The Hayflick Limit May Determine the Effective Clonal Diversity of Naive T

The Hayflick Limit May Determine the Effective Clonal Diversity of Naive T Cells Wilfred Ndifon and Jonathan Dushoff This information is current as J Immunol 2016; 196:4999-5004; Prepublished online 13 of September 25, 2021. May 2016; doi: 10.4049/jimmunol.1502343 http://www.jimmunol.org/content/196/12/4999 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

The Hayﬂick Limit May Determine the Effective Clonal Diversity of Naive T Cells

Wilfred Ndifon*,†,‡ and Jonathan Dushoffx

Having a large number of sufficiently abundant T cell clones is important for adequate protection against diseases. However, as shown in this paper and elsewhere, between young adulthood and >70 y of age the effective clonal diversity of naive CD4/CD8 T cells found in human blood declines by a factor of >10. (Effective clonal diversity accounts for both the number and the abundance of T cell clones.) The causes of this observation are incompletely understood. A previous study proposed that it might result from the emergence of certain rare, replication-enhancing mutations in T cells. In this paper, we propose an even simpler explanation: that it results from the loss of T cells that have attained replicative senescence (i.e., the Hayflick limit). Stochastic numerical simulations of naive T cell population dynamics, based on experimental parameters, show that the rate of homeostatic ∼ T cell proliferation increases after the age of 60 y because naive T cells collectively approach replicative senescence. This leads to Downloaded from a sharp decline of effective clonal diversity after ∼70 y, in agreement with empirical data. A mathematical analysis predicts that, without an increase in the naive T cell proliferation rate, this decline will occur >50 yr later than empirically observed. These results are consistent with a model in which exhaustion of the proliferative capacity of naive T cells causes a sharp decline of their effective clonal diversity and imply that therapeutic potentiation of thymopoiesis might either prevent or reverse this outcome. The Journal of Immunology, 2016, 196: 4999–5004. http://www.jimmunol.org/ ndividuals must maintain a large diversity of T cell clones to available TCRs results in severely impaired immune regulation remain protected against a vast array of pathogens to which and the development of colitis in mice (15). I they are constantly exposed (1, 2). The CD8 subset of T cells For human naive T cells, TCR sequence data (9, 11, 16) indicate contributes to the elimination of pathogens by killing infected that their effective clonal diversity (defined as the reciprocal of the cells displaying pathogen-derived Ags on their surface, whereas probability that two randomly chosen T cells belong to the same CD4 T cells are key regulators of these and other immune clone) (17) declines sharply after the age of ∼70 years. Note that responses (3). The magnitude of the T cell response to a newly in contrast to measures of diversity that account only for the encountered Ag depends strongly on the frequencies of naive number of different T cell clones, effective clonal diversity ac- T cell clones that have specificity for that Ag (4, 5). However, counts also for clone frequencies. It is important because the by guest on September 25, 2021 the clonal diversity of naive T cells decreases with age (6–11), magnitude of the T cell response to an Ag depends not only on resulting in the loss of Ag-specific clones. This leads to a failure of whether specific clones are present but also on the frequencies of the immune system to recognize certain Ags, including pathogen such clones as well as how efficiently they are recruited into the derived (12), and a higher probability that recognized Ags will response (4, 5). subsequently acquire mutations that abrogate their recognition The mechanistic basis for the sharp decline of effective clonal (13). Indeed, in certain strains of mice, as little as a 50% reduction diversity observed in old age is incompletely understood. A pos- in T cell diversity is sufficient to create large holes in the space of sible explanation is that the epithelial tissue of the human thymus recognized Ags (14), thus impairing their ability to control certain starts to involute at the age of ∼1 year (18), which causes the infections. Although T cell cross-reactivity can in principle number of new T cells that are subsequently produced by the compensate for such holes, this does not happen in many of the thymus to decrease exponentially with age (9, 11, 19). However, cases that have been studied (2). Thus, an