immune senescence Flashcards
introduction
ageing associated with progressive decline in immune system function
all components of immune response affected = haematopoiesis, T cells, B cells, innate immunity
considerable clinical challenges, particularly in ageing populations = increased incidence of acute/chronic infection, malignancy, autoimmune disease
important that we better understand mechanisms = may aid development of therapies to reverse/lessen senescence, may aid induction of better vaccine-related immunity in older adults
decline in haematopoiesis
decline in size and proliferative potential of LT-HSC pool
intrinsic defects in HSCs combined with extrinsic defects in stromal cells of niche
evidence for intrinsic defects in HSCs
bone marrow chimera experiments
reconstituted irradiated mice with mixture of young and old HSCs - over time, haematopoietic cells purely/predominately derived from young cells
as cells develop in same niche/microenvironment = old HSCs must show intrinsic defects that reduce function
intrinsic defects
reduced expression of adhesion molecules (e.g. VCAM, a4 + a5 integrins) = reduced adhesion to stromal cells of niche = reduced quiescence, protection from exhaustion
reduced homing to endosteal region, relocate to further sites = less protected from environmental insults/autoimmunity/inflammation/oxidative stress
Dykstra et al (2011)
- transplanted irradiated mice with mixture of young and old HSCs expressing different fluorescent proteins (CFP, GFP or dared) combined in known ratio = sacrificed animals 16-21h later and analysed fluorescence ratio in bone marrow sections using FC, comparing to pre-transplantation ratio
- old cells show 2-fold reduced homing efficiency
- also reduced bone marrow engraftment and reconstitution of haematopoietic cells (more than half of mice transplanted with old cells had <1% donor chimerism, only 2/19 had when transplanted with old cells)
extrinsic defects
progressive myeloid bias with age = less able to produce lymphoid progenitors and progeny
when young, HSCs divide asymmetrically –> 1 LT-HSC, 1 MPP, which differentiate into CLP and CMP in equal measure –> less able to produce CLP with age
also reduction in GH/IFG1 axis –> CAR cells differentiate into adipocytes = negative regulators of haematopoiesis
also results in loss of CXCL12 = contribute to nighttime waves of haemtopoiesis
myeloid bias and OPN
Osteopontin (OPN/Spp1)
• OPN is expressed by stromal cells in the bone marrow and exists in two isoforms, intracellular and secreted (soluble)
• Both isoforms favour lymphoid specification by different mechanisms; sOPN induces proliferation of lymphoid progenitors, while iOPN causes apoptosis of myeloid progenitors
• Loss of OPN with age due to changes in osteoblastic niche may contribute to the progressive skewing towards the myeloid lineage
Kanayama et al (2017) = found that iOPN inhibits myelopoiesis whilst sOPN promotes lymphopoiesis
• Found that irradiated WT mice transplanted with Spp1 KO bone marrow showed increased myeloid cell populations and decreased lymphoid cell populations in multiple organs 7 weeks after transplant
- Conclusion: OPN inhibits myelopoiesis and promotes lymphopoiesis
• Then compared phenotype of
1. irradiated mice transplanted with BM from Spp1 KO mice = no sOPN or iOPN
2. BM from LSL-iOPN mice transferred into irradiated WT mice = iOPN but no sOPN due to deletion of signal sequence that targets OPN to secretory vesicles
• Proportion of myeloid progenitors at week 3, and neutrophils/monocytes/macrophages at week 7, was higher in Spp1 KO chimeras than LSL-iOPN chimeras
• No significant difference in proportion of T + B cells iOPN does not expand lymphoid cells
• Conclusion: iOPN restricts myeloid cell development, sOPN expands lymphoid cells = coordinate to tip balance of myeloid vs lymphoid progenitors
myeloid bias and RANTES
Increased secretion of RANTES/CCL5 may also contribute to myeloid bias in old age
Ergen et al (2012)
1. Detected higher levels of RANTES expression in BM of older mice = cytometric bead arrays
2. More RANTES reduced lymphopoiesis and increased myelopoiesis
- Overexpressed RANTES-GFP in BM using retroviral vector, and transplanted into irradiated mice significant decrease in T cells, slight increase in myeloid cells, increased expression of regulators of myeloid differentiation (e.g. GATA1, GATA2) and reduced expression of regulators of lymphoid differentiation (e.g. Ikaros) (q-RTPCR)
- Treated ex vivo HSCs with RANTES before transplantation same skewing towards myelopoiesis and away from lymphopoiesis
3. RANTES KO mice more lymphocytes and fewer myeloid progenitors in CBC
failed to identify main source of RANTES in BM = what declines with age?
only in mice = translatable into humans?
