Dynamic tuning of lymphocytes: physiological basis, mechanisms, and function.

Dynamic tuning of cellular responsiveness as a result of repeated stimuli improves the ability of cells to distinguish physiologically meaningful signals from each other and from noise. In particular, lymphocyte activation thresholds are subject to tuning, which contributes to maintaining tolerance to self-antigens and persisting foreign antigens, averting autoimmunity and immune pathogenesis, but allowing responses to strong, structured perturbations that are typically associated with acute infection. Such tuning is also implicated in conferring flexibility to positive selection in the thymus, in controlling the magnitude of the immune response, and in generating memory cells. Additional functional properties are dynamically and differentially tuned in parallel via subthreshold contact interactions between developing or mature lymphocytes and self-antigen-presenting cells. These interactions facilitate and regulate lymphocyte viability, maintain their functional integrity, and influence their responses to foreign antigens and accessory signals, qualitatively and quantitatively. Bidirectional tuning of T cells and antigen-presenting cells leads to the definition of homeostatic set points, thus maximizing clonal diversity.

'Rinse and Replace': Boosting T Cell Turnover To Reduce HIV-1 Reservoirs.

Latent HIV-1 persists indefinitely during antiretroviral therapy (ART) as an integrated silent genome in long-lived memory CD4+ T cells. In untreated infections, immune activation increases the turnover of intrinsically long-lived provirus-containing CD4+ T cells. Those are 'washed out' as a result of their activation, which when coupled to viral protein expression can facilitate local inflammation and recruitment of uninfected cells to activation sites, causing latently infected cells to compete for survival. De novo infection can counter this washout. During ART, inflammation and CD4+ T cell activation wane, resulting in reduced cell turnover and a persistent reservoir. We propose accelerating reservoir washout during ART by triggering sequential waves of polyclonal CD4+ T cell activation while simultaneously enhancing virus protein expression. Reservoir reduction as an adjunct to other therapies might achieve lifelong viral control.

High production rates sustain in vivo levels of PD-1high simian immunodeficiency virus-specific CD8 T cells in the face of rapid clearance.

Programmed Death 1 (PD-1) expression by human/simian immunodeficiency virus (HIV/SIV)-specific CD8 T cells has been associated with defective cytokine production and reduced in vitro proliferation capacity. However, the cellular mechanisms that sustain PD-1(high) virus-specific CD8 T cell responses during chronic infection are unknown. Here, we show that the PD-1(high) phenotype is associated with accelerated in vivo CD8 T cell turnover in SIV-infected rhesus macaques, especially within the SIV-specific CD8 T cell pool. Mathematical modeling of 5-bromo-2' deoxyuridine (BrdU) labeling dynamics demonstrated a significantly increased generation rate of PD-1(high) compared to PD-1(low) CD8 T cells in all memory compartments. Simultaneous analysis of Ki67 and BrdU kinetics revealed a complex in vivo turnover profile whereby only a small fraction of PD-1(high) cells, but virtually all PD-1(low) cells, returned to rest after activation. Similar kinetics operated in both chronic and acute SIV infection. Our data suggest that the persistence of PD-1(high) SIV-specific CD8 T cells in chronic infection is maintained in vivo by a mechanism involving high production coupled with a high disappearance rate.

Memory phenotype CD4 T cells undergoing rapid, nonburst-like, cytokine-driven proliferation can be distinguished from antigen-experienced memory cells.

Memory phenotype (CD44(bright), CD25(negative)) CD4 spleen and lymph node T cells (MP cells) proliferate rapidly in normal or germ-free donors, with BrdU uptake rates of 6% to 10% per day and Ki-67 positivity of 18% to 35%. The rapid proliferation of MP cells stands in contrast to the much slower proliferation of lymphocytic choriomeningitis virus (LCMV)-specific memory cells that divide at rates ranging from <1% to 2% per day over the period from 15 to 60 days after LCMV infection. Anti-MHC class II antibodies fail to inhibit the in situ proliferation of MP cells, implying a non-T-cell receptor (TCR)-driven proliferation. Such proliferation is partially inhibited by anti-IL-7Rα antibody. The sequence diversity of TCRß CDR3 gene segments is comparable among the proliferating and quiescent MP cells from conventional and germ-free mice, implying that the majority of proliferating MP cells have not recently derived from a small cohort of cells that expand through multiple continuous rounds of cell division. We propose that MP cells constitute a diverse cell population, containing a subpopulation of slowly dividing authentic antigen-primed memory cells and a majority population of rapidly proliferating cells that did not arise from naïve cells through conventional antigen-driven clonal expansion.

