Tertiary Lymphoid Structures

Question 1

what are TLS?

Answer 1

TLS are found in sites of inflammation/autoimmunity – induced by insult
- They are not physiological or developmentally controlled – they are inducible structures
- Inducible aggregation of leukocytes within non-lymphoid organs - ectopic lymphoid structures
- Similar anatomy (cellular compartments) and spatial organisation to Secondary lymphoid organs (SLOs)
- Detected in target organs of infection, malignancy, autoimmunity

Question 2

how are TLS organised?

Answer 2

They don’t have organisation to be called an organ
- TLS are not encapsulated like an organ
- They are within a tissue

Similar anatomy to GC of lymph node
- Segregated T and B zones, germinal centres with FDCs
- Produce autoantibodies
- can express high endothelial venules

Question 3

when can TLS be good?

Answer 3

In case of infection, TLS important against pathogen e.g. against H. pylori:
- TLS generates quick response at the site
- They are transient and protective in infection

In cancer, they can be good:
- TLS have good prognosis with immune checkpoint blockade – effective immune response against cancer requires TLS

Question 4

when can TLS be bad?

Answer 4

In autoimmunity or chronic inflammation, TLS are chronic and pathogenic
- Associated with poor prognosis, implicated in autoantibody production and associated with lymphoma development

Question 5

how are TLS compared to SLOs?

Answer 5

Not as big as lymph node and not as organised
- But some organisation, similar to follicle – T and B cell zones, FDCs
- Can produce plasma cells and memory B cells

Question 6

why are TLS also known as ectopic lymphoid structures?

Answer 6

as they occur in non-lymphoid locations e.g. in sites of infection

Question 7

what diseases have TLS been implicated in?

Answer 7

associated with inflammation and autoimmunity
- TLS also documented in H pylori infection - forms IgA-producing plasma cells specific to the bacteria
- also found in Hep C infection, influenza A infection
- chronic allograft rejection
- atherosclerosis
- allergic lung disease
- breast cancer

Question 8

how are organs defined?

Answer 8

collection of cells, extracellular structures and fluids joined as an operational unit to serve a common function
- anatomy of organ is designed by resident stromal cells to provide shape and compartmentalisation
- e.g. secondary lymphoid organs: spleen, lymph nodes

Question 9

what are the characteristics of secondary lymphoid organs (SLO)?

Answer 9

SLO = lymph nodes, spleen
- sites of activation of naive immune cells during an immune response - platform for immune cell clustering for efficient adaptive response to pathogen
- display T and B cell areas
- hundreds of lymph nodes around the body at strategic sites - efficient control of pathogen dissemination
- evolves with development of adaptive response
- encapsulated and have independent vascularisation

Question 10

how do SLOs develop?

Answer 10

Developmentally controlled by LTi and LTo interaction
- Pre-natal formation
- Cells that induce SLO are lymphoid tissue inducers (LTi) and lymphoid tissue organisers (LTo)
- Lti is an embryonic immune cell population
- Lto is a stromal/fibroblast cell type
- Lti and Lto interact to initiate dev of SLO

Question 11

what are the characteristics of TLS?

Answer 11

TLS induced upon inflammation or infection in adult life:
- Not driven by Lti and Lto – post-embryonic development
- complex aggregates of leukocytes and stromal cells - resemble SLO
- Adult lymphocytes can take function of Ltis and fibroblast cells in tissue can mimic Lto function
- Then they interact to form TLS
- not encapsulated and lack independent vascular network
- accumulation of immune cells at sites of infection
- generates a localised immune response in tissues

Question 12

how are TLS developed?

Answer 12

1. form in the presence of lymphocytes hat are absence during SLO formation
2. TLS do not develop as separate encapsulated organ, and instead occur in inflamed tissue in repsonse to requirement for accumulation of lymphocytes in response to antigen
3. activation of resident vascular structure and upregulation of homing molecules to enable recruitment is a pre-requisite of TLS assembly

Question 13

what cell types initiate TLS?

Answer 13

inflammatory cytokine, IL-17-secreting T cells
LTi-like CD4 T cells
- these cells are attracted to the inflammatory site by chemokines CXCL13 and CCL21
- within the inflamed lesions, resident stromal cells contribute to the organisation of lymphoid aggregates
- IL-7 and lymphotoxin-a1b2 regulate the chemokine profile needed for B and T cell recruitment, organisation into clusteres and control of angiogenesis

Question 14

what enables maintenance of TLS?

Answer 14

persistent antigen presentation by FDCs and B cells
- defined CD4 T helper effector subsets acquire Tfh characteristics
- committment towards Tfh aids activity of TLS
- TFh relay immunological instruction to B cells which ensure continued action of TLS - CD40L-CD40
- In SLO – require lymphotoxin beta and TNF for maintenance – lymphotoxin controls CXCL13 release
- In TLS, these aren’t required, other cytokines can support maintenance e.g. IL-22 can educate fibroblasts to produce CXCL13 for GC repsonse

Question 15

what is the function of TLS?

