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Evidence for Increased T Cell Turnover and Decreased Thymic Output in HIV Infection

January 2002
The Journal of Immunology 167(11):6663-8

January 2002
167(11):6663-8

DOI:10.4049/jimmunol.167.11.6663

Source
PubMed

Authors:

Michael Betts

University of Pennsylvania

Brenna Hill

U.S. Army Medical Research Institute of Infectious Diseases

Susan Little

University of California, San Diego

Show all 15 authorsHide

The effects of HIV infection upon the thymus and peripheral T cell turnover have been implicated in the pathogenesis of AIDS. In this study, we investigated whether decreased thymic output, increased T cell proliferation, or both can occur in HIV infection. We measured peripheral blood levels of TCR rearrangement excision circles (TREC) and parameters of cell proliferation, including Ki67 expression and ex vivo bromodeoxyuridine incorporation in 22 individuals with early untreated HIV disease and in 15 HIV-infected individuals undergoing temporary interruption of therapy. We found an inverse association between increased T cell proliferation with rapid viral recrudescence and a decrease in TREC levels. However, during early HIV infection, we found that CD45RO-CD27high (naive) CD4+ T cell proliferation did not increase, despite a loss of TREC within naive CD4+ T cells. A possible explanation for this is that decreased thymic output occurs in HIV-infected humans. This suggests that the loss of TREC during HIV infection can arise from a combination of increased T cell proliferation and decreased thymic output, and that both mechanisms can contribute to the perturbations in T cell homeostasis that underlie the pathogenesis of AIDS.

T cell Ki67 expression and age. The percentage of Ki67 CD45RO CD27 high (naive) and CD45RO (memory) CD4 and CD8 T cells is shown for healthy individuals aged between 23 and 88 years.

…

Correlation between TREC levels and BrdU incorporation in treated HIV-infected individuals after therapy interruption. Shown are the relationships between the fold change in the percentage of BrdU CD4 and BrdU CD8 T cells, and the fold change in CD4 and CD8 T cell TREC levels in the interval between interruption of therapy and its resumption with high viral load. Best-fit exponential regression curves are shown.

…

Early HIV infection and TREC levels. TREC levels in sorted CD4 and CD8 T cells from uninfected individuals, (E) and untreated individuals with early (3-12 mo after seroconversion) HIV infection (F) are shown. Best-fit exponential regression curves for uninfected individuals are shown.

…

T cell Ki67 expression in early HIV infection. The percentage of CD4 and CD8 CD45RO CD27 high (naive) and CD45RO (memory) T cells that are Ki67 in uninfected individuals () and individuals with early HIV infection () are shown. The top, bottom, and line through the middle of the box correspond to the 75th percentile, 25th percentile, and 50th percentile (median), respectively. The whiskers extend from the 10th percentile to the 90th percentile.

…

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of June 14, 2013.

This information is current as

Decreased Thymic Output in HIV Infection

Evidence for Increased T Cell Turnover and

Richard T. Davey and Richard A. Koup

Michael W. Baseler, Philip Keiser, Douglas D. Richman,

Christian Yoder, Joseph W. Adelsberger, Randy A. Stevens,

Little, Richard Lempicki, Julia A. Metcalf, Joseph Casazza,

Daniel C. Douek, Michael R. Betts, Brenna J. Hill, Susan J.

http://www.jimmunol.org/content/167/11/6663

2001; 167:6663-6668; ;J Immunol

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Evidence for Increased T Cell Turnover and Decreased

Thymic Output in HIV Infection

Daniel C. Douek,

‡

Michael R. Betts,* Brenna J. Hill,* Susan J. Little,

Richard Lempicki,

储

Julia A. Metcalf,

†

Joseph Casazza,* Christian Yoder,

Joseph W. Adelsberger,

储

Randy A. Stevens,

储

Michael W. Baseler,

储

Philip Keiser,

Douglas D. Richman,

Richard T. Davey,

†

and Richard A. Koup*

The effects of HIV infection upon the thymus and peripheral T cell turnover have been implicated in the pathogenesis of AIDS.

In this study, we investigated whether decreased thymic output, increased T cell proliferation, or both can occur in HIV infection.

We measured peripheral blood levels of TCR rearrangement excision circles (TREC) and parameters of cell proliferation, in-

cluding Ki67 expression and ex vivo bromodeoxyuridine incorporation in 22 individuals with early untreated HIV disease and in

15 HIV-infected individuals undergoing temporary interruption of therapy. We found an inverse association between increased

T cell proliferation with rapid viral recrudescence and a decrease in TREC levels. However, during early HIV infection, we found

that CD45RO

ⴚ

CD27

high

(naive) CD4

ⴙ

T cell proliferation did not increase, despite a loss of TREC within naive CD4

ⴙ

T cells. A

possible explanation for this is that decreased thymic output occurs in HIV-infected humans. This suggests that the loss of TREC

during HIV infection can arise from a combination of increased T cell proliferation and decreased thymic output, and that both

mechanisms can contribute to the perturbations in T cell homeostasis that underlie the pathogenesis of AIDS. The Journal of

Immunology, 2001, 167: 6663–6668.

cell depletion of CD4

⫹

in HIV infection can arise as a

result of increased destruction and/or reduced production

of T cells through a number of mechanisms, none of

which are mutually exclusive, and for each of which there is ex-

perimental evidence (1–3). T cells can be destroyed by direct or

indirect virus-induced mechanisms, or through Ag-speciﬁc CTL-

mediated lysis (4–9). Reduced T cell production can result from

diminished peripheral expansion of pre-existing T cells or inhibi-

tion of de novo generation of naive T cells from thymocytes or

hematopoietic progenitor cells (8, 10–18). Sequestration of cells in

lymphoid tissues may also affect peripheral T cell numbers (19–

21). The recovery of T cell numbers during highly active antiret-

roviral therapy (HAART)

can occur through a number of different

mechanisms, which may result in the reconstitution of qualitatively

different immune function. Peripheral expansion or redistribution

of pre-existing T cells (17–20, 22) will result in a T cell repertoire

that reﬂects that already marred by HIV infection, whereas de novo

generation of new naive T cells from the thymus (23–27) will

reconstitute a more diverse T cell repertoire (28, 29).

In vivo bromodeoxyuridine (BrdU) incorporation studies in

SIV-infected monkeys showed increased turnover in all T cell pop-

ulations, with memory T cells affected more than naive (7, 30).

Studies using expression of the Ki67 nuclear Ag as a marker of cell

proliferation indicated that total T cell turnover increased in naive

and memory subsets during infection (31). This suggested that

CD4

⫹

T cell loss was due to interference of the virus with “T cell

renewal capacity” rather than with peripheral production, and that

redistribution accounted for increased CD4

⫹

T cell numbers dur

ing treatment (31). However, a more recent study (32) also using

Ki67 showed that turnover rate, but not proliferation, increased in

CD4

⫹

T cells, suggesting their increased death and decreased re

newal (32). In vivo labeling with deuterated glucose conﬁrmed

some of these ﬁndings, showing that HIV infection caused a de-

crease in memory (but not naive) CD4

⫹

and CD8

⫹

T cell half-life

with a compensatory increase in production of CD8

⫹

but not

CD4

⫹

T cells (33).

The measurement of TCR rearrangement excision circles

(TREC) has been used to assess thymic output in individuals with

and without HIV infection (23, 26, 34–37), and after hematopoi-

etic stem cell transplantation (29, 38, 39). In the majority of indi-

viduals with untreated HIV-infection, TREC levels were below

normal, but increased after viral suppression with HAART (23, 26,

37). This was taken to indicate that the thymus, in both adults and

children, is suppressed by HIV infection, but contributes to T cell

*Vaccine Research Center and

†

Laboratory of Immunoregulation, Clinical and Mo

lecular Retrovirology Section, National Institute of Allergy and Infectious Diseases,

‡

Department of Experimental Transplantation and Immunology, Medicine Branch,

National Cancer Institute, and

Critical Care Medicine Department, Warren Mag

nusen Clinical Center, National Institutes of Health, Bethesda, MD 20892;

Depart

ment of Medicine, University of California, San Diego, CA 92103;

储

Science Appli

cations International Corporation-Frederick, Clinical Services Program, Frederick

Cancer Research and Development Center, Frederick, MD 21702;

Department of

Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX

75390; and **San Diego Veterans Affairs Medical Center, La Jolla, CA 92093

Received for publication June 5, 2001. Accepted for publication September 24, 2001.

