Clearing Out the Dead Wood: Rejuvenating Humoral Immunity through Ablation Strategy
The degenerative aging of the immune system is responsible for an enormous burden of disease and disability, from the pain of recurrent Herpes zoster and postherpetic neuralgia, to elevated rates of chronic urinary tract infections, to complications in wounds, pressure sores, ulcers, and surgical incisions. Most prominently, it underlies the meteoric rise in mortality from respiratory infections with age: influenza, pneumonia, and septicemia rise from being negligible causes of death in healthy middle-aged adults in the USA, to emerge amongst the top 10 causes of death in adults over the age of 55, with mortality rates climbing with each successive year of aging. And in addition to increasing the morbidity and mortality specifically attributable to particular infections, the dysregulation of immune function by immunosenescence is widely acknowledged to exacerbate multiple chronic age-related illnesses, and to contribute to functional decline and frailty in aging people.(1)
While vaccine manufacturers and public health officials have rightly advocated for expansion of population vaccine coverage as a measure to blunt the burden of infectious disease in the elderly, the effectiveness of this strategy is itself limited by immunosenescence, which progressively diminishes the adaptive immune system's response to vaccination with age:
[Preventive] elimination of clinical disease is often unrealistic for older adult populations with diminished immunity and impaired vaccine responses. The goals of immunization in older adults are to prevent serious illness, hospitalization, and death, but benefits relating to exacerbation of underlying chronic illness, functional decline, and frailty are other worthy endpoints ... These endpoints are more difficult to measure and harder to specifically attribute to the organism(s) targeted by vaccines, often leading to conflicting evidence of vaccine efficacy in older adults. For example, although some investigators have found that administering the influenza vaccine to community-dwelling individuals aged 65 and older leads to significant reductions in risk of hospitalization for pneumonia or influenza and death, others have documented little or no effect, particularly in those aged 70 and older with significant comorbidities.(1)
The solution to age-related suffering and death from specific infections, autoimmunity, and inflammation is the application of rejuvenation biotechnology to the aging immune system itself.
The clearest and longest-established contributor to immune senescence is the decline in adaptive immunity mediated by T lymphocytes,(2) the biomedical remediation of which has therefore been the focus of SENS Foundation's investments in immunological rejuvenation research.(9,10) The existence, nature, and causes of age-related deficits in B-cell structure and function have long been less clear, but emerging evidence has recently led to a consensus of their reality, although the mechanisms have not yet been definitively established.(3) Among the key questions are whether and to what degree this decline in humoral immunity attributable to the degenerative aging process is mediated through the introduction of intrinsic defects to the B-cells themselves, vs. alterations in the systemic environment of the aging body. The strategy for the development of rejuvenation biotechnology for this arm of the aging immune system will be determined by the answers to these questions.
Now, a conceptually simple single experiment performed by Doron Melamed and colleagues at the Technion-Israel Institute of Technology(4) has simultaneously provided powerful evidence for the existence of intrinsic defects in an accumulating population of long-resident B-cells in biologically aged hosts, and for a relatively straightforward intervention to substantially restore youthful humoral immunity in such organisms.
