Measuring T cell output or reconstitution (thymopoiesis) following

hematopoietic stem cell transplantation (HSCT) or highly active

antiretroviral therapy (HAART)

 

Evaluating T cell immune competence in patients with primary

immunodeficiencies, with idiopathic T cell deficiencies, or receiving

immunotherapy or cancer vaccines

 

Assessing T cell recovery following thymus transplants for

DiGeorge's syndrome

T cell reconstitution is a critical feature of the recovery of the adaptive

immune response and has 2 main components:  thymic output of new

T cells and peripheral homeostatic expansion of preexisting T cells.

It has been shown that though thymic function declines with age,

substantial output is still maintained into late adult life.(1) In many

clinical situations, thymic output is crucial to the maintenance and

competence of the T cell effector immune response.

 

Thymic function can be determined by T cell receptor excision circle

(TREC) analysis. TRECs are episomal DNA by-products of T cell

receptor (TCR) rearrangement, which are nonreplicative. TRECs

are expressed only in T cells of thymic origin and each cell contains

a single copy of TREC. Hence, TREC analysis provides a very

specific assessment of T cell recovery (eg, during human immuno-

deficiency virus [HIV] treatment or after hematopoietic stem cell

transplantation) or T cell competence. There are several TRECs

generated during the process of TCR rearrangement and the TCR

delta deletion TREC (deltaREC psi-J-alpha signal joint TREC) has

been shown to be the most accurate TREC for measuring thymic

output.(2) This assay measures this specific TREC using quantitative,

real-time polymerase chain reaction (PCR).

  

Clinical use of TRECs in HIV and antiretroviral therapy:

HIV infection leads to a decrease in thymic function. Adult patients

treated with highly active antiretroviral therapy (HAART) show a rapid

and sustained increase in thymic output.(1)

 

Clinical use of TRECs in hematopoietic stem cell transplantation

(HSCT) and primary immunodeficiency (PID):

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.(3-5)  

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.(4) Also, recovery

of T cell function and diversity can take up to 12 months, although

this can be more rapid in pediatric patients. However, recovery

of T cell function is only possible when there is numerical

reconstitution of T cells. T cells, along with the other components

of adaptive immunity, are key players in the successful response

to vaccination post-HSCT.(6)  

 

Recently, it has been shown in patients who received HSCT for

severe combined immunodeficiency (SCID) that T cell recovery

early after stem cell transplant is crucial to long-term T cell

reconstitution.(7) Patients who demonstrated impaired reconstitution

were shown to have poor early grafting, as opposed to immune

failure caused by accelerated loss of thymic output or long-term

graft failure. In this study, the numbers of TRECs early after HSCT

were most predictive for long-term reconstitution. This data

suggests that frequent monitoring of T cell immunity and TREC

numbers after HSCT can help identify patients who will fail to

reconstitute properly, which would allow additional therapies to

be instituted in a timely manner.(7) It would be reasonable

to extrapolate such a conclusion to other diseases that are also

treated by HSCT.

 

TREC counts and thymic output in adults:

Since the adult thymus involutes after puberty and is progressively

replaced by fat with age, thymus-dependent T cell recovery has

been assumed to be severely limited in adults. However, with

TREC analysis it has been shown that the change in thymic function

in adults is a quantitative phenomenon rather than a qualitative one

and thymic output is not totally eliminated.(1,8,9) Thus, after HSCT

or HAART, the remaining thymic tissue can be mobilized in adults

to replenish depleted immune systems with a potentially broader

repertoire of naive T cells. Douek, et al have shown that there is a

significant contribution by the thymus to immune reconstitution after

myeloablative chemotherapy and HSCT in adults.(8) In fact, this

data shows that there is both a marked increase in the TREC numbers

and a significant negative correlation of TREC counts with age

posttransplant.

 

In addition to the specific clinical situations elucidated above, the

TREC count can be helpful in identifying patients with primary

immunodeficiencies and assessing their T cell immune competence.

It can also be used as a measure of immune competence in patients

receiving immunotherapy or cancer vaccines, where maintenance

of thymic function (ie, T cell output) is integral to the immune response

against cancer.

T & B ABSOLUTE COUNTS

      T-cells (CD3)

             0-2 months: 2,500-5,500 cells/uL*

             3-5 months: 2,500-5,600 cells/uL*

             6-11 months: 1,900-5,900 cells/uL*

             12-23 months: 2,100-6,200 cells/uL*

             2-5 years: 1,400-3,700 cells/uL*

             6-11 years: 1,200-2,600 cells/uL*

             12-17 years: 1,000-2,200 cells/uL*

             > or =18 years: 582-1,992 cells/uL

      Helper cells (CD4)  

             0-2 months: 1,600-4,000 cells/uL*

             3-5 months: 1,800-4,000 cells/uL*

             6-11 months: 1,400-4,300 cells/uL*

             12-23 months: 1,300-3,400 cells/uL*

             2-5 years: 700-2,200 cells/uL*

             6-11 years: 650-1,500 cells/uL*

             12-17 years: 530-1,300 cells/uL*

             > or =18 years: 401-1,532 cells/uL

      Suppressor cells (CD8)

             0-2 months: 560-1,700 cells/uL*

             3-5 months: 590-1,600 cells/uL*

             6-11 months: 500-1,700 cells/uL*

             12-23 months: 620-2,000 cells/uL*

             2-5 years: 490-1,300 cells/uL*

             6-11 years: 370-1,100 cells/uL*

             12-17 years: 330-920 cells/uL*

             > or =18 years: 152-838 cells/uL

*Shearer WT, Rosenblatt HM, Gelman RS, et al:  Lymphocyte

subsets in healthy children from birth through 18 years of age: The

Pediatric AIDS Clinical Trials Group P1009 study. J Allergy Clin Immunol   

2003;112(5):973-980

 

