TREC ir KREC ‐ žymenys T ir B ląstelių neogenezei po aloKKLT

Mindaugas StoškusVULSK HOTC MRMS

Intro

• T cell reconstitution is a critical feature of the recovery of the adaptive immune response.

• Though thymic function declines with age, a reasonable output is still maintained into late adult life.

» Douek DC, McFarland RD, Keiser PH, et al: Changes in thymicfunction with age and during the treatment of HIV infection. Nature 1998;396:690694

• In many clinical situations, thymic output is crucial to the maintenance and competence of the T cell effectorimmune response.

T‐cell reconstitution after alloHSCT

• Unlike innate immunity, which recovers early following HSCT, reconstitution of the adaptiveimmunity is a prolonged, if ever complete, process.

• Early T cell recovery is associated with a better clinical outcome.

• T cell reconstitution following HSCT depends on two major pathways

1. Thymic‐independent pathway

• Peripheral clonal expansion, within the recipient of donor‐derived mature T cells from the graft– Transient protection early post‐HSCT as these cells respond and proliferate quickly when challenged with previously encountered pathogens• Only of limited importance in the long‐term due to a restricted repertoire and are potentially alloreactive– GVHD is most likely caused by the alloreactive donor selected T cells that were co transplanted with the graft

Przybylski, G.K.; J. Appl. Genet. 2007, 48, 397–404

2. Thymic‐dependent pathway

• A long‐term process of de novo generation of naïve T cells differentiated in the recipient thymus– Fundamental role of the recipient thymus– Monitoring recent thymic emigrants (RTE) could be of value in HSCT as it would help determine factors affecting immune recovery

– A quantitative and noninvasive biomarker for estimating thymic output was proposed• First described by Douek et al. Nature 1998, 396, 690–695

– Measurement of the extrachromosomal DNA excision circles that are generated following T cell receptor (TCR) gene rearrangement in the thymus, the so called T cell receptor excision circles (TRECs).

TRECs to assay thymic function

• Extrachromosomal DNA byproducts of TCR rearrangement, which are nonreplicative.

• Genereated only in T cells of thymic origin and each cell is thought to contain a single copy of TREC.

• Provides a very specific assessment of T‐cell recovery ﴾e.g., after HSCT﴿ or numerical T‐cell competence.

....&KRECs

• Kappa‐deleting Recombination Excision Circle– generated during B cell development as a result of light chain rearrangements

– represent stable episomal, non‐replicative DNA

• Correlate with bone marrow B cell output.• Mensen et al. Journal of Translational Medicine 2013, 11:188

TCRDTCRG

TCRBTCRA

KrengerW, Blazar BR, Holländer GA. Blood. 2011 Jun 23;117(25):6768‐76

TCRB

Politikos I, Boussiotis VA. Blood. 2014 Nov 20;124(22):3201‐11

TCRA

Politikos I, Boussiotis VA. Blood. 2014 Nov 20;124(22):3201‐11

TRECs

• TCR rearrangement has been reported in all TCR genes ‐ TCRD, TCRG, TCRB and TCRA

– Following rearrangement in each gene, one or more TRECs are generated resulting in many different TRECs

• A good candidate TREC must be detectable in peripheral blood and not be affected by the different rearrangement possibilities

– TCRD and TCRG genes undergo rearrangements in very early stages, therefore their TRECs are extensively diluted before they enter the peripheral blood.

– Similarly, TRECs derived from TCRB rearrangements undergo dilution in the thymus so their concentration in the periphery is very low compared to other TRECs generated from later rearrangements.

Optimal TREC• Rearrangement of TCRA requires the deletion of the TCRD gene that is interspersed with TCRA along the same chromosomal location 14q11.

• Occurs late, thus TRECs are less diluted by thymocyte expansion.• 70% of TCRD deletion rearrangements result in a deltaREC‐psiJalpha signal joint TREC (sjTREC) and coding joint.– Coding joint is found in the final rearrangement of V‐J signal TREC but

might also be found on another allele of genomic DNA and there is no possibility of distinguishing between them.

• deltaREC ‐psiJalpha signal joint TREC (sjTREC) is the optimal target for measurement in clinical setting.

» Hazenberg MD, Verschuren MC, Hamann D, et al: T cell receptor excision circles as markers for recent thymic emigrants: basic aspects, technical approach, and guidelines for interpretation. J Mol Med 2001;79:631640

TCRA

Politikos I, Boussiotis VA. Blood. 2014 Nov 20;124(22):3201‐11

Variabilities in the methods used for TREC estimation

TREC result interpretation

• Should be carefully interpreted to avoid erroneous conclusions– Since sjTREC levels are also influenced by:

• Longevity of naïve T cells• Peripheral expansion or apoptosis of T cells and intracellular degradation

sjTREC / DJ TREC ratio

• A novel approach that allows diminishing the effect of peripheral expansion of T‐cells– sjTREC and DJ TRECs are measured simultaneously and the sjTREC/ DJ TREC ratio is determined.• This methodology is more informative, it is laborious, expensive and time‐consuming as implies multiple multiplex reactions preceeded by preamplification of DJ TRECs .