The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance

with 18 U.S.C. Section 1734 solely to indicate this fact.

This work was supported by the National Institutes of Health (Grants AI35522 and

AI43638 to R.A.K. and Grant AI43638), by the University of California (San Diego)

Center for AIDS Research (Grants AI36214 and AI291674), by the Research Center

for AIDS and HIV infection of the San Diego Veterans Affairs Healthcare System (to

D.D.R.), by the Leukemia and Lymphoma Society of America (Translational Re-

search Grant 6540-00), and by amFAR (Grant 02680-28-RGV to D.C.D.). This

project has also been funded in part with Federal funds from the National Cancer

Institute under Contract NO1-CO-56000.

Address correspondence and reprint requests to Dr. Daniel C. Douek, Vaccine Re

search Center, Room 3509, National Institute of Allergy and Infectious Diseases,

National Institutes of Health, 40 Convent Drive, Bethesda, MD 20892; E-mail ad-

dress: ddouek@mail.nih.gov

Abbreviations used in this paper: HAART, highly active antiretroviral therapy;

BrdU, bromodeoxyuridine; TREC, TCR rearrangement excision circles.

at NIH Library, National Institutes of Health on June 14, 2013http://www.jimmunol.org/Downloaded from

reconstitution during HAART. In this study, we sought to deter-

mine whether increased T cell turnover, decreased thymic output,

or both occur in HIV infection. We measured peripheral blood

TREC levels and parameters of CD4

⫹

and CD8

⫹

T cell prolifer

ation, including Ki67 expression and ex vivo BrdU incorporation,

in 22 individuals with early untreated HIV disease (3–12 mo after

infection), and in 15 successfully treated HIV-infected individuals

who underwent temporary interruption of therapy.

Materials and Methods

Human subjects

Twenty-two patients with early HIV infection (3–12 mo after seroconver-

sion) were seen at the University of Texas Southwestern Medical Center

and had not been on antiretroviral drugs at the time of blood draw. CD4

⫹

T cell counts were 220-1080 cells/

␮

l (mean 602) and viral loads were

⬍400 to ⬎7.5 ⫻ 10

RNA copies/ml. Fifteen patients were asymptomatic

HIV-infected adults with baseline CD4

⫹

T cell counts of ⬎350 cells/

␮

who had been on continuous HAART for a minimum period of 1 year, with

viral loads consistently below the limits of detection for at least that period

of time (40). On day 0, patients discontinued all antiretroviral drugs, and

resumed drugs when any of the following three conditions was met: the

CD4

⫹

T cell count declined at least 25% from the mean of three baseline

determinations, their viral load increased to 5000 RNA copies/ml, or the

patient resumed drug treatment independently. Viral loads were measured

by Amplicor assay (Roche, Basel, Switzerland). Studies were approved by

the Institutions’ review boards, and patients gave informed consent.

Measurement of TREC in MACS-sorted cells

Quantiﬁcation of TREC in sorted CD4

⫹

and CD8

⫹

T cells was performed

by quantitative PCR with an ABI7700 system (PerkinElmer/Cetus, Nor-

walk, CT) as previously described (29). PBMC were separated into CD4

⫹

and CD8

⫹

cells using MACS microbeads (Miltenyi Biotec, Auburn, CA).

Cells were lysed in proteinase K (Boehringer Mannheim, Indianapolis, IN)

and PCR was performed on 5

␮

l of cell lysate (50,000 cells). A standard

curve was plotted, and TREC values for samples were calculated by the

ABI7700 software. Samples were analyzed in duplicate. TREC levels are

expressed as TREC per microgram of DNA (1

␮

g of genomic DNA is

equivalent to 150,000 cells). Cell lysates have been checked for consis-

tency of DNA content using

␤

-actin and CCR5 control PCR; interassay

variability was found to be less than 13% of mean for the same sample in

20 different assays (data not shown).

Ex vivo BrdU uptake analysis

Blood samples were incubated with 100

␮

M BrdU for4hat37°C. Cell

surface staining was performed using Abs to CD3, CD45RO, CD4, and/or

CD8 (BD Biosciences, San Jose, CA). Cells were treated with OptiLyse

(Immunotech, Westbrook, ME) for 10 min at room temperature, then with

1% paraformaldehyde and 1% Tween 20 in PBS for 15 min at 37°C.

Cellular DNA was denatured with 100U DNase-I (Boehringer Mannheim)

for 30 min and was then stained with anti-BrdU-FITC (BD Biosciences).

Events (50,000–100,000) were collected ﬂow cytometrically, resulting in a

sensitivity of 0.01% BrdU

⫹

events, and were analyzed in parallel with

unlabeled cells from the same individual and this value was subtracted

from the value obtained for BrdU-labeled cells. Data are expressed as the

fold change in the percentage of BrdU

⫹

cells to avoid large baseline dif

ferences in absolute values between individuals. However, similar statis-

tical signiﬁcance was obtained when the fold change in absolute BrdU

⫹

cells was used (data not shown).

Naive T cell Ki67 analysis and FACS sorting

Analysis was performed by surface staining cells for either CD4/CD45RO/

CD27 or CD8/CD45RO/CD27 (BD Biosciences), followed by ﬁxation/

permeabilization and intracellular staining for Ki67 (BD Biosciences).

Cells were analyzed by four-color ﬂow cytometry using a FACSCalibur.

Ki67 expression was measured in both CD45RO

⫺

CD27

high

(naive) and

CD45RO

⫹

(memory/effector) CD4

⫹

and CD8

⫹

T cells using Paint-a-Gate

cluster analysis. For FACS sorting of naive and memory T cells, cells were

stained as above (but without Ki67), and were sorted for TREC analysis

using a FACSort (BD Biosciences).

Statistical analyses

Two-tailed Mann-Whitney U test and Spearman’s rank correlation coefﬁ-

cients were performed using SAS and Prism software (SAS Institute, Cary,

NC). Analysis of covariance was used to assess differences in CD4

⫹

and

CD8

⫹

T cell TREC between HIV-infected individuals and the healthy

controls, adjusting for age. A p value of ⬍0.05 was considered signiﬁcant.

Results

TREC levels measure thymic output

TREC frequency in total and in naive T cells has been shown to

decrease with age, after thymectomy, and in HIV infection (23, 26,

34–36, 41). However, a recent mathematical model has suggested

that this decrease in TREC solely reﬂects a theoretical increase in

the naive T cell division rate, and not decreased thymic output (42).

Therefore, we sought to test this experimentally by measuring

changes in T cell division with age using Ki67 expression as a sur-

rogate marker of cell proliferation. Fig. 1 shows that Ki67 expression

does not increase in either CD4

⫹

or CD8

⫹

CD45RO

⫺

CD27

high

(naive) T cells in healthy individuals aged between 23 and 88 years

of age (during which period the most rapid drop in naive T cell

TREC is seen; Refs. 23 and 26). Furthermore, it has recently been

shown that although TREC decrease after thymectomy, there is no

increase in CD27

high

(naive) or CD45RO

⫹

memory T cell Ki67

expression (41). The fact that Ki67 may be raised in nonprolifer-

ating or activated cells (3, 43) does not affect this analysis because

the aim in this study was to exclude any increases in proliferation.

Thus, a decrease in TREC levels can indeed reﬂect a decrease in

thymic output.