intriguing recent study computer simulations show that neither this exponential decrease showed that reducing clonal diversity by restricting the number of in the rate of thymopoeisis nor the age-associated loss of T cells (20) is sufficient to elicit a dramatic decline of effective clonal diversity (21). An alternative explanation was recently proposed *African Institute for Mathematical Sciences, Muizenberg 7945, Cape Town, (21): that certain rare mutations might arise suddenly in particular South Africa; †African Institute for Mathematical Sciences, Legon, Accra, Ghana; ‡Stellenbosch University, Matieland 7602, Stellenbosch, South Africa; and xDepartment T cells and allow them to outgrow other T cells, thus contributing of Biology, McMaster University, Hamilton, Ontario L8S 4K1, Canada to a sharp decrease in overall effective clonal diversity. ORCID: 0000-0003-0506-4794 (J.D.). In this paper, we propose a simpler explanation, based on the fact Received for publication November 4, 2015. Accepted for publication April 18, 2016. that a differentiated cell can undergo only a limited number of This work was supported by a grant from the Canadian International Development mitotic divisions under normal conditions (22). Specifically, dif- Research Center to the African Institute for Mathematical Sciences. ferentiated diploid cells (e.g., T cells) that are in the resting (or Address correspondence and reprint requests to Dr. Wilfred Ndifon, African Institute quiescent) phase of the cell cycle occasionally enter the prolifer- for Mathematical Sciences, 5-7 Melrose Road, Muizenberg 7945, Cape Town, South ation phase during which they divide. During cell division, the Africa. E-mail address: [email protected] DNA replication machinery found inside each cell copies the The online version of this article contains supplemental material. cell’s chromosomes, except certain base pairs found at chromo- Abbreviations used in this article: resp., respectively; s.e., standard error; TREC, somal ends called telomeres (23). Hence, each cell division TCR excision circle. shortens the telomeres (24, 25) until they reach a critical length Copyright 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 (26, 27) that causes the cell to become replicatively senescent. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1502343 5000 NAIVE T CELL PROLIFERATION COMPROMISES DIVERSITY

(Note that there are other ways in which cells can become se- #2 nucleotide differences from the cluster seed were added to the cluster. nescent without first exhausting their replicative capacity; e.g., see These steps were repeated until all sequences were clustered. We used an Ref. 28). Cells with such critically shortened telomeres are said to adaptation of Simpson’s index applicable to replicate sequence datasets (52) to quantify the effective diversity of all inframe CDR3 amino acid have reached the Hayflick limit (22, 29). Evidence that telomeres sequences corresponding to the cluster seeds. We defined effective diver- shorten as cells proliferate was initially obtained in human fibro- sity as the number of equally abundant sequences that will give a particular blasts (22, 29) and later confirmed in other cell types, including value of Simpson’s index, which is mathematically equivalent to the re- naive T cells (30, 31). Although the enzyme telomerase can in ciprocal of this index (17). principle extend telomeres during cell proliferation (32), its ex- Numerical simulation of T cell population dynamics pression level is low in naive T cells and insufficient to prevent Human naive T cell population dynamics were simulated, starting at the age telomeres from shortening in activated T cells (33). Note that of 20 y and continuing until 100 y. We sampled each T cell clone i from the hematopoietic stem cell aging resulting in telomere attrition (34) geometric distribution G(i)=w (1 2 w)i/(1 2 [1 2 w]S) (21), with bias might also contribute to the emergence of peripheral naive T cells parameter w = 0.01. The maximum size of the T cell population was set to 5 with shorter telomeres. The fact that the emergence of these K =53 10 cells, whereas the total number of possible clones was set to S 3 3 K S T cells in humans is accompanied by the loss of TCR excision =1 10 , as in previous work (21). With these values of and , each clone had an average starting size of K/S = 500, corresponding to an initial circles (TRECs) indicates that homeostatic proliferation is an number of cell divisions of log2(500) = 9. This is in line with the empirical important cause of their shorter telomeres (35). estimate of 11 T cell divisions by the