Antigen-stimulated CD4 T-cell expansion is inversely and log-linearly related to precursor number.

Antigen-driven expansion of specific CD4 T cells diminishes, on a per cell basis, as infused cell number increases. There is a linear relation between log precursor number and log factor of expansion (FE), with a slope of ∼-0.5 over a range from 3 to 30,000 precursors. Cell number dependence of FE is observed at low precursor number, implying that the underlying process physiologically regulates antigen-driven T-cell expansion. FE of small numbers of transgenic precursors is not significantly affected by concomitant responses of large numbers of cells specific for different antigens. Increasing antigen amount or exogenous IL-2, IL-7, or IL-15 does not significantly affect FE, nor does FE depend on Fas, TNF-α receptor, cytotoxic T-lymphocyte antigen-4, IL-2, or IFN-γ. Small numbers of Foxp3-deficient T-cell receptor transgenic cells expand to a greater extent than do large numbers, implying that this effect is not mediated by regulatory T cells. Increasing dendritic cell number does result in larger FE, but the quantitative relation between FE and precursor number is not abrogated. Although not excluding competition for peptide/MHC complexes as an explanation, fall in FE with increasing precursor number could be explained by a negative feedback in which increasing numbers of responding cells in a cluster inhibit the expansion of cells of the same specificity within that cluster.

Feedback regulation of proliferation vs. differentiation rates explains the dependence of CD4 T-cell expansion on precursor number.

The mechanisms regulating clonal expansion and contraction of T cells in response to immunization remain to be identified. A recent study established that there was a log-linear relation between CD4 T-cell precursor number (PN) and factor of expansion (FE), with a slope of ∼-0.5 over a range of 3-30,000 precursors per mouse. The results suggested inhibition of precursor expansion either by competition for specific antigen-presenting cells or by the action of other antigen-specific cells in the same microenvironment as the most likely explanation. Several molecular mechanisms potentially accounting for such inhibition were examined and rejected. Here we adopt a previously proposed concept, "feedback-regulated balance of growth and differentiation," and show that it can explain the observed findings. We assume that the most differentiated effectors (or memory cells) limit the growth of less differentiated effectors, locally, by increasing the rate of differentiation of the latter cells in a dose-dependent manner. Consequently, expansion is blocked and reversed after a delay that depends on initial PN, accounting for the dependence of the peak of the response on that number. We present a parsimonious mathematical model capable of reproducing immunization response kinetics. Model definition is achieved in part by requiring consistency with available BrdU-labeling and carboxyfluorescein diacetate succinimidyl ester (CFSE)-dilution data. The calibrated model correctly predicts FE as a function of PN. We conclude that feedback-regulated balance of growth and differentiation, although awaiting definite experimental characterization of the hypothetical cells and molecules involved in regulation, can explain the kinetics of CD4 T-cell responses to antigenic stimulation.

Progressive CD4+ central memory T cell decline results in CD4+ effector memory insufficiency and overt disease in chronic SIV infection.