TREC levels and BrdU uptake after interruption of therapy

It has recently been shown that after total thymectomy, TREC

levels begin to fall after only 3 mo (41). Therefore, in HIV infec-

tion, any decrease in TREC related to decreased thymic output

would not be expected to be observed until at least 3 mo after

seroconversion. Consequently, decreases in TREC before 3 mo of

HIV infection would reﬂect primarily increased T cell

proliferation.

To examine this, we initially studied T cell TREC levels in 15

HIV-infected individuals who had been successfully treated with

HAART, had undetectable viral loads, and then underwent inter-

ruption of therapy (40, 44). As previously reported, recrudescence

of viral replication occurred in all the patients within 28 days of

interruption of therapy. This allowed us to longitudinally assess

changes in TREC and proliferation during a rapid rise in HIV

levels—a situation reminiscent of acute HIV infection. As viral

load rose, both CD4

⫹

and CD8

⫹

T cell TREC levels decreased

concomitantly. We then performed ex vivo BrdU incorporation to

determine whether this fall in TREC was, in part, secondary to

increased T cell proliferation. The correlation between the S-phase

BrdU fraction and viral load has been previously described (44). In

FIGURE 1. T cell Ki67 expression and age. The percentage of Ki67

⫹

CD45RO

⫺

CD27

high

(naive) and CD45RO

⫹

(memory) CD4

⫹

and CD8

⫹

cells is shown for healthy individuals aged between 23 and 88 years.

6664 THYMIC OUTPUT IN HIV INFECTION

at NIH Library, National Institutes of Health on June 14, 2013http://www.jimmunol.org/Downloaded from

the interval between interruption and resumption of therapy, there

was a signiﬁcant negative correlation between the change in the

percentage of BrdU

⫹

CD4

⫹

T cells and the change in CD4

⫹

T cell

TREC levels (r ⫽⫺0.6, p ⫽ 0.02, and 95% conﬁdence interval ⫽

⫺0.86 to ⫺0.09; Fig. 2). Thus, as CD4

⫹

T cell proliferation in-

creased with a concomitant increase in viral load (40), TREC lev-

els decreased. The change in the percentage of BrdU

⫹

CD8

⫹

cells also varied inversely with the change in CD8

⫹

T cell TREC

levels. However, this relationship was not statistically signiﬁcant

for this sample size (r ⫽⫺0.3, p ⫽ 0.3, and 95% conﬁdence

interval ⫽⫺0.72–0.31), and a larger study sample would be re-

quired to establish a statistically signiﬁcant relationship. It is a

possibility that preferential redistribution of TREC-containing

cells out of the peripheral circulation could cause the decrease in

TREC. Of course, these data do not rule out a concomitant de-

crease in thymic output; it is simply not possible to differentiate the

effects of thymic output and T cell proliferation. However, bearing

in mind the delayed effects of thymectomy on TREC levels (41),

the rapid fall in TREC during an acute rise in HIV load more likely

reﬂects increased T cell proliferation than decreased thymic

output.

TREC levels and Ki67 expression in early HIV infection

Inhibition of thymic function should become detectable by 3 mo

after HIV infection. To differentiate experimentally between thy-

mic inhibition and increased peripheral T cell proliferation, it is

necessary to measure an independent marker of T cell proliferation

in naive and memory T cells, and to measure or calculate the

TREC content of the naive T cell pool. If naive T cell proliferation

is constant, then changes in TREC levels reﬂect changes in the

supply of naive T cells. Therefore, we measured TREC levels and

Ki67 expression in naive and memory CD4

⫹

and CD8

⫹

T cell

subsets in a separate group of 22 patients with early (3–12 mo after

seroconversion) untreated HIV infection. Ki67 expression was

used, rather than BrdU incorporation, because these were cryopre-

served samples. It should be stressed, however, that the number of

Ki67

⫹

cells and the S-phase fraction (BrdU

⫹

cells after a 4-h in

vitro pulse) are not equivalent measures of T cell activation, and

many more cells express Ki67 than are actually in S-phase at any

particular instant in time. The longevity of Ki67 expression after

mitosis remains unclear.

Fig. 3 shows that both CD4

⫹

and CD8

⫹

T cell TREC were

signiﬁcantly lower than in uninfected age-matched controls (p ⫽

0.008 and 0.001, respectively). For measurement of Ki67 expres-

sion in naive and memory T cells, subsets were very carefully

deﬁned so that the naive subset would contain few cells outside the

CD27

⫹

CD45RO

⫺

population. Fig. 4 shows that the percentage of

Ki67

⫹

CD4

⫹

and Ki67

⫹

CD8

⫹

CD45RO

⫹

(memory) T cells was

signiﬁcantly increased in HIV-infected individuals compared with

uninfected individuals (5.7-fold and 6.9-fold, respectively; p ⬍

0.0001 for both). We also conﬁrmed that the percentage of

Ki67

⫹

CD45RO

⫹

(memory) T cells was signiﬁcantly higher than

that of naive T cells within the infected and uninfected groups

(CD4, 17.4-fold and p ⬍ 0.0001; CD8, 5.2-fold and p ⫽ 0.0003;

CD4, 1.9-fold and p ⫽ 0.0031; CD8, 2.7-fold and p ⫽ 0.0017,

infected and uninfected, respectively). However, although the per-

centage of Ki67

⫹

CD8

⫹

CD45RO

⫺

CD27

high

(naive) T cells was

FIGURE 3. Early HIV infection and TREC levels. TREC levels in

sorted CD4

⫹

and CD8

⫹

T cells from uninfected individuals, (E) and un-

treated individuals with early (3–12 mo after seroconversion) HIV infec-

tion (F) are shown. Best-ﬁt exponential regression curves for uninfected

individuals are shown.

FIGURE 4. T cell Ki67 expression in early HIV infection. The percent-

age of CD4

⫹

and CD8

⫹

CD45RO

⫺

CD27

high

(naive) and CD45RO

⫹

(memory) T cells that are Ki67

⫹

in uninfected individuals (⫺) and indi-

viduals with early HIV infection (⫹) are shown. The top, bottom, and line

through the middle of the box correspond to the 75th percentile, 25th per-

centile, and 50th percentile (median), respectively. The whiskers extend

from the 10th percentile to the 90th percentile.

FIGURE 2. Correlation between TREC levels and BrdU incorporation

in treated HIV-infected individuals after therapy interruption. Shown are

the relationships between the fold change in the percentage of

BrdU

⫹

CD4

⫹

and BrdU

⫹

CD8

⫹

T cells, and the fold change in CD4

⫹

and

CD8

⫹

T cell TREC levels in the interval between interruption of therapy

and its resumption with high viral load. Best-ﬁt exponential regression

curves are shown.

6665The Journal of Immunology

at NIH Library, National Institutes of Health on June 14, 2013http://www.jimmunol.org/Downloaded from

also signiﬁcantly increased in HIV-infected individuals (3.6-fold,

p ⫽ 0.0002), the percentage of Ki67

⫹

CD4

⫹

CD45RO

⫺

CD27

high

(naive) T cells did not differ signiﬁcantly from uninfected individ-

uals (p ⫽ 0.064), and was in fact slightly lower. Thus, even though

Ki67 expression may indicate T cell proliferation and/or activa-

tion, these data suggested that the decrease in CD4

⫹

T cell TREC

during HIV infection could not be the result of increased turnover

of naive CD4

⫹

T cells.