Answer 15

• recapitulates cellular, molecular and structural organisation of SLO
• supports function of GC - B cell affinity maturation and differentiation to memory cells and plasma cells via antigen-driven selection process

Question 16

how does the GC response occur in TLS?

Answer 16

antibody fine tuning via somatic hypermutation and class switching - effects antigen recognition and antibody effector mechanism
- activation induced cytidine deaminase (AID) is expressed in TLS tissue at mRNA and protein level
- AID is implicated in autoimmunity and transplant rejection
- AID controls local affinity maturation - shown by restricted profile of V domain usage, hypermutation of V regions and oligoclonal expansion for infiltrating B cells
- class switching is active in TLS
- FDCs show antigen to B cells, Tfh support B cells

Question 17

how do TLS drive autoimmunity?

Answer 17

In autoimmunity, TLS is near autoantigen, and can educate B cells to produce autoantibodies to autoantigen
TLS perpetuates autoimmune process
More cytokines -> further damage and co-stim upregulation -> more autoantigen release -> perpetuates cycle

Question 18

why can TLS be targeted?

Answer 18

TLS doesn’t map completely onto SLO – can be therapeutic target
- Also has different microenviroment
- Cytokine in SLO needed for GC response, the same cytokine isn’t necessarily needed for TLS GC response
- SLO has low IFNy, TLS has high IFNy due to chronic inflammation

Question 19

in what diseases have TLS been associated with poor prognosis?

Answer 19

Sjorgen’s, RA, ulcerative colitis
- associated with poor prognosis, tissue damage, autoantibody production, lymphoma development
- TLS therefore promising target

Question 20

how can TLS be targeted?

Answer 20

target composition of TLS:
- anti-inflammatory cytokine e.g. anti-IL-22, anti-IL-23 which are known to be important for TLS formation and maintenance, anti-TNF
- lymphocyte depletion therapy e.g. rituxumab to prevent TLS formation
- co-stim blockade: in GC response, ICOS and CD40 are needed, so these can be targeted
- antibodies to homeostatic cytokines/chemokines CXCL13, IL-7 BAFF
- enhance Treg pathway - induce immunosuppressive environment and tolerance
- give anti-inflammatory cytokines like IL-27

Question 21

what are the key homeostatic cytokines?

Answer 21

BAFF for B cell survival
IL-7 for T cell survival
- These are essential for SLO and health of lymphocytes – these are homeostatic cytokines
- Anti-BAFF therapies in SLE can block TLS and chronic disease

Question 22

why is it hard to target TLS?

Answer 22

But in these diseases with TLS, there haven’t been many responses to therapies
- Hard to disturb the structure, even with B cell depletion
- After therapy, B cells repopulate

Question 23

why is depleting B cells insufficient?

Answer 23

Blocking/depleting B cells with anti-cd20 is insufficient, as BAFF production is still occurring:
- BAFF is produced by non-immune cells e.g. fibroblasts
- microenvironment is still active, so after therapy, patients relapse as environment is conducive for lymphocyte recruitment
- Stromal cells required for survival of T/B cells
- this is why when therapies are stopped, the niche for B cell survival persists, so patients relapse

Question 24

how do immune cells rely on the stroma in SLO and TLS?

Answer 24

stromal compartment is crucial for function and survival of immune cells
- Produce lymphoid chemokines/cytokines e.g. CXCL13, IL-7, BAFF
- Enables lymphocyte organization - Regulate lymphocyte survival
- Permit antigen-specific humoral response

Question 25

what population of stromal cells are key in SLO and TLS?

Answer 25

fibroblasts in TLS are similar to those found in SLO - network of stromal cells which are key for TLS immune cell function - These cells provide help to lymphocytes for function

Question 26

what do fibroblasts do in lymph nodes?

Answer 26

they control the organisation of survival of lymphocytes in distinct areas - fibroblast reticular cell network produce CCL19 and CCL21, along with IL-7 to maintain T cell survival and proliferation - provides niche in cortical region to interact with DCs - FDCs release CXCL13 which recruit CXCR5+ cells to B cell follicles

Question 27

what other stromal cells are important in GC response?

Answer 27

In SLO: CRCs - CXCL12-expressing reticular cell MRCs - marginal reticular cell - important for antigen delivery

Question 28

what stromal cell populations are important in TLS?

Answer 28

In TLS, stromal cell populations display comparable function to those in SLO - induce B and T cell segregation e.g. FRC-like fibroblast (T zone organiser), FDC-like fibroblast (B zone, GC organiser) - these explain the survival niche that enables these structures to persist

Question 29

how do lymphoid tissue-like stromal cells become primed to form TLS?