age of 20 y, which we obtained by Proliferating naive T cells leave the naive T cell pool by assuming that each T cell loses ∼75 bp of telomere/division (31), and a ∼ 3 differentiating into memory cells or by attaining replicative total of 20 40 bp = 800 bp are lost by the age of 20 y (48). As an illustration of how w affects clonal distributions, we note that the ex- senescence and subsequently being eliminated through both Ag- pected size of clone 1 in the initial T cell population is K 3 G(1)= 5 3 105 3 Downloaded from dependent (36) and -independent (28, 37) mechanisms. For ex- 0.01 3 (1 2 0.01)1/(1 2 [1 2 0.01]1000) = 4950 cells when w = 0.01, but it is ample, replicatively senescent fibroblasts express signals that only 525 cells when w =1024. Note that we obtain qualitatively similar 24 mark them for elimination, notably through phagocytosis (28). (In results even if w is as small as 10 , representing an approximately un- this study, we assume that replicatively senescent T cells also biased clonal distribution. Also, note that increasing K will not change our results as long as S is also increased to maintain the starting clone size. follow the same fate and that they are eliminated with 100% ef- Other parameters used in the simulation are described in Results. The

ficiency. Later, we will show that our conclusions are robust to a simulation algorithm was implemented in R (53) and is available from http://www.jimmunol.org/ 1000-fold reduction of this efficiency.) Because the overall size of W. Ndifon. the naive T cell population is regulated such that it stays ap- Time to reach replicative senescence with a constant T cell proximately constant (38, 39), a substantial T cell loss will trigger proliferation rate compensatory homeostatic proliferation of the remaining cells to b fill the newly freed space. For example, Goldrath et al. (40) found Without a progressive increase in the proliferation rate t and because of homeostatic regulation of the T cell population’s size (38), bt will be that transgenic T cells transferred into C57BL/6 mice in which approximately equal to the T cell turnover rate, d . In this case, a T cell that . t 95% of naive T cells had been removed through sublethal ir- is produced in the thymus at time ti will have undergone on average dt (t – ti) radiation underwent a massive proliferation to fill the empty ho- cell divisions at time t. Taking the distribution of ti to be exp(2rti)/r (18, meostatic space. In humans, T cell proliferation also increases 19), the average number of cell divisions for all T cells can be approxi- by guest on September 25, 2021 after lymphopenia (41). Naive T cells that continue to proliferate mated as Z ‘ will eventually reach the Hayflick limit. As these cells are elim- dt 1 d9 ¼ ðt 2 t Þe2rti dt ¼ d t 2 : ð Þ r i i t r 1 inated, others will further proliferate to fill the space left empty 0 and subsequently disappear. We propose that over time this dynamic will cause a chain reaction of cell loss, which will suddenly From equation 1, the expected time at which the average number of cell divisions for all T cells will be equal to the Hayflick limit H can be ap- drive most T cell clones to extinction. In the following, we will proximated as tc = H/dt +1/r. Substituting the values of H, r, and dt used in demonstrate, using stochastic numerical simulations based on the simulations into this equation, we find that tc = ∼125 y. experimental data (18, 21, 22, 28, 29, 31, 38, 42–48), that this A mathematical estimate of the T cell proliferation rate model provides a plausible explanation for the sharp decline of effective clonal diversity observed in elderly humans. We develop a simple mathematical instance of our simulations of T cell population dynamics. We assume that naive T cells divide synchronously Materials and Methods at defined cell cycling time intervals. The number of T cells that are in the resting phase at the time interval t is denoted Nt. Resting T cells with state x Data analysis enter the proliferation phase at the average rate bt. By “state” we mean characteristics of a T cell (e.g., its Ag specificity and expressed cytokines) We adapted our published bioinformatics pipeline (46) to analyze a large, that are important for its homeostasis. A T cell with state x that enters the previously published TCR sequence database (16) (dbGap accession proliferation phase either divides, thereby producing two daughter cells number phs000787.v1.p1). The data were obtained via high-throughput with the same state, or is lost with a probability m (x), if it has already TCR sequencing of samples of naive CD4 and CD8 T cells