Naive CD4

⫹

T cell activation in early HIV infection

Although we found no increase in naive CD4

⫹

T cell Ki67 ex

pression in early HIV infection, other studies have shown it to be

increased (31). The phenotypic deﬁnition of naive T cells as

CD45RO

⫺

CD27

high

by ﬂow cytometry reveals that HIV-infected

subjects contain more T cells with a phenotype between that of

true naive and effector/memory cells than uninfected individuals

(Fig. 5). These have been termed “transitional” cells (45). There-

fore, we reanalyzed the data shown in Fig. 4, incorporating a small

proportion of transitional CD4

⫹

T cells into the naive T cell gate,

and then measuring Ki67 expression. We found that the inclusion

of only 5% more transitional cells apparently increased naive

CD4

⫹

T cell Ki67 expression 4.7-fold (range 4–16) in the HIV-

infected subjects, but only 1.7-fold (range 1.3–2.2) in the unin-

fected subjects. This difference was statistically signiﬁcant (p ⫽

0.01), and leads to the misleading conclusion that naive CD4

⫹

cell Ki67 expression is increased in HIV-infected individuals. An

example of the effect of inclusion of transitional T cells on naive

CD4

⫹

T cell Ki67 expression is shown in Fig. 5. These T cells may

have recently been naive cells that are transitioning to activated

cells and that will proliferate. Thus, accurate measurement of ac-

tivation and proliferation in naive CD4

⫹

T cells requires rigorous

phenotypic deﬁnition of this population by ﬂow cytometry.

Naive T cell TREC levels in early HIV infection

Therefore, to determine whether thymic output was decreased in

early HIV infection in the context of unchanged naive CD4

⫹

T cell

proliferation (and also to exclude memory T cell expansions as a

cause of decreased total T cell TREC), we calculated naive T cell

TREC from the total measured TREC and the percentage of naive

T cells determined by ﬂow cytometry. Our calculation assumed

that the contribution of TREC from memory T cells was negligi-

ble. This is a valid assumption, as we have measured TREC in

highly FACS-puriﬁed CD4

⫹

T cell populations from 12 individ

uals and have found that CD45RO

⫹

(memory) T cells have, on

average, only 2% of the TREC content of CD45RO

⫺

CD27

high

(naive) T cells in the same individual. Furthermore, as an example

of the concordance between calculated and actual measured naive

T cell TREC, we FACS sorted CD45RO

⫺

CD27

high

(naive) T cells

in one individual, measured TREC directly, and compared this

result with TREC calculated from unsorted T cells and the naive T

cell percentage, as shown in Tables I and II.

We found that both CD4

⫹

and CD8

⫹

naive T cell TREC in

early HIV infection were signiﬁcantly lower than in uninfected

age-matched controls ( p ⫽ 0.006 and 0.001, respectively; Fig. 6a).

Because the percentage of Ki67

⫹

CD8

⫹

CD45RO

⫺

CD27

high

(na

ive) T cells was increased in HIV-infected individuals, the reduced

naive CD8

⫹

TREC levels could have been solely or partly due to

increased naive CD8

⫹

T cell proliferation. However, the percent

age of Ki67

⫹

CD4

⫹

CD45RO

⫺

CD27

high

(naive) T cells was not

increased, and therefore, the decrease in TREC within

CD45RO

⫺

CD27

high

(naive) CD4

⫹

T cells could not have been

due to increased proliferation of cells in this compartment. This

suggests that the decreased naive CD4

⫹

T cell TREC levels caused

by HIV infection should be reversible when virus replication is

suppressed. To conﬁrm this, we longitudinally followed, after ini-

tiation of HAART, the CD45RO

⫺

CD27

high

(naive) CD4

⫹

T cell

TREC levels of ﬁve patients who had low TREC before therapy.

After initiation of HAART, which reduced viral loads to undetect-

able levels, CD45RO

⫺

CD27

high

(naive) CD4

⫹

T cell TREC rose

to normal levels, suggesting that changes in thymic output were

responsible for the changes in TREC within the naive CD4

⫹

cells before and after HAART (Fig. 6b).

Discussion

There is a considerable body of evidence that HIV can infect the

thymus, both in vitro and in vivo, and compromise its function

(10–14, 16, 46, 47). Recent studies of CD4

⫹

T cell depletion and

reconstitution in HIV infection have tended to appreciate the mul-

tifactorial nature of immune homeostasis—the composition of na-

ive and memory/effector pools in blood and lymphoid tissue, the

role of the thymus, and the effect of clinical stage of the disease

FIGURE 5. CD45RO

⫺

CD27

high

(naive) CD4

⫹

T cell Ki67 expression

increases with the inclusion of transitional T cells. Flow cytometric anal-

ysis is shown for two 29-year-old individuals, with the uninfected one on

the left and the HIV-infected one on the right. The panels show the deﬁ-

nition of naive T cells by CD27 and CD45RO and then the percentage of

Ki67

⫹

cells within that naive population. The top panels are tightly gated

on CD4

⫹

CD27

high

CD45RO

⫺

naive small lymphocytes, the middle panels

have a wider gate including transitional cells, and the bottom panels show

the result for a wider small lymphocyte gate. The percent Ki67

⫹

naive

CD4

⫹

T cells is shown in each panel.

Table I. TREC levels per 10

cells in CD45RO

⫺

CD27

high

(naive) and CD45RO

⫹

(memory) T cell subsets measured in sorted T cells in 12

individuals

Subject

TREC per 100,000 CD4

⫹

T cells

1 2 3 456789101112

CD45RO

⫺

CD27

high

9,106 5,271 24,072 4,224 356 5,960 906 8,256 418 496 1,427 2,209

CD45RO

⫹

13 0 30 34 0 168 90 968 0 0 0 0

6666 THYMIC OUTPUT IN HIV INFECTION

at NIH Library, National Institutes of Health on June 14, 2013http://www.jimmunol.org/Downloaded from

(32, 33, 42, 46, 48). The use of BrdU incorporation, Ki67 expres-

sion, stable isotope incorporation, and phenotypic deﬁnition of T

cell populations has led to a general consensus that CD4

⫹

and

CD8

⫹

T cell activation and turnover are increased in HIV infec

tion, that HIV infection leads to increased death of CD4

⫹

T cells,

that there is a defect in the renewal/replacement mechanisms for

CD4

⫹

T cells, that these replacement mechanisms have both a

peripheral and thymic component, and that naive CD4

⫹

T cell

recovery correlates with thymic size (1, 3, 7, 15, 27, 30–33, 42, 44,

45, 49).

The measurement of peripheral blood TREC provides insight

into both thymic function and T cell proliferation. If naive T cell

TREC levels decrease in the absence of an increase in prolifera-

tion, it may be concluded that there is a decrease in the supply of

new TREC

⫹

cells, most likely from the thymus. In this study, we

aimed to determine whether decreased thymic output, as well as

increased T cell turnover, occur in HIV infection. In individuals

experiencing a rebound of viral replication after interruption of

therapy, the rapid fall we observed in TREC levels clearly reﬂects

the increase in T cell proliferation. Although not impossible, it is

unlikely that the fall in TREC reﬂects a marked decrease in thymic

output, as it has recently been shown that TREC levels only begin

to decrease 3 mo after total thymectomy in HIV-uninfected indi-

viduals (41).

Therefore, we reasoned that if HIV infection suppressed thymic

output, we would be able to detect this effect at least 3 mo after

infection. Indeed, our results from the analysis of individuals with

early HIV infection 3–12 mo after seroconversion suggest that

decreased thymic output begins to affect T cell homeostasis by this

stage of the disease. TREC levels were decreased in both CD4

⫹

and CD8

⫹

T cell subsets, and were also signiﬁcantly decreased

when the CD45RO

⫺

CD27

high

(naive) T cell TREC were calcu

lated. As both CD45RO

⫹

(memory) and CD45RO

⫺

CD27

high

(naive)

CD8

⫹

T cell populations had higher Ki67 expression in HIV-infected

individuals, it was not possible to distinguish between the effects of

increased proliferation and reduced thymic output for CD8

⫹

T cells.

However, the percentage of Ki67

⫹

CD4

⫹

CD45RO

⫺

CD27

high

(naive)

T cells did not increase. The ﬁnding that TREC within naive CD4

⫹

T cells were signiﬁcantly lower in infected individuals in the con-

text of unaltered naive CD4

⫹

T cell proliferation suggests that the

input of TREC

⫹

naive CD4

⫹

T cells into the peripheral naive T

cell pool from a “source” has decreased. The thymus is the most

likely source for such cells (50).