CAR cell depletion experiment
• Omatsu et al (2010)
- ablation of CAR cells using CXCL12-DTR knock-in mice leads to a decrease in the pool of HSC, which are more profoundly quiescent than WT HSCs
- HSC in CAR-depleted mice show increased expression of myeloid genes bone marrow contains reduced numbers of lymphoid and erythroid progenitors
age-related changes in B cell compartment
CXCL12 is essential for early B cell commitment = loss of CAR cells therefore causes in frequency of pro-B cells
transition from pro to pre-B cells is impaired due to reduction in VDJ recombination
reduced numbers of immature B cells reaching spleen results in accumulation of quiescent memory cells in peripheral niches = memory responses not compromised but new responses to pathogens are significantly attenuated
humeral responses are shorter in aged individuals + less protective due to low Ab titres, preponderance of IgM and impaired SHM = low affinity Abs
changes may be due to poor Th cell function = required for GC formation and promoting somatic mutations
effects on T cell compartment = newborn vs elderly
In the new-born = huge contribution from thymus, T cell repertoire is polyclonal with many different specificities, homeostatic proliferation contributes only a small amount to maintaining number of T cells in periphery, most T cells are naïve with few memory cells
In the elderly = thymic output providing new specificities to the repertoire is low, homeostatic expansion is largely responsible for maintaining T cell numbers when mature T cells go through cell division both progenies express exactly the same T cell receptor , most T cells have a memory phenotype, low TCR diversity
thymic involution
- Progressive loss of thymic mass and function from the neonatal period onwards, accelerated at puberty due to hormonal changes = permanent and irreversible
- In the human, the thymus increases in size with involution = degeneration of the cortex leads to infiltration with adipocytes = leads to impaired thymic function
TRECs
Analysis of TCR excision circles (TRECs) permits monitoring of thymic output over time
• Cells that have recently emigrated from the thymus can be identified by episomal pieces of DNA = T cell receptor excision circle (TREC)
• Released during VDJ recombination = occurs following upregulation of VDJ recombinase enzyme at CD44+ 25+ stage
• Have no origins of replication = as the cell divides, they are progressively diluted, and thus the proportion of TREC-marked cells in the periphery is a surrogate of thymic activity
• TRECs act as molecular signature that marks these cells, enabling us to track them as they leave the thymus and enter the periphery = gives us some idea of T cell output from the thymus
• What is more, TRECs released at different stages of rearrangement can be tracked using qPCR, providing even more precise information
impact of thymic involution
Impact of thymic involution
• Hakim et al (2005) = used quantitative PCR to track emergence of RTE from the thymus in patients treated with BMT = must reinitiate thymopoiesis to recover T cell populations
• TREC frequency approximately 2 years after transplant was strongly correlated with age of patients = age affects ability to reinitiate thymopoiesis
- 30 years old = 10% contain sjTREC as evidence they have just emigrated from the thymus
- Mid 60s = drops to 0.1%
• Clearly a precipitous decline in thymic output with age
• Surprises in study
- Thymic output never decreases to zero = still modest increases in CD4+ T cells even in oldest individuals
homeostatic expansion
- Achieved through homeostatic expansion = proliferation without activation
- Homeostatic proliferation maintains normal T cell numbers in response to lymphopenia (e.g. haemorrhage, HIV)
- Expansion is driven by exogenous cytokines of the common y chain family (e.g. IL-7 and IL-15), and by the TFs T-bet and Eo
- Proliferation is dependent on recognition of self-peptide/MHC complexes on immature DC in secondary lymphoid tissues, and is therefore analogous to thymic positive selection
- Homeostatic expansion is co-stimulation independent
decreased diversity of TCR
Thymic involution and homeostatic expansion results in a significantly decreased diversity of T cell repertoire
• Repertoire becomes progressively oligoclonal progressive immune compromise
• • Diversity of T cells can be measured using Spectra typing = uses mRNA from T cells to amplify cDNA across CDR3 region of TCRB by PCR = generates information about heterogeneity of relative frequencies of different CDR3 length products = used to determine TCR repertoire diversity
• Hakim (2005) = when output of thymus is high, there is a very high diversity in usage of T cell receptor chain genes; when output is low, diversity of T cells produced is also very low
• Naylor et al (2005)
- Isolated CD4+ T cells from PBMC samples sequenced random samples of TCR B-chains determined frequency of these sequences in subsequent blood sample by limiting dilution and PCR/oligonucleotide hybridisation
- estimated TCR VB repertoire diversity in young and middle-aged adults as 20 million different TCR-B chains pool severely contracted to 200,000 TCR-B specificities in elderly individuals (older than 70 years)