Answer 29

Model: priming, maturation and stabilisation of fibroblasts, to form TLS Acute inflammation leads to local production of cytokines by infiltrating leukocyte populations (initiators e.g. macrophages, DCs, resident stromal cells) - produce IL-6, IL-1 - this drives priming of local fibroblasts - upregulates VCAM, ICAM and inflammatory cytokines prolonged inflammation can drive production of LTa1b2, TNFa, LTa by haematopoietic inducer cells - this changes fibroblast phenotype and function, leading to production of lymphoid chemokines - FDCs differentiate from local fibroblasts via high levels of LTa3/a1/b2. TNF from B cells drive positive feedback loop

Question 30

how do lymphoid tissue-like stromal cells develop and mature in TLS?

Answer 30

Initiation of TLS formation, immune cells get recruited - organisation of resident stroma and haematopoeitic cells in T cell and B cell zones - enable priming and activation of T and B cells with antigen, leading to affinity maturation and plasma cell production in GC - Lots of BAFF/IL-7 so B/T cells stay there - Immune cells educate fibroblasts to produce more homeostatic cytokines – reciprocal interaction - Fibroblasts produce CXCL13, CCL19 - Perpetual interaction - Cytokines/chemokines lead to organisation and reach threshold of cells

Question 31

what happens if stromal cells are absent in TLS?

Answer 31

stromal cells are crucial for formation of TLS - in absence of stromal cells, there is failure to induce TLS

Question 32

why should the stroma be targeted in TLS?

Answer 32

combination therapy to target both microenvironmental stromal cells and immune cells to fully deplete the TLS - If immune cells deleted, stromal cells still stay activated – that’s why we need inhibitors of this cycle and silence both populations - Anti-CD40 can block immune cell and stromal cell interaction – more effective e.g. in sjorgen’s syndrome In RA and ulcerative colitis, those that can’t responds to anti-TNF tend to have TLS - More robust therapy needed

Question 33

how are TLS implicated in cancer?

Answer 33

TLS have been detected in tumour sections e.g. CRC, lung, sarcoma - TLS within a tumour contain a T cell zone containing DCs and fibroblast reticular cells, and a B cell zone with GC, plasma cells, antibodies forming immune complexes with tumour antigens and FDCs - the coordinated action of CD8 cells, CD4 cells and B cells generated from TLS enable in situ tumour killing via direct tumour cell killing with ADCC via macrophages and NK cells and direct cytotoxicity - associated with good prognosis and are protective

Question 34

how can memory formed from TLS be protective against cancer?

Answer 34

central memory T and B cells from TLS can circulate and protect against metastasis

Question 35

does the location of the TLS matter in the tumour? can this predict prognosis?

Answer 35

yes - their role in cancer depends on their location in/by the tumour - it impacts cancer progression if TLS is within the tumour, it has protective immune response to eliminate tumour cells - good prognosis if TLS is adjacent to tumour in the infamed invasive margin, it may serve as a niche for tumour progenitors and drive relapse e.g. in renal cell carcinoma - these TLS are rich in Tregs (immunosuppressive) - site for tumour progression - poor prognosis

Question 36

how are TLS context driven?

Answer 36

TLS are context driven - In hep C infection or H. pylori, TLS are positive – they get rid of infection and then resolve – they are transient - Can go wrong and be chronic in autoimmune - In cancer, TLS are very active

Question 37

how do TLS affect ICB?

Answer 37

In cancer, ICB is most effective in those with TLS - ICB leads to activation of CD8 T cells and TLS – enables organisation - Mounts local response to tumour - TLS+ tumours are in ICB responder patients and not in non-responders - Three groups found a strong B cell signature in TLS in responders to ICB suggesting a key role in anti-tumour immunity as compared to TLS with a poor B cell signature. TLS are good biomarker and can be used to predict ICB responders Also a biomarker for RA, if TLS present, anti-TNF won’t work

Question 38

how can TLS be used in cancer immunotherapy?

Answer 38

can try to induce TLS in immune-exhausted or immune-desert tumours in conjunction with ICB to make them more immunogenic location of TLS needs to be considered - ensure it forms within the tumour

Question 39

how could TLS be induced in cancer?

Answer 39

TLS agents like chemokines, cytokines, agonistic antibodies and engineered DCs could express T cell TFs like T-BET or CCL21 to turn tumours with immune cold phenotype into an immune hot tumour - ICB like anti-PD1 could sustain TLS neogenesis - Oncolytic virus can cause lysis of tumour cell, release of neoantigens – we can give IL-22, IL-13 to induce TLS against neoantigen - Or make scaffold of active stroma of TLS with biomaterials and put into tumour – this could help activate immune cells - In immunosuppressed tumour give anti-angiogeneics with cytokine therapy – change stroma in tumour to mimic TLS stroma and give immune cells chance to kill tumour