However, some studies have found that the percentage of

Ki67

⫹

CD4

⫹

naive T cells increased in HIV-infected individuals

(1, 31). The discrepancy between our data and these studies could

be due a number of reasons; for example, our subjects had a higher

mean CD4 counts, and some Ki67

⫹

cells might be nondividing (3,

43). However, as we have shown, it is more likely that the incor-

poration of transitional T cells, cells that were naive and have now

become activated, into the phenotypically deﬁned naive T cell sub-

set accounts for the apparent increase in CD4

⫹

naive T cell acti

vation and/or proliferation in such studies.

Thus, our data show that by 3 mo after HIV infection, a decrease

in TREC within CD45RO

⫺

CD27

high

(naive) CD4

⫹

T cells is clearly

detectable in the absence of an increase in CD45RO

⫺

CD27

high

(na

ive) CD4

⫹

T cell proliferation, which suggests a decrease in thy

mic output. It is possible that the decrease in TREC could be due

to members of the naive pool entering cell cycle, losing TREC due

to dilution, and then reverting to a naive phenotype. However,

there is no evidence in humans for activated or memory CD4

⫹

cells reverting to a CD45RO

⫺

CD27

high

phenotype. The effect of

HIV on thymic function was further conﬁrmed with the observa-

tion that in those individuals with low TREC, CD45RO

⫺

CD27

high

(naive) CD4

⫹

T cell TREC increased with suppression of virus on

HAART. This suggests that the thymus can recover from its sup-

pression during HIV infection. It is important to note that an in-

crease in naive T cell TREC can only occur in the context of active

thymic output. Therefore, even if decreases in CD4

⫹

T cell TREC

occurred due to proliferation and the thymus remained unaffected

by HIV, the increase in naive T cell TREC during HAART indi-

cates that the thymus contributes to immune reconstitution. An

appreciation of the relative roles of the thymus and peripheral T

cell pool in immune reconstitution in HIV infection may provide a

framework for the rational design of interventions that accelerate

and improve the nature of T cell reconstitution in HIV-infected

individuals.

Acknowledgments

We thank Dr. J. Sullivan for samples and Drs. L. Picker and Z. Grossman

for advice.

FIGURE 6. CD45RO

⫺

CD27

high

(naive) T cell TREC in early HIV in

fection and its treatment. a, CD45RO

⫺

CD27

high

(naive) T cell TREC lev

els in sorted CD4

⫹

and CD8

⫹

T cells from uninfected individuals (E) and

individuals with early HIV infection (F), calculated from the percentage of

CD45RO

⫺

CD27

high

(naive) T cells present in each population. Best-ﬁt

exponential regression curves for uninfected individuals are shown. b, In-

creases during HAART in CD45RO

⫺

CD27

high

(naive) CD4

⫹

T cell TREC

in ﬁve patients indicated in a who had low TREC before therapy. CD4

⫹

cell counts per microliter at the start of treatment for the ﬁve patients were:

1, 408; 2, 521; 3, 850; 4, 761; and 5, 416.

Table II. TREC levels per 10

cells in CD45RO

⫺

CD27

high

(naive) and CD45RO

⫹

(memory) T cell subsets

measured and back-calculated in one individual

% CD45RO

⫺

CD27

high

Naive CD4

⫹

T Cells

MACS-Sorted

CD4

⫹

T Cell

TREC

Calculated Naive

CD4

⫹

T Cell

TREC

FACS-Sorted Naive

CD4

⫹

T Cell TREC

FACS-Sorted Memory

CD4

⫹

T Cell TREC

34.06% 692 2,032 2,054 6

6667The Journal of Immunology

at NIH Library, National Institutes of Health on June 14, 2013http://www.jimmunol.org/Downloaded from

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6668 THYMIC OUTPUT IN HIV INFECTION

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Are homeostatic mechanisms aiding the reconstitution of the T-cell pool during lymphopenia in humans?

Article

Full-text available

Nov 2022

A timely recovery of T-cell numbers following haematopoietic stem-cell transplantation (HSCT) is essential for preventing complications, such as increased risk of infection and disease relapse. In analogy to the occurrence of lymphopenia-induced proliferation in mice, T-cell dynamics in humans are thought to be homeostatically regulated in a cell density-dependent manner. The idea is that T cells divide faster and/or live longer when T-cell numbers are low, thereby helping the reconstitution of the T-cell pool. T-cell reconstitution after HSCT is, however, known to occur notoriously slowly. In fact, the evidence for the existence of homeostatic mechanisms in humans is quite ambiguous, since lymphopenia is often associated with infectious complications and immune activation, which confound the study of homeostatic regulation. This calls into question whether homeostatic mechanisms aid the reconstitution of the T-cell pool during lymphopenia in humans. Here we review the changes in T-cell dynamics in different situations of T-cell deficiency in humans, including the early development of the immune system after birth, healthy ageing, HIV infection, thymectomy and hematopoietic stem cell transplantation (HSCT). We discuss to what extent these changes in T-cell dynamics are a side-effect of increased immune activation during lymphopenia, and to what extent they truly reflect homeostatic mechanisms.

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Jul 2021

D Gibb

It is intriguing that, unlike adults with HIV-1, children with HIV-1 reach a greater CD4+ T cell recovery following planned treatment cessation. The reasons for the better outcomes in children remain unknown but may be related to increased thymic output and diversity of T cell receptor repertoires. HIV-1 infected children from the PENTA 11 trial tolerated planned treatment interruption without adverse long-term clinical, virological, or immunological consequences, once antiretroviral therapy was re-introduced. This contrasts to treatment interruption trials of HIV-1 infected adults, who had rapid changes in T cells and slow recovery when antiretroviral therapy was restarted. How children can develop such effective immune responses to planned treatment interruption may be critical for future studies. PENTA 11 was a randomized, phase II trial of planned treatment interruptions in HIV-1-infected children (ISRCTN 36694210). In this sub-study, eight patients in long-term follow-up were chosen with CD4+ count>500/ml, viral load <50c/ml at baseline: four patients on treatment interruption and four on continuous treatment. Together with measurements of thymic output, we used high-throughput next generation sequencing and bioinformatics to systematically organize memory CD8+ and naïve CD4+ T cell receptors according to diversity, clonal expansions, sequence sharing, antigen specificity, and T cell receptor similarities following treatment interruption compared to continuous treatment. We observed an increase in thymic output following treatment interruption compared to continuous treatment. This was accompanied by an increase in T cell receptor clonal expansions, increased T cell receptor sharing, and higher sequence similarities between patients, suggesting a more focused T cell receptor repertoire. The low numbers of patients included is a limitation and the data should be interpreted with caution. Nonetheless, the high levels of thymic output and the high diversity of the T cell receptor repertoire in children may be sufficient to reconstitute the T cell immune repertoire and reverse the impact of interruption of antiretroviral therapy. Importantly, the effective T cell receptor repertoires following treatment interruption may inform novel therapeutic strategies in children infected with HIV-1.

Bezlotoxumab prevents extraintestinal organ damage induced by Clostridioides difficile infection

Article

Full-text available

Aug 2022

Clostridioides difficile is the most common cause of infectious antibiotic-associated diarrhea, with disease mediated by two major toxins TcdA and TcdB. In severe cases, systemic disease complications may arise, resulting in fatal disease. Systemic disease in animal models has been described, with thymic damage an observable consequence of severe disease in mice. Using a mouse model of C. difficile infection, we examined this disease phenotype, focussing on the thymus and serum markers of systemic disease. The efficacy of bezlotoxumab, a monoclonal TcdB therapeutic, to prevent toxin mediated systemic disease complications was also examined. C. difficile infection causes toxin-dependent thymic damage and CD4⁺CD8⁺ thymocyte depletion in mice. These systemic complications coincide with changes in biochemical markers of liver and kidney function, including increased serum urea and creatinine, and hypoglycemia. Administration of bezlotoxumab during C. difficile infection prevents systemic disease and thymic atrophy, without blocking gut damage, suggesting the leakage of gut contents into circulation may influence systemic disease. As the thymus has such a crucial role in T cell production and immune system development, these findings may have important implications in relapse of C. difficile disease and impaired immunity during C. difficile infection. The prevention of thymic atrophy and reduced systemic response following bezlotoxumab treatment, without altering colonic damage, highlights the importance of systemic disease in C. difficile infection, and provides new insights into the mechanism of action for this therapeutic. Abbreviations: Acute kidney injury (AKI); Alanine Transaminase (ALT); Aspartate Aminotransferase (AST); C. difficile infection (CDI); chronic kidney disease (CKD); combined repetitive oligo-peptides (CROPS); cardiovascular disease (CVD); Double positive (DP); hematoxylin and eosin (H&E); immunohistochemical (IHC); multiple organ dysfunction syndrome (MODS); phosphate buffered saline (PBS); standard error of the mean (SEM); surface layer proteins (SLP); Single positive (SP); wild-type (WT).