- limitation = limiting dilution systems always provide upper estimate because assay system may not be sensitive enough to detect a single TCR-B chain = good to replicate w another method
• results in reduced ability to mount immune responses to new pathogens
clinical impact of thymic involution
• decreased diversity of the T cell repertoire and declining thymic output results in progressive immune compromise with age = important for vaccinations
• Thymic involution also results in imbalance of T cell subsets
- homeostatic expansion applies more to effector T cells than regulatory T cells
- effector T cells rapidly go into cell division in response to T cell loss, whereas regulatory T cells are much more reticent to do so
- predominance of effector T cells leads to increased incidence of autoimmunity
• low thymic output results in poor recovery of immune function following depletion of peripheral T cells
impact of involution on recovery from chemo
• Mackall et al (1995)
- Looked at impact of chemo on different aged patients
- In 3-year-old child = before chemotherapy, immune system is composed of a population of naïve T cells (CD45RA positive) and memory T cells (CD45RO positive)
- Onset of chemotherapy induces rapid decline in T cell numbers = small population of cells that survive are almost entirely memory T cells
- Two months after cessation, we start to see a recovery = almost entire T cell repertoire regenerated by 6 months
- In 23-year-old = chemo induces rapid decline in T cell numbers begin to see recovery only over 8 months after cessation, and still deficiency at 15 months (though we do see reappearance of some naïve T cells)
- Elderly individuals show very poor recovery from chemotherapy = thymus makes negligible contribution, very small proportion of naïve T cells
- Immune system not even began to recover even 24 months after cessation
Mackall et al (1997) = prolonged T cell subset imbalance after chemo
• Compared CD4+ and CD8+ T cell regeneration in patients following intensive chemotherapy
• Used mathematical modelling to provide estimates of doubling times = significant quantitative differences between subtypes = imbalance in recovery
- 98% of CD8+ T cells recovered by 3 months post-therapy
- Only 35% of CD4+ T cells recovered by 3 months still incomplete at up to 12 months
• However, CD8+ cells are completely reliant on CD4+ T cells for T cell help = abundance of CD8+ cells is of little benefit to the individual
• Mechanism unclear = but likely that thymic-independent pathways of CD8+ T cell generation are more efficient than CD4+ similar expansion of other clinical settings associated with impaired thymopoiesis (e.g. HIV, post-BMT)
AIDS
Impact on T cell recovery after indinavir treatment for AIDS
• Vast majority of new T cells produced by homeostatic expansion not by thymus = very few naïve T cells recovered
possible reasons for involution
Why does involution occur?
Hypotheses include
1. General wear and tear of the stroma, as thymus is very active early in life
- BUT why does thymus succumb more than other organs (e.g. gut)?
- Exhaustion of the MTS24+ stem cell pool
- Why would thymus be more vulnerable to exhaustion of SCs than other tissues? - Secondary to insufficiency of bone marrow progenitors
• Investigated by Mackall et al (1998)
• Took old mouse that had already undergone thymic involution lethally irradiated it rescued with bone marrow from young mouse sufficient to restore architecture of thymus?
• Used immunocytochemical staining using ER-TR4 and ER-TR5 antibodies which preferentially bind to CTEC and MTEC
• NO provision of a significant pool of young progenitors did not restore age-related histological abnormalities/involution/rejuvenate thymus - Direct adaptation to reduce the risk of malignancy
- Thymus supports the greatest rate of cellular proliferation of any organ
- Proliferation is associated with recombination of the germ line genes and significant risk of mutation (very error-prone process) high risk of leukaemia
- BUT similar level of proliferation of B cells, also requiring recombination of receptor chain genes, in bone marrow no involution here! - The cortical epithelium is the target of chronic autoimmunity, as a consequence of positive selection
- Positive selection in the thymus results in a repertoire that is inherently self-reactive
- Self-reactivity is functionally silent in the periphery as self-peptides are not normally presented in the context of inflammation
- Activated T cells may mount a response to cTEC upon recirculation through the thymus = killing of cTEC cells = autoimmunity
- Evidence for autoimmune hypothesis
- Thymic involution is progressive from birth onwards, correlating with increased exposure to pathogens
- Strains of mice prone to autoimmunity display a significantly enhanced rate of thymic involution (particularly NOD mouse) - Widely accepted hypothesis = the thymus represents part of the ‘disposable soma’
- Neonatal thymus contains 1011 thymocytes, of which 20-25% arise de novo each day