isolated t reached the Hayflick limit (22, 29). from the blood of nine individuals aged 20–83 y. Five biological replicates New T cells with state x are produced by the thymus at the rate et(x). In of each donor’s CD4 and CD8 T cell samples were analyzed, resulting in a addition, T cells have an average turnover rate d (e.g., because of apo- total of 90 paired-end nucleotide sequence read datasets. We used the t ptosis) at time interval t. Therefore, the total number of resting T cells paired-end read merger software (49) to merge the paired-end reads into changes from one time interval to the next one as single reads. We then mapped individual reads to reference TCRBV and Z Z TCRBJ gene segments found in ImMunoGeneTics (50) using the Smith– Waterman pairwise sequence alignment algorithm (51). This mapping Ntþ1 ¼ Nt 1 2 dt þ bt ftðxÞ 1 2 2mtðxÞ dx þ εðxÞdx; ð2Þ provided the translation start site for each nucleotide sequence, allowing us to reconstruct the corresponding amino acid sequence. where ft(x) is the probability density of T cells with state x found at time The CDR3 region of each sequence was defined as starting from the interval t. This equation implies that the T cell proliferation rate is given by second conserved cysteine found near the 39 end of TCRBV, and ending at 9 R the 5 conserved phenylalanine of TCRBJ. We clustered CDR3 nucleotide d þ N 2 εðxÞdx N 2 sequences assigned the same Vb and Jb gene segments to correct up to 2 t tþ1 t 1 bt ¼ ; ð3Þ nucleotide misincorporation errors that occurred during the sequencing 1 2 Æ2mtæ reaction, using the following steps: 1) the sequence with the most identical C copies was chosen to seed a new cluster, and 2) unclustered sequences with where Æmt æ (,1/2) denotes the expected value of mt(x). The Journal of Immunology 5001

Results We proposed earlier that the Hayflick limit determines the sharp decline of effective clonal diversity observed in individuals aged . 70 y. In this study, we use numerical simulations to further assess the deductive validity of this proposal. We begin by examining the empirical variation of effective clonal diversity with age. Effective clonal diversity declines sharply in old age We use a large database of CDR3 amino acid sequences (16) from the TCR b-chain to estimate how the effective number of naive CD4 and CD8 T cell clones found in human blood varies with age (Materials and Methods). T cell clonal diversity is mostly determined by variability of the TCR, the largest contribution to which is made by the CDR3 region of the TCR b-chain (54). Thus, we use the effective diversity of CDR3 amino acid sequences as a lower bound for the effective clonal diversity of T cells. (This estimate is a lower bound because it does not capture the diversity contained outside of the CDR3 region of the TCR b-chain. Nev- ertheless, the relative change in this estimate should reflect the Downloaded from relative change in clonal diversity). By analogy with the estimation FIGURE 1. Effective clonal diversity of naive T cells declines with age. of effective species diversity in ecology (17), we define the effective We used five replicate TCRB CDR3 amino acid sequence datasets (16) diversity of CDR3 amino acid sequences as the reciprocal of the from both naive CD4 (left panel) and CD8 (right panel) T cells to estimate probability that any two sequences randomly chosen from the same effective clonal diversity (Materials and Methods). r denotes the correla- individual are identical. We find that effective clonal diversity de- tion coefficient between effective clonal diversity and age. Each p value creases significantly with age, for both naive CD4 (correlation co- was computed by permuting randomly the corresponding estimates of http://www.jimmunol.org/ efficient: 20.70, p = 0.0043; Fig. 1, left panel)andnaiveCD8 effective clonal diversity and then comparing the resulting correlation (correlation coefficient: 20.95, p = 0.0049; Fig. 1, right panel) coefficient to that obtained using unpermuted estimates. T cells. For naive CD4 T cells, we estimate that there are ∼2.0 3 106 (standard error [s.e.] 7.5 3 105) CDR3 sequences in young adults aged 20–35 y but only ∼1.7 3 105 (s.e. ∼3.6 3 104)inel- We initialize each simulation with a population of K naive T cells, derly individuals aged 70–83 y (Fig. 1, left panel). Similarly, there each sampled randomly from S possible distinct T cell clones. We are ∼2.0 3 106 (s.e. ∼2.1 3 105) naive CD8 T cell CDR3 sequences sample individual clones from the distribution G(i), where i, i = 1,..., in young adults but only ∼7.5 3 104 (s.e. ∼5.8 3 104) in the elderly S, is a clonal index, and