Systemic Immune Dysfunction in Cancer Patients Driven by IL6 Induction of LAG3 in Peripheral CD8+ T Cells

Article

May 2022

Many cancer patients do not develop a durable response to the current standard of care immunotherapies, despite substantial advances in targeting immune inhibitory receptors. A potential compounding issue, which may serve as an unappreciated, dominant resistance mechanism, is an inherent systemic immune dysfunction that is often associated with advanced cancer. Minimal response to inhibitory receptor (IR) blockade therapy and increased disease burden have been associated with peripheral CD8+ T-cell dysfunction, characterized by suboptimal T-cell proliferation and chronic expression of IRs (eg. Programmed Death 1 [PD1] and Lymphocyte Activation Gene 3 [LAG3]). Here, we demonstrated that approximately a third of cancer patients analyzed in this study have peripheral CD8+ T cells that expressed robust intracellular LAG3 (LAG3IC), but not surface LAG3 (LAG3SUR) due to A Disintegrin and Metalloproteinase domain-containing protein 10 (ADAM10) cleavage. This associated with poor disease prognosis and decreased CD8+ T-cell function, which could be partially reversed by anti-LAG3. Systemic immune dysfunction was restricted to CD8+ T cells, including, in some cases, a high percentage of peripheral naïve CD8+ T cells, and was driven by the cytokine IL6 via STAT3. These data suggest that additional studies are warrented to determine if the combination of increased LAG3IC in peripheral CD8+ T cells and elevated systemic IL6 can serve as predictive biomarkers and identify which cancer patients may benefit from LAG3 blockade.

Early lire imprints the hierarchy of T cell clone sizes

Article

Feb 2021
eLife

The adaptive immune system responds to pathogens by selecting clones of cells with specific receptors. While clonal selection in response to particular antigens has been studied in detail, it is unknown how a lifetime of exposures to many antigens collectively shape the immune repertoire. Here, using mathematical modeling and statistical analyses of T cell receptor sequencing data, we develop a quantitative theory of human T cell dynamics compatible with the statistical laws of repertoire organization. We find that clonal expansions during a perinatal time window leave a long-lasting imprint on the human T cell repertoire, which is only slowly reshaped by fluctuating clonal selection during adult life. Our work provides a mechanism for how early clonal dynamics imprint the hierarchy of T cell clone sizes with implications for pathogen defense and autoimmunity.

Mucosal‐associated invariant T cells: A cryptic coordinator in HIV‐infected immune reconstitution

Article

Mar 2022

Human immunodeficiency virus type 1 (HIV‐1) infection causes considerable morbidity and mortality worldwide. Although antiretroviral therapy (ART) has largely transformed HIV infection from a fatal disease to a chronic condition, approximately 10%~40% of HIV‐infected individuals who receive effective ART and sustain long‐term viral suppression still cannot achieve optimal immune reconstitution. These patients are called immunological non‐responders, a state associated with poor clinical prognosis. Mucosal‐associated invariant T (MAIT) cells are an evolutionarily conserved unconventional T cell subset defined by expression of semi‐invariant αβ T cell receptor (TCR), which recognizes metabolites derived from the riboflavin biosynthetic pathway presented on major histocompatibility complex (MHC)‐related protein‐1 (MR1). MAIT cells, which are considered to act as a bridge between innate and adaptive immunity, produce a wide range of cytokines and cytotoxic molecules upon activation through TCR‐dependent and TCR‐independent mechanisms, which is of major importance in defense against a variety of pathogens. In addition, MAIT cells are involved in autoimmune and immune‐mediated diseases. The number of MAIT cells is dramatically and irreversibly decreased in the early stage of HIV infection and is not fully restored even after long‐term suppressive ART. In light of the important role of MAIT cells in mucosal immunity and because microbial translocation is inversely associated with CD4+ T cell counts, we propose that MAIT cells participate in the maintenance of intestinal barrier integrity and microbial homeostasis, thus further affecting immune reconstitution in HIV‐infected individuals. This article is protected by copyright. All rights reserved.

Determining role of thymus in immune pathogenesis of autoimmune, oncological and infectious diseases

Article

Feb 2023

V. A. Kozlov

Research data presented in the article are based, mainly, on the concept that thymus, together with bone marrow represent the central organ of immune system being the source of all T cell populations that, following their migration from thymus to periphery, participate in development of immune response to any antigens of viral, bacterial and tissue origin, and to any allergens. This difference is principal, as opposed to the bone marrow which produces other members of immune response, i.e., dendritic cells, macrophages, B cells. E.g., the bone marrow also generates the cells which migrate to thymus where they undergo differentiation to the T cells. Over last 50 years, a plethora of data was accumulated on the leading role of immune system in pathogenesis of virtually all socially significant human diseases affecting the modern mankind, including infectious and malignant disorders, atherosclerosis, autoimmune and allergic diseases. Moreover, current studies show that the aberrant functions of different T cell populations play the leading role in pathogenesis of these diseases. These T cell disturbances in peripheral areas of different organs are proven to develop, mainly in the thymic area. Hence, thymus is a producing organ of T cells with altered functional activities which promote pathogenetic changes in these disorders. Currently, the entire set of immunotherapeutic approaches is aimed for correction of disturbances among the same T cells subpopulations at periphery, without taking into account thymic mechanisms which have induced these disturbances before their emigration from thymus. One should, therefore, develop novel methods and approaches to correct these alterations within thymic area.

SARS-CoV-2 infection of thymus induces loss of function that correlates with disease severity

Article

Full-text available

Feb 2023
J ALLERGY CLIN IMMUN

Background: Lymphopenia, particularly restricted to the T-cell compartment, has been described as one of the major clinical hallmarks in COVID-19 patients and proposed as a disease severity indicator. Although several mechanisms fostering COVID-19-related lymphopenia have been described, including cell apoptosis and tissue homing, the underlying causes of the decline in T-cell count and function are still not completely understood. Objective: Given that viral infections can directly target thymic microenvironment and impair the process of T-cell generation, we sought to investigate the impact of SARS-CoV-2 on thymic function. Methods: We performed molecular quantification of T-cell receptor excision circles (TRECs) and kappa-deleting recombination excision circles (KRECs) to assess, respectively, T- and B-cell neogenesis in SARS-CoV-2-infected patients. We developed in vitro culture system of primary human thymic epithelial cells (TECs) to mechanistically investigate the impact of SARS-CoV-2 on TEC function. Results: We showed that COVID-19 patients had reduced thymic function, which was inversely associated with the severity of the disease. We found that angiotensin-converting enzyme 2 (ACE2), through which SARS-CoV-2 enters the host cells, was expressed by thymic epithelium, and in particular by medullary TECs. We also demonstrated that SARS-CoV-2 can target TECs and downregulate critical genes and pathways associated with epithelial cell adhesion and survival. Conclusions: Our data demonstrate that the human thymus is a target of SARS-CoV-2 and thymic function is altered upon infection. These findings expand our current knowledge of the effects of SARS-CoV-2 infection on T-cell homeostasis and suggest that monitoring thymic activity may represent a useful marker to predict disease severity and progression.