KrengerW, Blazar BR, Holländer GA. Blood. 2011 Jun 23;117(25):6768‐76

KrengerW, Blazar BR, Holländer GA. Blood. 2011 Jun 23;117(25):6768‐76

Alternative approaches

Clinical Applications of TREC

• Assessment thymic output where T cells are essential for clinical evaluation and/or are involved in disease pathophysiology:– Congenital immunodeficiency– HIV infection– Autoimmune diseases – HSCT– As a measure of immune competence

• in patients receiving immunotherapy or cancer vaccines– where maintenance of T cell outputis integral to the immune response against cancer.

Gaballa et al. Int. J. Mol. Sci. 2016, 17, 1705

TRECs & SCID• Heterogeneous group of severe genetic disorders of the

immune system • Characterized by T cell maturation arrest with or without B cell involvement.

• The disease is fatal unless prompt measures are taken at very early age

• TREC has proven to be the best non‐invasive, feasible screening tool for SCID and has been implemented clinically in the USA.

• .....most likely become a worldwide screening tool for SCID

• The numbers of TRECs early after HSCT were most predictive for longterm reconstitution.

TRECs in HIV and Antiretroviral Therapy

• A decrease in thymic output in HIV‐infected patients compared to age‐matched healthy controls was reported.

• After initiation of treatment and suppression of viral load a rapid increase in TREC was observed

• Adult patients treated with highly active antiretroviral therapy ﴾HAART﴿ show a rapid and sustained increase in thymic output.

• Douek et al .Nature 1998;396:690694

TRECs in autoimmune diseases?

• A marked reduction of TREC levels was shown in 51 rheumatoid arthritis adult patients compared to 47 healthy controls

• However, no reduction in TREC levels was found in another study of 70 children with juvenile idiopathic arthritis (JIA) and 110 healthy age‐matched controls.

• Decreased significantly in patients with multiple sclerosis indicating that the thymus might play an important role in the pathogenesis of the disease.

TRECs in HSCT• Frequent monitoring of T cell immunity and TREC numbers after

HSCT can help identify patients who will fail to reconstitute properly.– Following HSCT, there is a period of prolonged immunodeficiency that

varies depending on the nature and type of stem cell graft used and the conditioning regimen, among other factors.• This secondary immunodeficiency also includes defects in thymopoiesis.

» It has been shown that numerical T cell recovery is usually achieved by day 100 posttransplant, though there is an inversion of the CD4:CD8 ratio that can persist for up to a year.

» Also, recovery of T cell function and diversity can take up to 12 months, although this can be more rapid in pediatric patients.

– Identification of such patients would allow additional therapies to be intoduced in a timely manner.

TREC levels after HSCT

Impact on pre‐transplant thymicfunction of the recipient

• Least studied possible effect on TREC levels

• Patients with high TRECs have better OS, decreased incidence of severe GVHD (acute and chronic) and lower incidence of several opportunistic infections

• Clave, E. et al Blood 2005, 105, 2608–2613.

• High levels of sjTRECs before HSCT and a reduced risk of death mainly due to a reduction in the incidence of relapse

• Saglio, F. et al. Biol. Blood Mar. Trans. 2015, 21, 1099–1105.

• Unfortunately, in both studies pre‐transplant TREC levels were not correlated to TREC levels post HSCT or other thymic activity parameters.

Other factors affecting TREC levels• Changes in absolute counts of lymphocyte subsets due to

– Variety of biological factors, including hormones, the environment, and temperature.

– Diurnal ﴾circadian﴿ variations • Progressive increase in CD4 T cell count throughout the day, while CD8 T cells

and CD19+ B cells increase between 8:30 am and noon, with no change between noon and afternoon.

– Natural killer ﴾NK﴿ cell counts, on the other hand, are constant throughout the day ﴾10)• It is generally accepted that lower CD4 T cell counts are seen in the morning

compared with the evening and during summer compared to winter.

• Show an inverse correlation with age– though there can be substantial variations in TREC copies relative to T

cell count within a given age group.

General recommendations for TREC level monitoring

• Pretransplant or a pretreatment TREC baseline should be determined– ...so that appropriate comparisons can be made between the pre‐ and post‐

HSCT treatment specimens. – Substantial variability between individuals in TREC copies.

• The best comparison is made to the patient's own baseline specimen rather than the age‐ and/or disease‐matched reference range.

• Assessment in CD3+ subset generally gives more Consistent results

• Preferred PB sample volumes:– Adults: 10 ml– Chidren: 5 ml for > 1 year old, 3 ml for < =1 year old

• Timing and consistency in timing of blood collection are critical when serially monitoring patients for lymphocyte subsets.

Limitations of TREC assessment• Cannot be taken as a direct measure of thymic output

• ....because are diluted by peripheral T cell division and intracellular degradation.

• the longevity of naive T cells in the periphery precludes TREC from being regarded as recent thymic emigrants.

• Is not informative in adult patients over age 60• due to physiological decline in thymic function in the sixth and seventh

decades of life.

• Depends on T cell counts• in patients with profound lymphopenia it may be impossible to perform the

assay

• Temperature and time. • Ideally, samples should be delivered in 20‐25 C• Deliverd with 24Hr.

• Transportation delays may result in significant TREC degradation.