G(i) is the expected frequency of clone i. by guest on September 25, 2021 (Fig. 1, right panel). This indicates that there is a ∼ 10 (respectively Empirical data indicate that different clones have very different a priori production frequencies (60), with the fold difference between [resp.] ∼ 30) fold decline in the effective clonal diversity of naive the highest and lowest frequencies being on the order of 104 CD4 (resp. CD8) T cells in elderly individuals compared with (46, 47). This implies that G(i) must be highly biased. The one- young adults. For CD8 T cells, this decline is ∼ 125 fold when one parameter geometric distribution fits reasonably well the experi- outlying elderly individual (who has an exceptionally high effective mentally measured frequencies of T cell clones expressing different number of CDR3 sequences) is removed from the analysis. TCR b-chains (61). Therefore, to investigate the effects of bias, we Simulations predict a sharp decline of effective clonal diversity. To set G(i) equal to the following geometric distribution with bias assess our proposed explanation for the sharp decline of effective parameter w (21): G(i)=w (1 2 w)i/(1 2 [1 2 w]S). This distri- clonal diversity observed in old age, we simulate the population bution is approximately uniform (i.e., unbiased) for very small w, dynamics of naive T cells found in the peripheral lymphoid organs but it is biased in favor of clones with small indices for larger w.We of a human (e.g., the blood and spleen), starting in early adulthood use a value of w for which the ratio of the highest to the lowest ∼ 2 ( 20 y), when the total size of the T cell population is expected to clone frequencies is ∼104—i.e., w =12 104/(1 S).Wewillshow be under homeostatic control (38). Because the thymic epithelium that similar results are produced by using a smaller value of w,for involutes before adulthood (18), the rate at which new T cells are which G(i) is approximately uniform (i.e., unbiased). produced by the thymus decreases exponentially with age so that In addition, we assume that the naive T cell population changes it becomes negligible after ∼40 y (9, 11, 19). Therefore, the dy- at defined time intervals because of emigration from the thymus, namics of the adult human T cell population is largely determined elimination of replicatively senescent T cells, and homeostatic by changes to existing T cells and not by thymic production of turnover (including T cell death because of other causes). At each new T cells (55). time interval t, a clone i, of size K/S, enters the T cell population Note that in the following analyses we do not assume that with probability et(i). The initial clone size is determined by both thymopoesis ceases after the age of 40 y. Previous studies have intrathymic and extrathymic cell division. Accordingly, we set approximated the rate of thymopoiesis by measuring the abundance the number of such cell divisions equal to the base 2 logarithm of of TRECs. Mathematical modeling indicates that TREC abundance the initial clone size. Experiments suggest that et(i) decreases depends not only on thymopoeisis but also on T cell proliferation and exponentially with time (9, 18, 19). Hence, we define 2t=T 21 death (56, 57). Nevertheless, with the exception of one study (58), etðiÞ¼te GðiÞ, with T = 33.33 yr (18) and t = 0.0017 d most others (9, 35, 55, 59) indicate that TREC abundance declines (21). In addition, experiments indicate that the daily turnover rate with age in accordance with the rate of thymic involution described of naive T cells in adult humans ranges from ∼0.1 to ∼0.2% (42– by Steinmann et al. (18). Like previous researchers (21, 57), we 44). A much lower estimate of the T cell turnover rate was pre- assume that this rate of thymic involution implies a comparable rate viously reported (62), but a mathematical model that fits the same of decline of thymopoiesis beginning in early adulthood. experimental data much better yields an estimate that is consistent 5002 NAIVE T CELL PROLIFERATION COMPROMISES DIVERSITY with those cited above (45). Therefore, we assume that T cells are deed, the average number of proliferations per T cell per decade 21 lost from the population at the average rate dt = 0.15% day . doubles between the ages of 30 and 70 y and it subsequently T cells are also lost if they have divided .50 times, which is the oscillates (Fig. 2A). This is accompanied by a dramatic increase in Hayflick limit (22, 29). To simulate homeostasis (38), by main- the abundance of a small subset of T cells, whose total relative taining the T cell population’s size at time t, Nt, at the carrying abundance