributes to naive T cell generation in healthy children, we examined the cause of reduced naive T cell numbers in children with DS. Compared with aged matched controls, the total number of signal joint TCR excision circles (sjTREC) per ml blood was reduced in DS. Reduced frequencies and absolute numbers of protein tyrosine kinase 7-positive recent thymic emigrants, but similar levels of naive T cell apoptosis and Ag-driven activation in DS, suggested that reduced thymic output and not increased peripheral loss of naive T cells caused the reduced sjTREC numbers. We found no support for defective peripheral generation of naive T cells in DS. In DS the naive T cells responded to IL-7 and, based on Ki-67 expression, had similar proliferation rates as in healthy controls. sjTREC content per naive CD8(+) T cells was not increased, but even decreased, pointing to increased survival or peripheral generation of naive T cells in DS. In conclusion, we show in this study that reduced thymic output, but not reduced peripheral generation nor increased loss of naive T cells, results in the low naive T cell numbers found in DS.

CD27 deficiency is associated with combined immunodeficiency and persistent symptomatic EBV viremia.

BACKGROUND: CD27 is a lymphocyte costimulatory molecule that regulates T-cell, natural killer (NK) cell, B-cell, and plasma cell function, survival, and differentiation. On the basis of its function and expression pattern, we considered CD27 a candidate gene in patients with hypogammaglobulinemia. OBJECTIVE: We sought to describe the clinical and immunologic phenotypes of patients with genetic CD27 deficiency. METHODS: A molecular and extended immunologic analysis was performed on 2 patients lacking CD27 expression. RESULTS: We identified 2 brothers with a homozygous mutation in CD27 leading to absence of CD27 expression. Both patients had persistent symptomatic EBV viremia. The index patient was hypogammaglobulinemic, and immunoglobulin replacement therapy was initiated. His brother had aplastic anemia in the course of his EBV infection and died from fulminant gram-positive bacterial sepsis. Immunologically, lack of CD27 expression was associated with impaired T cell-dependent B-cell responses and T-cell dysfunction. CONCLUSION: Our findings identify a role for CD27 in human subjects and suggest that this deficiency can explain particular cases of persistent symptomatic EBV viremia with hypogammaglobulinemia and impaired T cell-dependent antibody generation.

A generalized mathematical model to estimate T- and B-cell receptor diversities using AmpliCot.

The efficiency of the adaptive immune system is dependent on the diversity of T- and B-cell receptors, which is created by random rearrangement of receptor gene segments. AmpliCot is an experimental technique that allows the measurement of the diversity of the T- and B-cell repertoire. This procedure has the advantage over other cloning and sequencing techniques of being time- and expense-effective. In previous studies, receptor diversity, measured with AmpliCot, has been inferred assuming a second-order kinetics model. The latter implies that the relation between diversity and concentration × time (Cot) values is linear. We show that a more detailed model, involving heteroduplex and transient-duplex formation, leads to significantly better fits of experimental data and to nonlinear diversity-Cot relations. We propose an alternative fitting procedure, which is straightforward to apply and which gives an improved description of the relationship between Cot values and diversity.