Primary simian immunodeficiency virus (SIV) infections of rhesus macaques result in the dramatic depletion of CD4(+) CCR5(+) effector-memory T (T(EM)) cells from extra-lymphoid effector sites, but in most infections, an increased rate of CD4(+) memory T cell proliferation appears to prevent collapse of effector site CD4(+) T(EM) cell populations and acute-phase AIDS. Eventually, persistent SIV replication results in chronic-phase AIDS, but the responsible mechanisms remain controversial. Here, we demonstrate that in the chronic phase of progressive SIV infection, effector site CD4(+) T(EM) cell populations manifest a slow, continuous decline, and that the degree of this depletion remains a highly significant correlate of late-onset AIDS. We further show that due to persistent immune activation, effector site CD4(+) T(EM) cells are predominantly short-lived, and that their homeostasis is strikingly dependent on the production of new CD4(+) T(EM) cells from central-memory T (T(CM)) cell precursors. The instability of effector site CD4(+) T(EM) cell populations over time was not explained by increasing destruction of these cells, but rather was attributable to progressive reduction in their production, secondary to decreasing numbers of CCR5(-) CD4(+) T(CM) cells. These data suggest that although CD4(+) T(EM) cell depletion is a proximate mechanism of immunodeficiency, the tempo of this depletion and the timing of disease onset are largely determined by destruction, failing production, and gradual decline of CD4(+) T(CM) cells.

Pathogenesis of HIV infection: what the virus spares is as important as what it destroys.

Upon transmission to a new host, HIV targets CCR5+ CD4+ effector memory T cells, resulting in acute, massive depletion of these cells from mucosal effector sites. This depletion does not initially compromise the regenerative capacity of the immune system because naive and most central memory T cells are spared. Here, we discuss evidence suggesting that frequent activation of these spared cells during the chronic phase of HIV infection supplies mucosal tissues with short-lived CCR5+ CD4+ effector cells that prevent life-threatening infections. This immune activation also facilitates continued viral replication, but infection and killing of target T cells by HIV are selective and the impact on effector-cell lifespan is limited. We propose, however, that persistent activation progressively disrupts the functional organization of the immune system, reducing its regenerative capacity and facilitating viral evolution that leads to loss of the exquisite target cell-sparing selectivity of viral replication, ultimately resulting in AIDS.

Perspectives of (/memorandum for) systems thinking on COVID-19 pandemic and pathology.

Is data-driven analysis sufficient for understanding the COVID-19 pandemic and for justifying public health regulations? In this paper, we argue that such analysis is insufficient. Rather what is needed is the identification and implementation of over-arching hypothesis-related and/or theory-based rationales to conduct effective SARS-CoV2/COVID-19 (Corona) research. To that end, we analyse and compare several published recommendations for conceptual and methodological frameworks in medical research (e.g., public health, preventive medicine and health promotion) to current research approaches in medical Corona research. Although there were several efforts published in the literature to develop integrative conceptual frameworks before the COVID-19 pandemic, such as social ecology for public health issues and systems thinking in health care, only a few attempts to utilize these concepts can be found in medical Corona research. For this reason, we propose nested and integrative systemic modelling approaches to understand Corona pandemic and Corona pathology. We conclude that institutional efforts for knowledge integration and systemic thinking, but also for integrated science, are urgently needed to avoid or mitigate future pandemics and to resolve infection pathology.

An integrative systems biology view of host-pathogen interactions: The regulation of immunity and homeostasis is concomitant, flexible, and smart.

The systemic bio-organization of humans and other mammals is essentially "preprogrammed", and the basic interacting units, the cells, can be crudely mapped into discrete sets of developmental lineages and maturation states. Over several decades, however, and focusing on the immune system, we and others invoked evidence - now overwhelming - suggesting dynamic acquisition of cellular properties and functions, through tuning, re-networking, chromatin remodeling, and adaptive differentiation. The genetically encoded "algorithms" that govern the integration of signals and the computation of new states are not fully understood but are believed to be "smart", designed to enable the cells and the system to discriminate meaningful perturbations from each other and from "noise". Cellular sensory and response properties are shaped in part by recurring temporal patterns, or features, of the signaling environment. We compared this phenomenon to associative brain learning. We proposed that interactive cell learning is subject to selective pressures geared to performance, allowing the response of immune cells to injury or infection to be progressively coordinated with that of other cell types across tissues and organs. This in turn is comparable to supervised brain learning. Guided by feedback from both the tissue itself and the neural system, resident or recruited antigen-specific and innate immune cells can eradicate a pathogen while simultaneously sustaining functional homeostasis. As informative memories of immune responses are imprinted both systemically and within the targeted tissues, it is desirable to enhance tissue preparedness by incorporating attenuated-pathogen vaccines and informed choice of tissue-centered immunomodulators in vaccination schemes. Fortunately, much of the "training" that a living system requires to survive and function in the face of disturbances from outside or within is already incorporated into its design, so it does not need to deep-learn how to face a new challenge each time from scratch. Instead, the system learns from experience how to efficiently select a built-in strategy, or a combination of those, and can then use tuning to refine its organization and responses. Efforts to identify and therapeutically augment such strategies can take advantage of existing integrative modeling approaches. One recently explored strategy is boosting the flux of uninfected cells into and throughout an infected tissue to rinse and replace the infected cells.