- Less than 5% of cells survive selection (cells die by neglect in situ in cortex and are removed by macrophages, or are actively deleted in negative selection): no other organ is so energetically wasteful
- There is significant selection pressure to dispose of the thymus to conserve energy
- The original selection pressure on homo sapiens was to dispense with energetically unfavourable tissues once immunity to local pathogens had been established and reproductive age had been reached
IL-22 in preventing involution
IL-22 drives endogenous transient thymic regeneration in mice enhances survival and proliferation of thymocytes and TECs
Dudakov et al (2012)
- Compared thymic function in WT and IL-22KO mice
- No difference in total thymic cellularity or composition at baseline
- Following sublethal irradiation or HSCT, IL22KO mice displayed significantly impaired thymic regeneration compared to WT (significantly reduced numbers of all developing thymocytes, TECs and non-TECs) persisted up to 98 days after insult - Found that levels of intrathymic IL-22 increased by 2-3 fold in irradiated WT mice vs control mice that were not irradiated, as measured by ELISA
- Deduced that ILC subtype called lymphoid tissue-inducer cells (LTi) was responsible for IL-22 secretion
- Frequency increased significantly after TBI
- KO of RORyT (crucial for LTi development) no effect on baseline intrathymic IL-22, but no significant increase in intrathymic IL-22 following TBI = crucial for IL-22 upregulation following thymic damage - Secretion of IL-22 by LTi is dependent on IL-23 secreted by thymic DCs, which sense tissue damage via DAMPs
- Also showed that administration of recombinant IL-22 to mice after SL-TBI significantly increased thymic cellularity (and numbers of all thymocyte and TEC subsets) at days 7 and 28 similar effects following HSCT
Can we use IL-22 therapeutically? experiments have shown that recombinant IL-22 is not effective at rejuvenating thymus in old mice so likely not
study showed no effect in non-irradiated mice
hormone treatment for regeneration
Sutherland et al (2005)
- Androgen ablation results in complete restoration of aged male thymus in mice, with distinct cortical and medullary architecture
Brad administration followed by immunofluorescence staining w anti-BRDU antibodies revealed significant increase in proliferation
FACS profiles of annexin V staining revealed significant reduction in apoptotic cells
- Sex steroid ablation for treatment of prostatic carcinoma (LHRH agonists) results in an increase in naïve TREC+ cells in peripheral blood (RT-PCR)
- Will never be used clinically = only useful in men, many wouldn’t see castration as useful treatment for thymic involution
reprogramming to produce TECs
Bredenkamp et al (2014)
• Working from principles of Waddington’s epigenetic landscape, they reprogrammed primary mouse embryonic fibroblasts (MEFs) into functional TECs through transduction with FOXN1 gene (master TF for thymus production)
• Used Cre-lox technology
- Crossed mice expressing tamoxifen-inducible Cre allele expressed under Rosa26 promoter, with mice in which FOXN1 cDNA was knocked into Rosa26 locus under control of CAG promoter, with LoxP-flanked STOP cassette inserted between CAG promoter and FOXN1 cDNA
• Following tamoxifen administration, iFOXN1 MEFs expressed TEC-specific genes at levels comparable to foetal TECs = e.g. Dll4, Cccl25, KitL (RT-PCR)
• When implanted under the kidney capsule of syngeneic adult mice, induced cells spontaneously formed a functional thymus able to select a normal T cell repertoire = histology, FC
• Potential clinical applications as reprogramming strategies are improved
innate immunity
Neutrophils
• Impairment in neutrophil function likely a major contributor to increased incidence of infections in older adults
• Reduced chemotaxis and migration, compromised phagocytosis, reduced production of ROS, reduced GM-CSF signalling, impaired signal transduction
• Simell et al (2011) = evidence for reduced phagocytic killing in elderly
- Isolated polymorphonuclear neutrophils from young subjects + elderly adults significantly reduced phagocytic killing of Strep pneumococcus opsonised with antibodies and complement in vitro reverse association between advancing age and killing efficiency
- Up to 2.8-fold decline in efficacy for some strains = significant
Macrophages/monocytes
• Skewing towards M2 (immunoregulatory) phenotype with old age
• Reduced TLR-induced pro-inflammatory cytokine production, reduced co-stimulatory molecule expression, reduced phagocytosis
Dendritic cells = reduced ability control tumour growth, reduced expression of co-stimulatory proteins etc
Natural killer cells = decreased cytotoxicity, reduced IFNy production, reduced cytokine and chemokine secretion
IL-7 to improve cell-mediated immunity in old people
Aspinall et al (2007)
- treated old female rhesus macaques w subcutaneous injection of IL-7 for 14 days
- increase in CD4+ and CD8+ T cells, naive T cells (CD45RA+) for both subsets and TREC levels (FC)
greater HAI titres, and influenza-specific memory CD8+ T cells following vaccination w inactivated influenza vaccine