Melamed's group studied the effects of the degenerative aging process on B-cell function using young adult (4 mo) and early-old (20 mo)
C57JBl/6 mice. Several models were used to evaluate the possible role of accumulations of long-extant peripheral B cells with age in suppressing B-cell lymphopoiesis and contributing the the overall levels of cell-intrinsic defects in the aging organism's B-lymphocyte population. One such model was transgenic (TG) mice with an inducible Cre/lox system allowing for conditional knockout of the gene encoding the receptor for B-Cell Activation Factor Receptor (Baff-r). Baff-r is a B-cell activation factor in the tumor necrosis factor (TNF) family whose signaling is essential for the survival of mature B lymphocytes, but is conveniently dispensable for generation of new ones. Activation of the recombinase system allowed the investigators to rapidly deplete animals of B-cell populations that had developed, matured, and aged normally. In additional experiments, the Israeli group further confirmed and expanded their findings in Baff-r-TG mice with similar B-cell depletion studies in aging wiltd-type (WT) mice subjected to depleting antibody mixture, and to the targeting of transgenic human CD20 in hCD20-TG mice.(4)
Restoration of Repressed B-Cell Production
Old and young animals' bone marrows contained similar numbers of B lymphocytes, but the proportions of mature versus precursor and and early B-cells were greatly skewed. In young animals animals, 75% of B-lymphocytes in the bone marrow were newborn and precursor cells (proB (B220+/CD43+/IgM-), preB (B220+/CD43-/IgM-), and immature B (B220lo/IgM+)); on old animals, the proportion of such cells had fallen to just 12%. Induced Baff-r knockout in these animals' B-cells cut the B-cell population in peripheral blood and spleen in half, but following this assault, newly-generated and precursor B-cells began to appear in bone marrow at similar frequencies in old mice as in young ones. These results were reinforce in later studies using another depletion model, which further demonstrated that levels of common lymphoid progenitors and multipotent primitive progenitors were depressed in the bone marrow of old mice, but rapidly recovered to youthful levels following an depletion.(4)
Renewal of Lymphopoeisis
These results suggested that the accumulating population of long-resident B lymphocytes in old animals was playing an important role in the age-related decline in B lymphopoiesis. To test this hypothesis further, the investigators depleted B-lymphocytes directly, using a mixture of antibodies, leading to a >80% depletion of peripheral B-cells without substantially reducing the total numbers of newborn and precursor B-lymphocytes in the periphery or the bone marrow. However, the efficiency of repopulation in old animals remained significantly depressed compared to youthful rates: while young mice largely reconstituted their peripheral blood and spleen B-cells within 5 d of depletion, old animals required >50 d to accomplish the same reconstitution -- a rate similar to that previously reported in old animals subjected to ablation using cyclophosphamide or irradiation. But with successive further rounds of B-cell depletion, the old animals began to reconstitute their B lymphocyte populations at increasingly rapid, more youthful rates. After subjecting aged animals to a first round of B-cell depletion and waiting for peripheral blood B-cell numbers to fall by ≥80%, repopulation time following a second round of depletion was reduced to just 18-30 d, falling further to only 8 d following a third round of depletion -- a rate similar to that required in young animals. Consistent with this, the absolute number of precursor B-cells in old animals progressively increased with each round of depletion, nearly equaling that of young animals by the third round.(4)
Re-Emergence of Naïve B-Lymphocytes
The higher frequencies of early-stage B-cells that appeared in the bone marrow and periphery of old animals after the depletion of long-resident B-cell populations was paralleled by rapid declines in the abnormally high numbers of antigen-experienced PanCD45+/B220lo B-cells that had accumulated in the spleens of aging mice. These cells were rapidly replaced by mature but antigen-naïve PanCD45+/B220+ B-cells.When peripheral B-cells were depleted in later experiments using a transgenic immunoglobulin reporter gene to mark newly-generated B-cells, lymphopoieisis was seen to be restored: the absolute number of B-cells in the bone marrow rapidly increased, and in parallel the frequency of newly-generated B220+ cells in the spleen rose >15-fold, and >90% of the reconstituting B cells were of a newly-generated population. Moreover, the population of mature splenic B-cells had shifted rapidly away from antigen-experienced PanCD45+/B220lo cells to naïve PanCD45+/B220+ cells, generating a repertoire not significantly different from that of young animals bearing the same reporter gene.(4)
Renovated Antibody Production
These studies demonstrated that old mice retain the capacity to perform lymphopoeisis at rates comparable to their youth, and to restore the youthful balance of B-cell subpopulations, and that the removal of accumulations of long-resident peripheral B-cells was sufficient to return these aspects of immunological aging to profiles similar to much younger animals. But the most important question, from the perspective of rejuvenation research, still remained. Would this intervention go beyond phenotypic changes in the lymphopoietic system, to rejuvenate the flagging B-cell function of old, immunosenescent animals?