TREC, IMMUNE RECONSTITUTION

      Pediatric

             0-2 months: not established

             6 months-18 years: >801 copies per million peripheral blood mononuclear cells

      Adult

             19-44 years: >227 copies per million CD3 T cells

             45-54 years: >111 copies per million CD3 T cells

             > or = 55 years: >78 copies per million CD3 T cells

TREC results are expressed as copies per million CD3 T cells for adult

specimens or per million peripheral blood mononuclear cells for pediatric

specimens.

TRECs generally show an inverse correlation with age, though

there can be substantial variations in TREC count within a given

age group.

 

Following HSCT, HAART, thymic transplants, etc, the TREC count

typically increases from absent or very low levels (below age-matched

reference range) to baseline levels or exceeds baseline levels,

showing evidence of thymic rebound, which is consistent with

recovery of thymic output and T cell reconstitution.  

 

When a patient is being monitored for thymic recovery posttransplant

treatment, this assay requires that a pretransplant (prior to myeloablative

or nonmyeloablative conditioning) or a pretreatment baseline

specimen be provided so that appropriate comparisons can be

made between the pre- and posttransplant treatment specimens.  

Since there is substantial variability between individuals in TREC

counts, the best comparison is made to the patient's own baseline

specimen rather than the reference range (which provides a guideline

for TREC counts for age-matched healthy controls).

 

Additionally, a single TREC measurement has very little value in

discerning thymic reconstitution in patients. Serial measurements

3 months apart for the 1st year and 6 months apart for the 2nd year

following transplant are recommended.

 

For HIV patients on HAART, TREC measurement can be used for

monitoring, along with other laboratory parameters (specified on

page 4, section on Initial Assessment and Monitoring of Therapeutic

Response in the Guidelines for the use of antiretroviral agents in

HIV-1 infected adults and adolescents, May 4, 2006, developed

by the DHHS Panel on Antiretroviral Guidelines for Adults and

Adolescents - a working group of the Office of AIDS Research

Advisory Council). These guidelines suggest monitoring CD4

count every 3 to 6 months and, at the same time, the TREC count

can be measured as well.  

 

A consultative report will be generated for each patient.

While indicative of thymic function and T cell recovery, TREC

results cannot be taken as a direct measure of thymic output

because the counts are diluted by peripheral T cell division

and intracellular degradation. In addition, the longevity of naive

T cells in the periphery precludes TRECs from being regarded

as recent thymic emigrants. The assay provides a quantitative

measure of TRECs, ie, TREC count per million cells; however,

this number should be regarded as a relative rather than absolute

number because of the caveats explained above.

 

Assay results are dependent on the patient's T-cell counts

and in patients with profound lymphopenia it may be impossible

to perform the assay if there are insufficient numbers of cells.

The assay has been optimized to be performed using 50,000

cells per reaction in triplicate for both the TREC and albumin

probes. The linearity of the assay is robust between 20,000 cells

and 125,000 cells.

 

Temperature and time are critical to the performance of

the assay. Temperatures that exceed or drop below

20 degrees C to 25 degrees C can dramatically affect the assay.

High temperatures can cause substantial hemolysis that will

interfere with the methodology used to perform the assay.

Transportation delays may result in significant TREC degradation.

 

The ordering of the test requires either completion of the test requisition

form or a pretest consultation to ensure that pertinent clinical information

required for test interpretation is gathered.

1.   Douek DC, McFarland RD, Keiser PH, et al:

      Changes in thymic function with age and during

      the treatment of HIV infection. Nature 1998;396:690-694

 

2.   Hazenberg MD, Verschuren MCM, 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;79:631-640

 

3.   Parkman R, Weinberg K:  Immunological reconstitution

      following hematopoietic stem cell transplantation.

      In Hematopoietic Cell Transplantation. 2nd edition.

      Edited by ED Thomas, KG Blume, SJ Forman.

      Blackwell Scientific, Oxford, UK, 1999, pp 704-711

 

4.   Weinberg K, Blazar BR, Wagner JE, et al:  Factors

      affecting thymic function after allogeneic hematopoietic

      stem cell transplantation. Blood 2001;97:1458-1466

 

5.   Weinberg K, Annett G, Kashyap A, et al:  The effect of

      thymic function on immunocompetence following

      bone marrow transplantation. Biol Blood Marrow

      Transplant 1995;1:18-23

 

6.   Auletta JJ, Lazarus HM:  Immune restoration following

      hematopoietic stem cell transplantation:  an evolving

      target. Bone Marrow Transplant 2005;35:835-857

 

7.   Borghans JA, Bredius RG, Hazenberg MD, et al:  

      Early determinants of long-term T cell reconstitution

      after hematopoietic stem cell transplantation for severe

      combined immunodeficiency. Blood 2006;108:763-769

 

8.   Douek DC, Vescio RA, Betts MR, et al:  Assessment

      of thymic output in adults after hematopoietic stem cell

      transplantation and prediction of T cell reconstitution.  

      Lancet 2000;355:1875-1881

 

9.   Jamieson BD, Douek DC, Killian S, et al:  Generation

      of functional thymocytes in the human adult. Immunity

      1999;10:569-575