is ∼100% at the age of 60 y. Importantly, effective capacity K, each T cell is replicated with probability rate bt = clonal diversity declines to less than a tenth of its initial value after (1 – Nt/K), producing another cell with the same clonality as the ∼70 y (Fig. 2C). The results are consistent with experimental data parent cell. (By carrying capacity we mean the maximum T cell (9) showing that naive CD4 T cell proliferation rate doubles after population size that an individual normally supports.) Further the age of 70 y coincident with a sharp decline of T cell diversity. details about the simulations are described in Materials and These results, which were obtained by assuming that there is a Methods. strong bias in the production frequencies of different T cell clones We quantify the effective clonal diversity of the T cell population (i.e., w = 0.01) consistent with empirical data (46, 47, 60), are using the reciprocal of the Gini–Simpson diversity index (17). We similar to results obtained without such bias (Supplemental Fig. 1). calculated the effective clonal diversity of the simulated T cell In addition, we obtain similar results as shown in Fig. 2 by population once every 90 d during the time period from t =20 assuming that replicatively senescent T cells are eliminated from to t = 100 y. In addition, we also calculated both the average rate the human body at the rate of only 0.1% day21, which is 1000- of T cell homeostatic proliferation and the average number of fold lower than we assumed earlier (Supplemental Fig. 2). In this T cell divisions at each of these time points. The results are plotted case, we see that the frequency of senescent cells increases over in Fig. 2. time, reaching a maximum at ∼80 y of age and cycling thereafter Downloaded from The results show that the average proliferation rate of individual (Supplemental Fig. 2D). Interestingly, T cell diversity still drops naive T cells bt increases with age (Fig. 2A), in agreement with sharply after ∼70 y in line with the results reported earlier data obtained from both humans (9) and rhesus macaques (7). The (Supplemental Fig. 2C). Thus, in our model, even if the process of increase in bt is initially very slow, but it eventually accelerates eliminating senescent cells has a very low efficiency, T cell di- after the age of ∼60 y, coincident with the approach of the average versity still drops sharply in accordance with empirical observa-

number of cell divisions toward the Hayflick limit (Fig. 2B). In- tions. Taken together, our data are consistent with the proposal http://www.jimmunol.org/ that the Hayflick limit, or a peripheral T cell maintenance limit, is an important determinant of the effective clonal diversity of naive T cells. Increased homeostatic proliferation is necessary for sharp decline of diversity. We note that an increased rate of homeostatic proliferation of T cells because of the loss of senescent T cells is an essential requirement for a significant decline in effective clonal diversity to occur around the eighth decade of life. Without this increase, the expected time at which the average number of cell by guest on September 25, 2021 divisions for all T cells will be equal to the Hayflick limit is estimated to be 125 y (Materials and Methods), which is .50 y longer than the value predicted by the simulations and observed empirically. Therefore, a sufficient increase in bt is necessary to produce a sharp decline of effective clonal diversity during the normal lifespan of a human. The increase in bt that is predicted by the simulations is further supported by a simple mathematical model of T cell population dynamics (Materials and Methods). First, the model makes the intuitive prediction that bt will increase as the size of the thymic output falls (equation 3). A corollary to this prediction is that a larger thymic output size will slow down the rate at which individual T cells reach the Hayflick limit and subsequently become extinct. Second, the model predicts that bt will also increase as the average probability that individual T cells have reached the Hayflick limit at time t increases. Indeed, bt is predicted to grow infinitely large in the limit as this average probability reaches the critical value 1/2. FIGURE 2. Simulations predict sharp decline of effective clonal diversity. We simulated the population dynamics of human naive T cells as Discussion described in the text and Materials and Methods, based on experimental Data obtained from a variety of species (6–11, 16) indicate that the estimates of the Hayflick limit (22, 29), the bias in thymic production of effective