Клинико-иммунологические фенотипы течения расстройств аутистического спектра

Article

Full-text available

Nov 2022

РАС страдает каждый 59-й ребенок. У пациентов с РАС чаще, чем в популяции в целом, присутствуют сопутствующие расстройства, которые могут усугубить течение основного заболевания или повлиять на постановку диагноза. Цель работы – выделить клинико-иммунологические фенотипы течения РАС. Материалом исследования служили образцы крови детей двух групп исследования: дети с РАС (n=100) и клинически здоровые (n=30). На основании наличия сопутствующих заболеваний дети были разделены на 3 фенотипа: судорожный, инфекционный, дермато-респираторный и желудочно-кишечный. В сыворотке крови определяли концентрации цитокинов IL-4, IL-6, IL-10, IFN-γ, IL-17А. Модифицированным методом ИФА с применением методологии Immunohealth™ определяли концентрацию spIgG к 111 пищевым антигенам (пАГ). Оценку показателей когнитивных и психофизиологических показателей у детей проводили с помощью анкеты АТЕС. В результате исследования выделены клинико-иммунологические фенотипы течения РАС, связанные с особым типом пищевой реактивности, цитокиновым профилем, клинической тяжестью психо-физиологических расстройств и сопутствующими коморбидными заболеваниями. Во всех четырех фенотипах установлена повышенная активность синтеза специфических антител, связанного с изученным пАГ гуморального иммунитета, повышенная концентрация суммарного spIgG к пАГ и концентрации spIgG к бобовым продуктам и казеину, значения С-реактивного белка. При этом в судорожном фенотипе (сопутствующие эпилепсия и судороги) максимальные концентрации spIgG установлены к паслёновым продуктам, повышена концентрация IL-10 и снижена концентрация IL-4, снижено содержание сывороточного железа и ферритина. В инфекционном фенотипе (часто болеющие дети) – к зерновым и бродильным продуктам, повышены концентрации IL-10 и IFN-γ и снижена концентрация IL-4, повышены абсолютное и относительное количество лимфоцитов и фибриноген. В дермато-респираторном фенотипе (кожные высыпания) – к молочным продуктам, повышены концентрации IL-4 и IL-17А. В желудочно-кишечном фенотипе зарегистрировано самое высокое количество реакций повышенных IgG к наибольшему спектру пАГ на фоне тенденции изменения в цитокиновом профиле в сторону повышения IFN-γ в соотношениях IFN=γ/IL-4 и IFN=γ/IL-10. Таким образом, выделенные фенотипы течения РАС связаны с влиянием пАГ и отражают особый вариант иммунологического воспалительного патогенеза, что позволяет персонифицировать элиминационные диеты, предложить мероприятия по коррекции и индивидуальной профилактике и, вероятно, рассчитать прогноз течения заболевания.

Human Immunodeficiency Virus and Allergic Disease

Chapter

Jan 2009

T Cell Turnover in SIV Infection

Article

Full-text available

Apr 1999

In their report of 20 February 1998 ([1][1]), H. Mohri et al . analyze the proliferation and death of T cells in normal macaques and in those infected with simian immunodeficiency virus (SIV). With the use of bromodeoxyuridine (BrdU) to label proliferating cells, they observed that the uptake of

Measuring Recent Thymic Emigrants in Blood of Normal and HIV-1–Infected Individuals before and after Effective Therapy

Article

Full-text available

Sep 1999
J EXP MED

The role of the thymus in HIV-1 pathogenesis remains unclear. We developed an assay to quantify the number of recent thymic emigrants in blood based on the detection of a major excisional DNA byproduct (termed α1 circle) of T cell receptor rearrangement. By studying 532 normal individuals, we found that α1 circle numbers in blood remain high for the first 10–15 yr of life, a sharp drop is seen in the late teen years, and a gradual decline occurs thereafter. Compared with age-matched uninfected control individuals, α1 circle numbers in HIV-1–infected adults were significantly reduced; however, there were many individuals with normal α1 circle numbers. In 74 individuals receiving highly active antiretroviral therapy, we found no appreciable effect on α1 circle numbers in those whose baseline values were already within the normal range, but significant increases were observed in those with a preexisting impairment. The increases in α1 circle numbers were, however, numerically insufficient to account for the rise in levels of naive T lymphocytes. Overall, it is difficult to invoke thymic regenerative failure as a generalized mechanism for CD4 lymphocyte depletion in HIV-1 infection, as α1 circle numbers are normal in a substantial subset of HIV-1–infected individuals.

HIV-1 and T Cell Dynamics after Interruption of Highly Active Antiretroviral Therapy (HAART) in Patients with a History of Sustained Viral Suppression

Article

Full-text available

Dec 1999
P NATL ACAD SCI USA

Identifying the immunologic and virologic consequences of discontinuing antiretroviral therapy in HIV-infected patients is of major importance in developing long-term treatment strategies for patients with HIV-1 infection. We designed a trial to characterize these parameters after interruption of highly active antiretroviral therapy (HAART) in patients who had maintained prolonged viral suppression on antiretroviral drugs. Eighteen patients with CD4+ T cell counts ≥ 350 cells/μl and viral load below the limits of detection for ≥1 year while on HAART were enrolled prospectively in a trial in which HAART was discontinued. Twelve of these patients had received prior IL-2 therapy and had low frequencies of resting, latently infected CD4 cells. Viral load relapse to >50 copies/ml occurred in all 18 patients independent of prior IL-2 treatment, beginning most commonly during weeks 2–3 after cessation of HAART. The mean relapse rate constant was 0.45 (0.20 log10 copies) day−1, which was very similar to the mean viral clearance rate constant after drug resumption of 0.35 (0.15 log10 copies) day−1 (P = 0.28). One patient experienced a relapse delay to week 7. All patients except one experienced a relapse burden to >5,000 RNA copies/ml. Ex vivo labeling with BrdUrd showed that CD4 and CD8 cell turnover increased after withdrawal of HAART and correlated with viral load whereas lymphocyte turnover decreased after reinitiation of drug treatment. Virologic relapse occurs rapidly in patients who discontinue suppressive drug therapy, even in patients with a markedly diminished pool of resting, latently infected CD4+ T cells.

HIV-1 directly kills CD4+ T cells by a Fas-independent mechanism

Article

Full-text available

Apr 1998
J EXP MED

The mechanism by which HIV-1 induces CD4(+) T cell death is not known. A fundamental issue is whether HIV-1 primarily induces direct killing of infected cells or indirectly causes death of uninfected bystander cells. This question was studied using a reporter virus system in which infected cells are marked with the cell surface protein placental alkaline phosphatase (PLAP). Infection by HIV-PLAP of peripheral blood mononuclear cells (PBMCs) and T cell lines leads to rapid depletion of CD4(+) T cells and induction of apoptosis. The great majority of HIV-induced T cell death in vitro involves direct loss of infected cells rather than indirect effects on uninfected bystander cells. Because of its proposed role in HIV-induced cell death, we also examined the Fas (CD95/Apo1) pathway in killing of T cells by HIV-1. Infected PBMCs or CEM cells display no increase in surface Fas relative to uninfected cells. In addition, HIV-1 kills CEM and Jurkat T cells in the presence of a caspase inhibitor that completely blocks Fas-mediated apoptosis. HIV-1 also depletes CD4+ T cells in PBMCs from patients who have a genetically defective Fas pathway. These results suggest that HIV-1 induces direct apoptosis of infected cells and kills T cells by a Fas-independent mechanism.