HIV-1 viremia prevents the establishment of interleukin 2-producing HIV-specific memory CD4+ T cells endowed with proliferative capacity.

CD4+ T cell responses are associated with disease control in chronic viral infections. We analyzed human immunodeficiency virus (HIV)-specific responses in ten aviremic and eight viremic patients treated during primary HIV-1 infection and for up to 6 yr thereafter. Using a highly sensitive 5-(and-6)-carboxyfluorescein diacetate-succinimidyl ester-based proliferation assay, we observed that proliferative Gag and Nef peptide-specific CD4+ T cell responses were 30-fold higher in the aviremic patients. Two subsets of HIV-specific memory CD4+ T cells were identified in aviremic patients, CD45RA- CCR7+ central memory cells (Tcm) producing exclusively interleukin (IL)-2, and CD45RA- CCR7- effector memory cells (Tem) that produced both IL-2 and interferon (IFN)-gamma. In contrast, in viremic, therapy-failing patients, we found significant frequencies of Tem that unexpectedly produced exclusively IFN-gamma. Longitudinal analysis of HIV epitope-specific CD4+ T cells revealed that only cells that had the capacity to produce IL-2 persisted as long-term memory cells. In viremic patients the presence of IFN-gamma-producing cells was restricted to periods of elevated viremia. These findings suggest that long-term CD4+ T cell memory depends on IL-2-producing CD4+ T cells and that IFN-gamma only-producing cells are short lived. Our data favor a model whereby competent HIV-specific Tcm continuously arise in small numbers but under persistent antigenemia are rapidly induced to differentiate into IFN-gamma only-producing cells that lack self-renewal capacity.

Immunological Paradigms, Mechanisms, and Models: Conceptual Understanding Is a Prerequisite to Effective Modeling.

Most mathematical models that describe the individual or collective actions of cells aim at creating faithful representations of limited sets of data in a self-consistent manner. Consistency with relevant physiological rules pertaining to the greater picture is rarely imposed. By themselves, such models have limited predictive or even explanatory value, contrary to standard claims. Here I try to show that a more critical examination of currently held paradigms is necessary and could potentially lead to models that pass the test of time. In considering the evolution of paradigms over the past decades I focus on the "smart surveillance" theory of how T cells can respond differentially, individually and collectively, to both self- and foreign antigens depending on various "contextual" parameters. The overall perspective is that physiological messages to cells are encoded not only in the biochemical connections of signaling molecules to the cellular machinery but also in the magnitude, kinetics, and in the time- and space-contingencies, of sets of stimuli. By rationalizing the feasibility of subthreshold interactions, the "dynamic tuning hypothesis," a central component of the theory, set the ground for further theoretical and experimental explorations of dynamically regulated immune tolerance, homeostasis and diversity, and of the notion that lymphocytes participate in nonclassical physiological functions. Some of these efforts are reviewed. Another focus of this review is the concomitant regulation of immune activation and homeostasis through the operation of a feedback mechanism controlling the balance between renewal and differentiation of activated cells. Different perspectives on the nature and regulation of chronic immune activation in HIV infection have led to conflicting models of HIV pathogenesis-a major area of research for theoretical immunologists over almost three decades-and can have profound impact on ongoing HIV cure strategies. Altogether, this critical review is intended to constructively influence the outlook of prospective model builders and of interested immunologists on the state of the art and to encourage conceptual work.

Immunologic and virologic evolution during periods of intermittent and persistent low-level viremia.