To answer this key question, the investigators subjected old (22 mo) WT mice to B-cell depletion, and then 70 d later challenged them, along with young controls and untreated old mice, with i.p. NP-CGG (Chicken Gamma Globulin). As shown by ELISA, the NP-targeting IgG1response to antigen exposure was greatly reduced in old as compared with young mice (66.5±19.8 units vs. 347.3±47.7). But prior B-cell depletion substantially rejuvenated antibody response, with titers rebounding to levels intermediate between those of young and old untreated mice (161.3±44.8, p=0.03 vs. young).
Further Research ... and Development
It remains to be demonstrated that this apparent rejuvenation effect extends to a gold-standard test of greater survival from infectious disease in treated animals. If that can be convincingly shown, then discovering the reasons for the remaining limits on B-cell function in animals treated with B-cell depletion will be important to further the progress to this research, and to using the proof-of-principle that the Israel-Technion team appears to have provided to develop intervention protocols suitable not only for animal testing, but for translation into rejuvenation therapies for aging humans.
Another question is how these new findings integrate with prior research on the aging haematopoietic system. Previous work has shown the role of the deranged signaling environment of the biologically aged organism in repressing non-immune functions of the haematopoietic stem cell niche, and the rejuvenation of the HSC niche by a youthful systemic environment. Other research has highlighted the role of intrinsic defects in the degenerative aging process of HSCs,(5-7) and the ability of the autophagy-enhancing (and, in mice, life-extending) drug rapamycin to partially restore immune function in aging mice.(8) The combination of this research on the systemic, cell-intrinsic, and population influences on degenerative aging of B-lymphocyte functions, and the many interventions already shown to partially rejuvenate aspects of it, gives significant grounds for optimism that interventions can be developed to effect a protocol -- or combination of protocols -- to effect the thoroughgoing rejuvenation of haematopoietic aging, and on timescales that many in the field might until recently have thought unrealistic.
For some time, SENS Foundation's research investments in immune rejuvenation have been centered on T-cell function, dedicated to the complementary strategies of engineering youthful thymic epithelium to restore youthful production of naïve T-cells, and ablation of anergic T-cells to open up the immunologic 'space' required for their expansion.(9,10) The finding that depletion of long-resident B-lymphocytes leads to a partial rejuvenation of B-cell precursor content, lymphopoieisis, and antibody production is an unexpected and striking parallel to the expected effects of ablating anergic T-cells. This new research strongly confirms the expectation that a comprehensive strategy of rejuvenation biotechnologies will need to include both arms of the adaptive immune system, and strongly suggests key features of the tools that will be needed to do so.
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7: Janzen V, Forkert R, Fleming HE, Saito Y, Waring MT, Dombkowski DM, Cheng T, DePinho RA, Sharpless NE, Scadden DT. Stem-cell ageing modified by the cyclin-dependent kinase inhibitor p16INK4a. Nature. 2006 Sep 28;443(7110):421-6. Epub 2006 Sep 6. PubMed PMID: 16957735.
8: Chen C, Liu Y, Liu Y, Zheng P. mTOR regulation and therapeutic rejuvenation of aging hematopoietic stem cells. Sci Signal. 2009 Nov 24;2(98):ra75. PubMed PMID: 19934433.
9: Rebo J, Causey K, Zealley B, Webb T, Hamalainen M, Cook B, Schloendorn J. Whole-animal senescent cytotoxic T cell removal using antibodies linked to magnetic nanoparticles. Rejuvenation Res. 2010 Apr-Jun;13(2-3):298-300. PubMed PMID: 20426617.
10: Nikolich-Zugich Lab. Rejuvenation of the aging t-cell pool by rebalancing T-cell repertoire. DG Cohort #1 Final Report. 2010 Nov 23.