Essential Role of the Thymus to Reconstitute Naive (CD45RA+) T-Helper Cells After Human Allogeneic Bone Marrow Transplantation

Article

Jul 1997

To contribute to the understanding of the role of the thymus in humans in the reconstitution of naive (CD45RA+) T cells after bone marrow transplantation (BMT), we compared T-cell regeneration in a unique situation, namely a thymectomized cancer patient (15 years old), with that of thymus-bearing patients after allogeneic BMT. These cases shared features of transplantation (total body irradiation, HLA-matched donors, and graft-versus-host disease prophylaxis with cyclosporine A) and all had an uncomplicated posttransplantation course. As shown by fluorescence-activated cell sorting analyses, the thymectomized host failed to reconstitute CD45RA+ T-helper cells even 24 months after BMT (11% CD45RA+ of CD4+ cells). In this patient, preferentially CD45RO+ cells contributed to the recovery of CD4+ cells (206 of 261/μL at 6 months and 463 of 558/μL at 24 months after BMT, CD45RA+ of CD4+ cells), whereas CD45RA+ cells remained low (<60/μL). In contrast, nine thymus-bearing hosts (5 children and 4 adults) examined between 6 and 24 months after BMT effectively reconstituted CD4+/CD45RA+ cells according to their normal age-related range (≥28% in adults and ≥50% in children). Five of these were analyzed sequentially at 6 and 9 months after BMT. Within this period, CD45RA+ cells increasingly contributed to the recovery of CD4+ cells (median, +21%), even when total CD4+ cells decreased. With respect to T-cytotoxic/suppressor cells, the thymectomized host retained the capacity to recover CD45RA+ cells (137 of 333/μL at 6 months and 596 of 1,046/μL at 24 months after BMT, CD45RA+ of CD8+ cells), a proportion similar to that seen in thymus-bearing hosts. These findings suggest that a thymus-independent pathway exists to regenerate CD45RA+ T-cytotoxic/suppressor cells, but residual thymus is essential to reconstitute naive (CD45RA+) T-helper cells after BMT in humans.

T-cell division in human immunodeficiency virus (HIV)-1 infection is mainly due to immune activation: a longitudinal analysis in patients before and during highly active antiretroviral therapy (HAART)

Article

Jan 2000

In human immunodeficiency virus (HIV)-1 infection, highly increased T-cell turnover was proposed to cause exhaustion of lymphocyte production and consequently development of AIDS. Here, we investigated cell proliferation, as measured by expression of the Ki-67 nuclear antigen, in peripheral blood CD4+ and CD8+ lymphocyte subpopulations before and during highly active antiretroviral therapy (HAART). In untreated HIV-1 infection, both the percentage and number of Ki-67+CD4+ and CD8+ lymphocytes were significantly increased, compared with values obtained from healthy individuals. A more than 10-fold increase in the percentage of dividing naive CD4+ T cells in the blood was found when the number of these cells were below 100 per μL.. HAART induced an immediate decline in Ki-67 antigen expression, despite often very low CD4+ T-cell numbers, arguing against increased proliferation being a homeostatic response. After approximately 24 weeks of HAART treatment, a transient increase in the number of proliferating cells was seen, but only in the CD4+CD27+ memory pool. In the CD8+ T-cell compartment, the number of dividing cells was elevated 20- to 25-fold. This increase was most notable in the CD27+ CD 45RO+ and CD27−CD45RO+ memory CD8+ T-cell pool, corresponding with the degree of expansion of these subsets. Reduction of plasma HIV-RNA load by HAART was accompanied by a decrease in numbers and percentages of dividing cells in all CD8+T-cell subsets. Taken together, our results indicate that peripheral T-cell proliferation is a consequence of generalized immune activation. (Blood. 2000;95:249-255)

Changes in thymic function with age and during the treatment of HIV infection

Article

Jan 1998
NATURE

Factors affecting thymic function after allogeneic hematopoietic stem cell transplantation

Article

Mar 2001

Kenneth Weinberg

Hematopoietic stem cell transplantation (HSCT) is followed by profound immunodeficiency. Thymic function is necessary for de novo generation of T cells after HSCT. Circulating CD45RA+ naive T-cell levels are predictive of antigen-specific T-cell responses in the absence of graft-versus-host disease (GVHD). These T cells may not represent recent thymic emigrants, since naive T cells may maintain this phenotype if not antigen-activated. To accurately measure thymic output after HSCT and determine the factors that influence thymic function, T-cell receptor excision circles (TRECs) were examined in CD4+ and CD8+ cells from a cross-section of patients following HSCT. TREC levels rose weeks after HSCT and could be detected in patients 6 years after HSCT. TREC levels correlated with the frequency of phenotypically naive T cells, indicating that such cells were not expanded progeny of naive T cells present in the donor graft. Chronic GVHD was the most important factor that predicted low TREC levels even years after HSCT. Patients with a history of resolved GVHD had decreased numbers of TREC, compared with those with no GVHD. Because few adults had no history of GVHD, it was not possible to determine whether age alone inversely correlated with TREC levels. Recipients of cord blood grafts had no evidence of decreased TREC induced by immunosuppressive prophylaxis drugs. Compared with unrelated donor grafts, recipients of matched sibling grafts had higher TREC levels. Collectively, these data suggest that thymopoiesis is inhibited by GVHD. Larger studies will be needed to determine the independent contributions of age and preparative regimen to post-transplant thymopoietic capacity.

Thymic Size and Lymphocyte Restoration in Patients with Human Immunodeficiency Virus Infection after 48 Weeks of Zidovudine, Lamivudine, and Ritonavir Therapy

Article

Jan 2000

Human immunodeficiency virus (HIV) infection is associated with progressive loss of circulating CD4+ lymphocytes. Treatment with highly active antiretroviral therapy (HAART) has led to increases in CD4+ T lymphocytes of naive (CD45RA+62L+) and memory (CD45R0+RA−) phenotypes. Thymic computerized tomography scans were obtained on 30 individuals with HIV disease to investigate the role of the thymus in cellular restoration after 48 weeks of HAART. Individuals with abundant thymic tissue had higher naive CD4+ T lymphocyte counts at weeks 2–24 after therapy than individuals with minimal thymic tissue. Individuals with abundant thymic tissue had significantly larger increases in naive CD4+ cells during the first 4 weeks of therapy. These individuals were also more likely to experience viral rebound despite comparable initial declines in plasma HIV-1 RNA. These findings suggest that there is a complex relationship among the thymus, viral replication, and lymphocyte restoration after application of HAART in HIV disease.

T-cell division in human immunodeficiency virus (HIV)-1 infection is mainly due to immune activation: A longitudinal analysis in patients before and during highly active antiretroviral therapy (HAART)

Article

Jan 2000

In human immunodeficiency virus (HIV)-1 infection, highly increased T-cell turnover was proposed to cause exhaustion of lymphocyte production and consequently development of AIDS. Here, we investigated cell proliferation, as measured by expression of the Ki-67 nuclear antigen, in peripheral blood CD4(+) and CD8(+) lymphocyte subpopulations before and during highly active antiretroviral therapy (HAART). In untreated HIV-1 infection, both the percentage and number of Ki-67(+) CD4(+) and CD8(+) lymphocytes were significantly increased, compared with values obtained from healthy individuals. A more than 10-fold increase in the percentage of dividing naive CD4(+) T cells in the blood was found when the number of these cells were below 100 per microL. HAART induced an immediate decline in Ki-67 antigen expression, despite often very low CD4(+) T-cell numbers, arguing against increased proliferation being a homeostatic response. After approximately 24 weeks of HAART treatment, a transient increase in the number of proliferating cells was seen, but only in the CD4(+) CD27(+) memory pool. In the CD8(+) T-cell compartment, the number of dividing cells was elevated 20- to 25-fold. This increase was most notable in the CD27(+) CD 45RO(+) and CD27(-) CD45RO(+) memory CD8(+) T-cell pool, corresponding with the degree of expansion of these subsets. Reduction of plasma HIV-RNA load by HAART was accompanied by a decrease in numbers and percentages of dividing cells in all CD8(+) T-cell subsets. Taken together, our results indicate that peripheral T-cell proliferation is a consequence of generalized immune activation. (Blood. 2000;95:249-255)

Evidence for Increased T Cell Turnover and Decreased Thymic Output in HIV Infection

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