BACKGROUND: HIV replication, HIV-specific T-cell responses and T-cell activation each contributes to disease outcome during untreated HIV infection. The interaction of these factors is not well understood, particularly in the setting of antiretroviral therapy. METHODS: This is a longitudinal study of antiretroviral-treated patients with plasma HIV RNA levels < 1000 copies/ml. Patients were divided into three groups: suppressed viremia, intermittent viremia ('blips') and persistent low-level viremia. HIV-specific immunity was measured using interferon-gamma ELISPOT. T-cell activation was defined by CD38 and HLA-DR co-expression. Drug resistance was quantified using a phenotypic susceptibility assay. RESULTS: The breadth and the magnitude of the HIV-specific CD8 T-cell response was greater in patients with either intermittent or persistent viremia compared to patients with suppressed viremia. In contrast, T-cell activation was significantly elevated only in those patients with persistent viremia. Patients with persistent low-level viremia had moderate levels of phenotypic antiretroviral drug resistance that increased over time. Virologic failure (confirmed increase in viral load > 1000 HIV RNA copies/ml) was primarily observed in the persistently viremic group. CONCLUSIONS: Antiretroviral-treated individuals with intermittent viremia appear to mount an effective HIV-specific T-cell response while not experiencing increases in the level of immune activation. This may limit viral evolution and emergence of drug resistance. In contrast, antiretroviral-treated individuals with persistent low-level viremia exhibit significant increases in overall immune activation and a substantial risk of subsequent treatment failure. It is likely that higher viremia and stronger immune activation act synergistically to accelerate the development of systemic drug resistance.

Pathogen-sensing and regulatory T cells: integrated regulators of immune responses.

We present the concept that pathogen-sensing and regulatory T cells (Treg) mutually regulate immune responses to conventional and tumor antigens through countervailing effects on dendritic cells (DC). Normally, conventional CD4 T cells recognizing their cognate antigen presented by a DC will respond only if the DC also receives a signal through its pathogen-sensing/danger/adjuvant recognition systems (the pathogen-sensing triad). However, in the absence of Tregs capable of interacting with the same DC, DCs are competent to present antigens, both foreign and self, even without the stimulation provided by the pathogen-sensing triad. Tregs recognizing an antigen presented by the DC that is also presenting antigen to a conventional CD4 T cell will prevent the activation of the CD4 T-cell responses, but a signal delivered by a member of the pathogen-sensing triad will overcome the inhibitory action of Tregs, thus allowing CD4 T-cell responses to go forward. These considerations take on special meaning for responses to "weak antigens" such as many of the antigens displayed by spontaneous human tumors.

Pathogen-sensing, regulatory T cells, and responsiveness-tuning collectively regulate foreign- and self-antigen mediated T-cell responses.

The concept that induction of T-cell-dependent immune responses requires both T-cell recognition of an epitope and recognition by the innate immune system of pathogen-associated molecular patterns needs to be extended to include the contribution of regulatory T cells and of responsiveness-tuning. Here we develop the hypothesis that both pathogen sensing and regulatory T cells act on antigen-presenting dendritic cells (DCs) to determine whether the DCs will be competent to activate T cells with cognate receptors. Tregs that recognize self-peptide/major histocompatibility complexes (MHCs) on the DC in question will serve to inactivate that DC, whereas sensing of pathogens or innate "danger" signals by the DC will oppose the action of the Tregs. The responsiveness of the T-cell compartment is further controlled by activation-threshold tuning in which responsiveness is adjusted to the strength of recurrent stimulation of T cells by self-peptide/MHCs in the periphery. Thus, a robust T-cell response depends on T-cell recognition of a peptide/MHC presented by a competent DC, competence depending on interactions with Tregs and the degree of innate stimulation through pathogen sensing, and on a combined TCR and accessory-signaling strength of stimulation that exceeds the activation threshold of the T cells, determined by its responsiveness tuning.

Pathogenic mechanisms in simian immunodeficiency virus infection.