clonal diversity of T cells declines with age. Using different T-cell clones (46, 47), and the rates of thymopoeisis (9, 18, 19, CDR3 amino acid sequences obtained from the TCR b-chain (16), 21), T cell turnover (42, 43, 45), and telomere attrition (31, 48). We set the we estimated the age-dependent change in the effective clonal bias in thymic production of different T cell clones to w = 0.01. We ran the diversity of naive CD4/CD8 T cells found in human blood (Fig. 1). simulations 100 times and plotted the dynamics of the upper and lower 25 percentile values of the following: (A) the mean number of proliferations We found that between young adulthood (20–35 y) and old age accrued by each T cell during the preceding decade; (B) the mean number (70–83 y), the effective clonal diversity of naive CD4 (resp. CD8) of T cell divisions accrued during the lifetime of each T cell and its an- T cells decreased by a factor of ∼10 (resp. ∼30) (Fig. 1). For CD8 cestors; and (C) the effective clonal diversity of the T cell population. The T cells, when one outlying elderly individual was not considered effective clonal diversity is plotted on a log scale. in the analysis, effective clonal diversity decreased by ∼125-fold. The Journal of Immunology 5003

In particular, the effective number of CD8 CDR3 amino acid se- rates alone may be insufﬁcient to cause the sharp decline of T cell quences was ∼2 million in young adults, but only ∼16,000 in the diversity observed in elderly humans. elderly (Fig. 1). The diversity of the CDR3 region might be af- Previous studies have implicated chronic exposure to pathogens fected by an individual’s gender as well as by chronic infections, such as CMV, EBV, and HIV in the accumulation of replicatively most notably CMV. We obtained the results reported in this paper senescent T cells during human aging (66). An increased frequency using CDR3 amino acid sequences collected by Goronzy and of such senescent cells might restrict further immune responses to coworkers (16), which came from four younger and ﬁve older pathogens (67). The present study indicates that senescent T cells individuals. The genders of these individuals are unknown, so we may also emerge frequently from the normal process of homeo- cannot evaluate the potential effect of gender on our estimates of static T cell proliferation. Furthermore, their emergence was diversity. Several of the older individuals tested negative for Abs shown to coincide with the marked decline of the effective clonal to CMV, so it is unlikely that the sharp drop in T cell diversity diversity of naive T cells that occurs in elderly humans. The re- that we measured in these individuals is mainly because of CMV. sults support a model in which exhaustion of the proliferative Because the CDR3 region is important for Ag recognition (2), capacity of naive T cells is a key trigger for the subsequent sharp our results imply that, compared with young adults, elderly in- decline of their effective clonal diversity (68). dividuals are much less able to recognize a diverse ensemble of Ags, including pathogen derived. Acknowledgments We predicted, using a population-dynamic modeling approach We thank Nir Friedman for his constructive comments on an earlier ver- based on experimental data (18, 21, 22, 28, 29, 31, 38, 42, 43, 46– sion of this manuscript.

48), that the markedly lower effective clonal diversity found in Downloaded from elderly humans results from the fact that each T cell can divide for Disclosures only a limited number of times before it reaches replicative se- The authors have no financial conflicts of interest. nescence (or the Hayflick limit) (29, 63) and is consequently eliminated (28, 36, 37). This, coupled to the facts that thymic production of new T cells declines substantially with age (11, 18, References 1. Messaoudi, I., J. A. Guevara Patinõ, R. Dyall, J. LeMaoult, and J. Nikolich- 19) and that T cells will undergo compensatory proliferation (38, http://www.jimmunol.org/ Zugich. 2002. Direct link between mhc polymorphism, T cell avidity, and di- 40, 64) to fill the empty homeostatic space created by the loss of versity in immune defense. Science 298: 1797–1800. senescent cells, leads to a sharp fall in effective clonal diversity 2. Nikolich-Zugich, J., M. K. Slifka, and I. Messaoudi. 2004. 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J. Immunol. 174: 7446–7452. divisions approaches the Hayflick limit (Fig. 2A, 2B). 10. Shifrut, E., K. Baruch, H. Gal, W. Ndifon, A. Deczkowska, M. Schwartz, and A previous study proposed an alternative explanation for the N. Friedman. 