PURPOSE OF REVIEW: Recent work in pathogenic simian immunodeficiency (SIV) infection of Asian macaques and in natural, nonpathogenic SIV infections of African nonhuman primate species has demonstrated that persistent activation has profound effects on CD4+ memory T-cell proliferation, differentiation and survival. Disease progression in pathogenic infection has been closely linked to these dynamics, reflecting a complex interplay of virus-mediated killing, the effects of systemic activation and host regenerative mechanisms. We review these recent advances. RECENT FINDINGS: Massive depletion of CD4+ effector-memory T cells invariably occurs during acute CCR5-tropic SIV infection, but is initially stabilized by new production of these cells from spared central memory precursors above the threshold required to maintain clinical immune competence. In pathogenic (but not natural, apathogenic) infections, a persistent state of immune activation, characterized by multiple, recurrent bursts of lymphocyte proliferation, differentiation, migration, death and functional modification of 'resting' cells, is associated with progressive depletion of central memory CD4+ T cells, and ultimately, a collapse of effector site CD4+ memory populations that is closely associated with overt immune deficiency. SUMMARY: The importance of maintaining the regenerative capacity of the central-memory compartment of CD4+ T cells is increasingly evident. Defining the physiologic and molecular mechanisms responsible for instability of the CD4+ central-memory T cell pool could enable new immunotherapeutic interventions.

The duration of exposure to HIV modulates the breadth and the magnitude of HIV-specific memory CD4+ T cells.

The impact of exposure to Ag on the development and maintenance of human CD4(+) memory T cells in general and HIV infection in particular is partially understood. In this study, we measured HIV-specific CD4(+) T cell proliferative responses against HIV proteins and derived peptides one year after highly active antiretroviral therapy initiation in 39 HIV-infected patients who initiated therapy at different times following infection. We show that a brief exposure to HIV of <1 month does not allow the generation of significant detectable frequencies of HIV-specific CD4(+) memory T cells. Patients having prolonged cumulative exposure to high viral load due to therapy failures also demonstrated limited HIV-specific CD4(+) T cell responses. In contrast, patients exposed to significant levels of virus for periods ranging from 3 to 18 mo showed brisk and broad HIV-specific CD4(+) T cell responses 1 year following the onset of therapy intervention. We also demonstrate that the nadir CD4(+) T cell count before therapy initiation correlated positively with the breadth and magnitude of these responses. Our findings indicate that the loss of proliferative HIV-specific CD4(+) T cell responses is associated with the systemic progression of the disease and that a brief exposure to HIV does not allow the establishment of detectable frequencies of HIV-specific memory CD4(+) T cells.

CD4 T cell depletion is linked directly to immune activation in the pathogenesis of HIV-1 and HIV-2 but only indirectly to the viral load.

The causal relationships among CD4 cell depletion, HIV replication, and immune activation are not well understood. HIV-2 infection, "nature's experiment" with inherently attenuated HIV disease, provides additional insights into this issue. We report the finding that in HIV-2 and HIV-1 patients with a comparable degree of CD4 depletion the imbalance in the relative sizes of the naive and memory T cell populations and the up-regulation of CD4 and CD8 cell activation markers (HLA-DR, CD38, CD69, Fas molecules) are similar, even though the viral load in the plasma of HIV-2-infected patients is two orders of magnitude lower than in HIV-1 patients and HIV-2 patients are known to have slower rates of CD4 T cell decline and a better clinical prognosis. Moreover, we found a similar increase in the frequency of cycling CD4 T cells (Ki67+), which was in strong correlation with the expression of activation markers. Finally, the level of T cell anergy, as assessed by the proliferative responses to CD3 stimulation and to a panel of microbial Ags, proved to be comparable in HIV-1 and HIV-2 patients with a similar degree of CD4 depletion despite large differences in viral load. Our data are consistent with a direct causal relationship between immune activation and CD4 cell depletion in HIV disease and an only indirect relation of these parameters to the virus replication rate. Invoking the concept of proximal immune activation and virus transmission, which links efficient transmission of virus to local cell activation and proliferation in response to Ags and inflammation, we propose an integrative interpretation of the data and suggest that strongly elevated immune activation induces CD4 cell depletion and not vice versa, with potential implications for the choice of treatment strategies.