2013. CD4+ T cell-receptor repertoire diversity is compromised in the spleen but not in the bone marrow of aged mice due to private and sporadic decline of effective clonal diversity observed in old age, namely clonal expansions. Front. Immunol. 4: 379. that it might be caused by replication-enhancing mutations that 11. Goronzy, J. J., W.-W. Lee, and C. M. Weyand. 2007. Aging and T-cell diversity. arise suddenly in certain T cells (21). This earlier proposal suggests Exp. Gerontol. 42: 400–406. 12. Yager, E. J., M. Ahmed, K. Lanzer, T. D. Randall, D. L. Woodland, and that opportunities for therapeutically preventing an eventual de- M. A. Blackman. 2008. Age-associated decline in T cell repertoire diversity cline of effective clonal diversity are quite limited (21). In con- leads to holes in the repertoire and impaired immunity to influenza virus. J. Exp. trast, the simpler explanation proposed in the current study paints Med. 205: 711–723. 13. Meyer-Olson, D., N. H. Shoukry, K. W. Brady, H. Kim, D. P. Olson, K. Hartman, a more hopeful picture: It might be possible to maintain a more A. K. Shintani, C. M. Walker, and S. A. Kalams. 2004. Limited T cell receptor clonally diverse naive T cell repertoire through therapeutic inter- diversity of HCV-specific T cell responses is associated with CTL escape. J. Exp. Med. 200: 307–319. ventions that increase thymopoiesis. 14. Nanda, N. K., R. Apple, and E. Sercarz. 1991. Limitations in plasticity of the It is worth noting that in addition to the Hayflick limit and T-cell receptor repertoire. Proc. Natl. Acad. Sci. USA 88: 9503–9507. replication-enhancing mutations (21), increased proliferation of 15. Nishio, J., M. Baba, K. Atarashi, T. Tanoue, H. Negishi, H. Yanai, S. Habu, S. Hori, K. Honda, and T. Taniguchi. 2015. Requirement of full TCR repertoire naive T cells might also occur as a result of differences in the for regulatory T cells to maintain intestinal homeostasis. Proc. Natl. Acad. Sci. reactivity of T cells with self-peptide MHC. In aged mice, the USA 112: 12770–12775. CD8 T cell repertoire is biased in favor of T cells with a high 16. Qi, Q., Y. Liu, Y. Cheng, J. Glanville, D. Zhang, J.-Y. Lee, R. A. Olshen, C. M. Weyand, S. D. Boyd, and J. J. Goronzy. 2014. Diversity and clonal se- avidity for self-peptide MHC (8). These T cells preferentially lection in the human T-cell repertoire. Proc. Natl. Acad. Sci. USA 111: 13139– undergo homeostatic proliferation, leading to a contraction of 13144. 17. Gotelli, N., and C. Anne. 2013. Measuring and estimating species richness, T cell repertoire diversity in aged mice (8). Numerical simulations species diversity, and biotic similarity from sampling data. Encycl. Biodivers. 5: (21) suggest, however, that this unequal homeostatic proliferation 195–211. 5004 NAIVE T CELL PROLIFERATION COMPROMISES DIVERSITY

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2 3 Simulations with low naïve clonal production bias also predict sharp decline of effective clonal 4 diversity. We simulated the population dynamics of human naïve T cells as described in the text and 5 Methods, with the bias in thymic production of different T-cell clones set to a very low value of 6 φ=0.0001. With this value of φ, different T-cell clones were produced by the thymus at approximately 7 the same rate. We ran the simulations 100 times and plotted the dynamics of the upper and lower 25 8 percentile values of: a) The mean number of proliferations accrued by each T cell during the 9 preceding decade, b) the mean number of T-cell divisions accrued during the lifetime of each T cell 10 and its ancestors, and c) the effective clonal diversity of the T-cell population. The effective clonal 11 diversity is plotted on a log scale.

1 13 Supplemental figure 2

14 15 Even an inefficient elimination of senescent cells from the body still leads to a sharp drop of T- 16 cell diversity. We simulated the population dynamics of human naïve T cells as described in the text 17 and Methods, with the bias in thymic production of different T-cell clones set to φ=0.01 and the daily 18 probability of senescent-cell elimination reduced from 1.0 to only 0.001. We ran the simulations 100 19 times and plotted the dynamics of the upper and lower 25 percentile values of: a) The mean number of 20 proliferations accrued by each T cell during the preceding decade, b) the mean number of T-cell 21 divisions accrued during the lifetime of each T cell and its ancestors, c) the effective clonal diversity 22 of the T-cell population, and d) the fraction of all cells that are replicatively senescent (i.e. that have 23 reached the Hayflick limit). The effective clonal diversity is plotted on a log scale.